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Sample records for tetrameric human thymidine

  1. 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...

  2. Expression of fully functional tetrameric human hemoglobin in Escherichia coli

    International Nuclear Information System (INIS)

    Hoffman, S.J.; Looker, D.L.; Roehrich, J.M.; Cozart, P.E.; Durfee, S.L.; Tedesco, J.L.; Stetler, G.L.

    1990-01-01

    Synthesis genes encoding the human α- and β-globin polypeptides have been expressed from a single operon in Escherichia coli. The α- and β-globin polypeptides associate into soluble tetramers, incorporate heme, and accumulate to >5% of the total cellular protein. Purified recombinant hemoglobin has the correct stoichiometry of α- and β-globin chains and contains a full complement of heme. Each globin chain also contains an additional methionine as an extension to the amino terminus. The recombinant hemoglobin has a C 4 reversed-phase HPLC profile essentially identical to that of human hemoglobin A 0 and comigrates with hemoglobin A 0 on SDS/PAGE. The visible spectrum and oxygen affinity are similar to that of native human hemoglobin A 0 . The authors have also expressed the α- and β-globin genes separately and found that the expression of the α-globin gene alone results in a marked decrease in the accumulation of α-globin in the cell. Separate expression of the β-globin gene results in high levels of insoluble β-globin. These observations suggest that the presence of α- and β-globin in the same cell stabilizes α-globin and aids the correct folding of β-globin. This system provides a simple method for expressing large quantities of recombinant hemoglobin and allows facile manipulation of the genes encoding hemoglobin to produce functionally altered forms of this protein

  3. 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

  4. 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

  5. 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...

  6. 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.

  7. 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.

  8. Crystallization and preliminary X-ray diffraction studies of the tetramerization domain derived from the human potassium channel Kv1.3

    International Nuclear Information System (INIS)

    Winklmeier, Andreas; Weyand, Michael; Schreier, Christina; Kalbitzer, Hans Robert; Kremer, Werner

    2009-01-01

    The tetramerization domain of human Kv1.3 was cloned, expressed, purified and crystallized. The crystals belonged to space group I4 and diffracted to 1.2 Å resolution using synchrotron radiation. The tetramerization domain (T1 domain) derived from the voltage-dependent potassium channel Kv1.3 of Homo sapiens was recombinantly expressed in Escherichia coli and purified. The crystals were first grown in an NMR tube in 150 mM potassium phosphate pH 6.5 in the absence of additional precipitants. The crystals showed I4 symmetry characteristic of the naturally occurring tetrameric assembly of the single subunits. A complete native data set was collected to 1.2 Å resolution at 100 K using synchrotron radiation

  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. 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

  11. 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

  12. 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.

  13. 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.

  14. Reversible tetramerization of human TK1 to the high catalytic efficient form is induced by pyrophosphate, in addition to tripolyphosphates, or high enzyme concentration

    DEFF Research Database (Denmark)

    Munch-Petersen, Birgitte

    2009-01-01

    of ATP is necessary for tetramerisation and how the reaction velocity is influenced by the enzyme concentration. The results show that only two or three of the phosphate groups of ATP are necessary for tetramerisation, and that kinetics and tetramerisation are closely related. Furthermore, enzyme...... concentration was found to have a pivotal effect on catalytic efficiency.......Thymidine kinase (TK1) is a key enzyme in the salvage pathway of deoxyribonucleotide metabolism catalyzing the first step in the synthesis of dTTP by the transfer of a gamma-phosphate group from a nucleoside triphosphate to the 5´-hydroxyl group of thymidine forming dTMP. Human TK1 is cytosolic...

  15. 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

  16. 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...

  17. 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...

  18. 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

  19. 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.

  20. 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...

  1. 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

  2. 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

  3. 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

  4. Regulation of p53 tetramerization and nuclear export by ARC.

    Science.gov (United States)

    Foo, Roger S-Y; Nam, Young-Jae; Ostreicher, Marc Jason; Metzl, Mark D; Whelan, Russell S; Peng, Chang-Fu; Ashton, Anthony W; Fu, Weimin; Mani, Kartik; Chin, Suet-Feung; Provenzano, Elena; Ellis, Ian; Figg, Nichola; Pinder, Sarah; Bennett, Martin R; Caldas, Carlos; Kitsis, Richard N

    2007-12-26

    Inactivation of the transcription factor p53 is central to carcinogenesis. Yet only approximately one-half of cancers have p53 loss-of-function mutations. Here, we demonstrate a mechanism for p53 inactivation by apoptosis repressor with caspase recruitment domain (ARC), a protein induced in multiple cancer cells. The direct binding in the nucleus of ARC to the p53 tetramerization domain inhibits p53 tetramerization. This exposes a nuclear export signal in p53, triggering Crm1-dependent relocation of p53 to the cytoplasm. Knockdown of endogenous ARC in breast cancer cells results in spontaneous tetramerization of endogenous p53, accumulation of p53 in the nucleus, and activation of endogenous p53 target genes. In primary human breast cancers with nuclear ARC, p53 is almost always WT. Conversely, nearly all breast cancers with mutant p53 lack nuclear ARC. We conclude that nuclear ARC is induced in cancer cells and negatively regulates p53.

  5. 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

  6. 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

  7. 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...

  8. 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

  9. 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

  10. 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

  11. Somatostatin reduces 3H-thymidine incorporation and c-myc, but not thyroglobulin ribonucleic acid levels in human thyroid follicular cells in vitro

    International Nuclear Information System (INIS)

    degli Uberti, E.C.; Hanau, S.; Rossi, R.; Piva, R.; Margutti, A.; Trasforini, G.; Pansini, G.; del Senno, L.

    1991-01-01

    The action of somatostatin (SRIH) on 3 H-thymidine (thy) incorporation and on c-myc and thyroglobulin RNA levels in a suspension of follicles from normal and goitrous human thyroid was examined. SRIH, at 10 - 7 M concentration, inhibited basal thy incorporation (maximally by 4 h lasting for up 24 h), which effect was greater in goiter than in normal thyroid and was also detected in growing adherent epithelial cells. Moreover, in a follicle suspension SRIH prevented TSH-stimulated thy incorporation, both in normal and in goitrous thyroid. Basal expression of c-myc RNA was not affected by SRIH in either tissue, whereas the TSH-stimulated c-myc RNA level was significantly reduced in goiter. No effect of SRIH was observed on basal or TSH-stimulated thyroglobulin RNA levels. SRIH did not alter basal cAMP concentrations in normal or goitrous follicles, but it significantly reduced TSH-stimulated cAMP accumulation both in normal thyroid and in goiter. Overall, our data indicate a direct inhibitory action of SRIH on growth, but not on differentiation, of human thyroid, probably by a mechanism not entirely cAMP dependent

  12. [{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.

  13. Significant human beta-cell turnover is limited to the first three decades of life as determined by in vivo thymidine analog incorporation and radiocarbon dating.

    Science.gov (United States)

    Perl, S; Kushner, J A; Buchholz, B A; Meeker, A K; Stein, G M; Hsieh, M; Kirby, M; Pechhold, S; Liu, E H; Harlan, D M; Tisdale, J F

    2010-10-01

    Diabetes mellitus results from an absolute or relative deficiency of insulin-producing pancreatic β-cells. The turnover rate of adult human β-cells remains unknown. We employed two techniques to examine adult human islet β-cell turnover and longevity in vivo. Subjects enrolled in National Institutes of Health clinical trials received thymidine analogs [iododeoxyuridine (IdU) or bromodeoxyuridine (BrdU)] 8 d to 4 yr prior to death. Archival autopsy samples from 10 patients (aged 17-74 yr) were employed to assess β-cell turnover by scoring nuclear analog labeling within insulin-staining cells. Human adult β-cell longevity was determined by estimating the cells' genomic DNA integration of atmospheric (14)C. DNA was purified from pancreatic islets isolated from cadaveric donors; whole islet prep DNA was obtained from a 15-yr-old donor, and purified β-cell DNA was obtained from two donors (ages 48 and 80 yr). (14)C levels were then determined using accelerator mass spectrometry. Cellular "birth date" was determined by comparing the subject's DNA (14)C content relative to a well-established (14)C atmospheric prevalence curve. In the two subjects less than 20 yr of age, 1-2% of the β-cell nuclei costained for BrdU/IdU. No β-cell nuclei costained in the eight patients more than 30 yr old. Consistent with the BrdU/IdU turnover data, β-cell DNA (14)C content indicated that the "birth date" of cells occurred within the subject's first 30 yr of life. Under typical circumstances, human β-cells and their cellular precursors are established by young adulthood.

  14. Adult Human Pancreatic Islet Beta-Cells Display Limited Turnover and Long Lifespan as Determined by In-Vivo Thymidine Analog Incorporation and Radiocarbon Dating

    Energy Technology Data Exchange (ETDEWEB)

    Perl, S; Kushner, J A; Buchholz, B A; Meeker, A K; Stein, G M; Hsieh, M; Kirby, M; Pechhold, S; Liu, E H; Harlan, D M; Tisdale, J F

    2010-03-15

    Diabetes mellitus results from an absolute or relative deficiency of insulin producing pancreatic beta-cells. The adult human beta-cell's turnover rate remains unknown. We employed novel techniques to examine adult human islet beta-cell turnover and longevity in vivo. Subjects enrolled in NIH clinical trials received thymidine analogues [iododeoxyuridine (IdU) or bromodeoxyuridine (BrdU)] 8-days to 4-years prior to death. Archival autopsy samples from ten patients (aged 17-74 years) were employed to assess beta-cell turnover by scoring nuclear analog labeling within insulin staining cells. Human adult beta-cell longevity was determined by estimating the cells genomic DNA integration of atmospheric carbon-14 ({sup 14}C). DNA was purified from pancreatic islets isolated from cadaveric donors; whole islet prep DNA was obtained from a 15 year old donor, and purified beta-cell DNA was obtained from two donors (age 48 and 80 years). {sup 14}C levels were then determined using accelerator mass spectrometry (AMS). Cellular 'birth date' was determined by comparing the subject's DNA {sup 14}C content relative to a well-established {sup 14}C atmospheric prevalence curve. In the two subjects less than age 20 years, 1-2% of the beta-cell nuclei co-stained for BrdU/IdU. No beta-cell nuclei co-stained in the eight patients more than 30 years old. Consistent with the BrdU/IdU turnover data, beta-cell DNA {sup 14}C content indicated the cells 'birth date' occurred within the subject's first 30 years of life. Under typical circumstances, adult human beta-cells and their cellular precursors are established by young adulthood.

  15. 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

  16. 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

  17. 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.

  18. 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

  19. [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)

  20. The mitogenic activities of bean proteins determined by assay of the incorporation of sup(3)H - thymidine by human lymphocytes

    International Nuclear Information System (INIS)

    Derbyshire, E.; Carvalho, M.T.V.; Vitti, D.M.S.; Costa, C.P. da

    1988-01-01

    The proteins in a saline extract from cotyledons of the bean cultivar Goiano precoce included a protein with electrophoretic mobility equal to that of a commercial preparation of bean mitogen. The crude extract stimulated the incorporation of sup(3)H-tymidine by cultures of human lymphocytes at concentrations of extracted protein from 30 mu g - 300 mu g/culture, and the existence of an optimal concentration in the vicinity of 175 mu g/culture was indicated by the data. The range of active concentrations and the optimal concentration of the heterogeneous extract were 12-15 times greater than the corresponding values obtained when the commercial mitogen was employed. Microscopic examinations showed the presence of blast cells and mitotic figures only in cultures which included seed extract or commercial mitogen. (author)

  1. Labelling of human resting lymphocytes by continuous infusion of (/sup 3/H)thymidine. 1. Characterization of cytoplasmic label

    Energy Technology Data Exchange (ETDEWEB)

    Schick, P; Trepel, F; Maisel, K H; Past, W; Reisert, I; Begemann, H; Pilgrim, C [Ulm Univ. (Germany, F.R.)

    1978-01-01

    After continuous /sup 3/H-TdR infusion in vivo or incubation with /sup 3/H-TdR in vitro human blood lymphocytes were examined by light-microscopic and electron-microscopic autoradiography. Using relatively long autoradiographic exposure times (50-300 days) not only nuclear but also cytoplasmic labelling was visualized, the cytoplasmic label being present in up to 96% of the cells. The cytoplasmic label was predominantly associated with the mitochondria and was removed from the cells nearly completely by treatment with DNase but not with RNase or cold perchloric acid. It is concluded that this cytoplasmic label mainly represents /sup 3/H-TdR incorporated into mitochondrial DNA which is continuously renewed in an average turnover time of 14 days or less. This value is compatible with a turnover time of 11 days for mitochondrial DNA in mammalian cells reported in the literature.

  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. 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

  4. 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.

  5. 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...

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. Dark proteins disturb multichromophore coupling in tetrameric fluorescent proteins

    NARCIS (Netherlands)

    Blum, Christian; Meixner, Alfred J.; Subramaniam, Vinod

    2011-01-01

    DsRed is representative of the tetrameric reef coral fluorescent proteins that constitute particularly interesting coupled multichromophoric systems. Either a green emitting or a red emitting chromophore can form within each of the monomers of the protein tetramer. Within the tetramers the

  11. 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

  12. 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.

  13. Effect of barbiturates on radiosensitivity of cells: a comparative study of electrophoretic mobility, colony forming ability and thymidine uptake on human amnion cells

    International Nuclear Information System (INIS)

    Lalwani, N.D.; Chaubal, K.A.

    1980-01-01

    Suspensions of human amnion cells were 60 Co γ-irradiated in the presence of phenobarbital or thiobarbital (50 μg/ml). The barbiturates protected the cells against the dose-dependent reduction in electrophoretic mobility (EPM) observed 4 hours after irradiation of untreated cells, although there was an initial decrease in the EPM of treated cells followed by recovery. Treated irradiated cells exhibited greater colony-forming ability than the untreated cells. Pentobarbital and phenobarbital had similar effects, but thiobarbital was not so effective. 3 H-TdR uptake increased within 4 hours of irradiation for the treated cells. The reproductive capacity of the cells was retained at doses as high as 500 rad. The results are discussed with reference to the effects of anaesthetics on cell membranes. (U.K.)

  14. Structure of a tetrameric galectin from Cinachyrella sp. (ball sponge)

    Energy Technology Data Exchange (ETDEWEB)

    Freymann, Douglas M., E-mail: freymann@northwestern.edu [Northwestern University, 303 East Chicago Avenue, Chicago, IL 60611 (United States); Nakamura, Yuka [Hokkaido University, 3-1-1 Minato-cho, Hakodate 041-8611 (Japan); Focia, Pamela J. [Northwestern University, 303 East Chicago Avenue, Chicago, IL 60611 (United States); Sakai, Ryuichi [Hokkaido University, 3-1-1 Minato-cho, Hakodate 041-8611 (Japan); Swanson, Geoffrey T. [Northwestern University, 303 East Chicago Avenue, Chicago, IL 60611 (United States)

    2012-09-01

    The structure of a tetrameric sponge galectin suggests a basis for glutamate receptor potentiation. The galectins are a family of proteins that bind with highest affinity to N-acetyllactosamine disaccharides, which are common constituents of asparagine-linked complex glycans. They play important and diverse physiological roles, particularly in the immune system, and are thought to be critical metastatic agents for many types of cancer cells, including gliomas. A recent bioactivity-based screen of marine sponge (Cinachyrella sp.) extract identified an ancestral member of the galectin family based on its unexpected ability to positively modulate mammalian ionotropic glutamate receptor function. To gain insight into the mechanistic basis of this activity, the 2.1 Å resolution X-ray structure of one member of the family, galectin CchG-1, is reported. While the protomer exhibited structural similarity to mammalian prototype galectin, CchG-1 adopts a novel tetrameric arrangement in which a rigid toroidal-shaped ‘donut’ is stabilized in part by the packing of pairs of vicinal disulfide bonds. Twofold symmetry between binding-site pairs provides a basis for a model for interaction with ionotropic glutamate receptors.

  15. Structure of a tetrameric galectin from Cinachyrella sp. (ball sponge)

    International Nuclear Information System (INIS)

    Freymann, Douglas M.; Nakamura, Yuka; Focia, Pamela J.; Sakai, Ryuichi; Swanson, Geoffrey T.

    2012-01-01

    The structure of a tetrameric sponge galectin suggests a basis for glutamate receptor potentiation. The galectins are a family of proteins that bind with highest affinity to N-acetyllactosamine disaccharides, which are common constituents of asparagine-linked complex glycans. They play important and diverse physiological roles, particularly in the immune system, and are thought to be critical metastatic agents for many types of cancer cells, including gliomas. A recent bioactivity-based screen of marine sponge (Cinachyrella sp.) extract identified an ancestral member of the galectin family based on its unexpected ability to positively modulate mammalian ionotropic glutamate receptor function. To gain insight into the mechanistic basis of this activity, the 2.1 Å resolution X-ray structure of one member of the family, galectin CchG-1, is reported. While the protomer exhibited structural similarity to mammalian prototype galectin, CchG-1 adopts a novel tetrameric arrangement in which a rigid toroidal-shaped ‘donut’ is stabilized in part by the packing of pairs of vicinal disulfide bonds. Twofold symmetry between binding-site pairs provides a basis for a model for interaction with ionotropic glutamate receptors

  16. 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

  17. 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)

  18. 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.

  19. 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.

  20. 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....

  1. 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

  2. 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.

  3. 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)

  4. Centrifugal partition chromatography enables selective enrichment of trimeric and tetrameric proanthocyanidins for biomaterial development.

    Science.gov (United States)

    Phansalkar, Rasika S; Nam, Joo-Won; Chen, Shao-Nong; McAlpine, James B; Leme, Ariene A; Aydin, Berdan; Bedran-Russo, Ana-Karina; Pauli, Guido F

    2018-02-02

    Proanthocyanidins (PACs) find wide applications for human use including food, cosmetics, dietary supplements, and pharmaceuticals. The chemical complexity associated with PACs has triggered the development of various chromatographic techniques, with countercurrent separation (CCS) gaining in popularity. This study applied the recently developed DESIGNER (Depletion and Enrichment of Select Ingredients Generating Normalized Extract Resources) approach for the selective enrichment of trimeric and tetrameric PACs using centrifugal partition chromatography (CPC). This CPC method aims at developing PAC based biomaterials, particularly for their application in restoring and repairing dental hard tissue. A general separation scheme beginning with the depletion of polymeric PACs, followed by the removal of monomeric flavan-3-ols and a final enrichment step produced PAC trimer and tetramer enriched fractions. A successful application of this separation scheme is demonstrated for four polyphenol rich plant sources: grape seeds, pine bark, cinnamon bark, and cocoa seeds. Minor modifications to the generic DESIGNER CCS method were sufficient to accommodate the varying chemical complexities of the individual source materials. The step-wise enrichment of PAC trimers and tetramers was monitored using normal phase TLC and Diol-HPLC-UV analyses. CPC proved to be a reliable tool for the selective enrichment of medium size oligomeric PACs (OPACs). This method plays a key role in the development of dental biomaterials considering its reliability and reproducibility, as well as its scale-up capabilities for possible larger-scale manufacturing. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Biochemical and molecular characterization of an azoreductase from Staphylococcus aureus, a tetrameric NADPH-dependent flavoprotein

    Science.gov (United States)

    Chen, Huizhong; Hopper, Sherryll L.; Cerniglia, Carl E.

    2018-01-01

    Azo dyes are a predominant class of colourants used in tattooing, cosmetics, foods and consumer products. A gene encoding NADPH-flavin azoreductase (Azo1) from the skin bacterium Staphylococcus aureus ATCC 25923 was identified and overexpressed in Escherichia coli. RT-PCR results demonstrated that the azo1 gene was constitutively expressed at the mRNA level in S. aureus. Azo1 was found to be a tetramer with a native molecular mass of 85 kDa containing four non-covalently bound FMN. Azo1 requires NADPH, but not NADH, as an electron donor for its activity. The enzyme was resolved to dimeric apoprotein by removing the flavin prosthetic groups using hydrophobic-interaction chromatography. The dimeric apoprotein was reconstituted on-column and in free stage with FMN, resulting in the formation of a fully functional native-like tetrameric enzyme. The enzyme cleaved the model azo dye 2-[4-(dimethylamino)phenylazo]benzoic acid (Methyl Red) into N,N-dimethyl-p-phenylenediamine and 2-aminobenzoic acid. The apparent Km values for NADPH and Methyl Red substrates were 0·;074 and 0·057 mM, respectively. The apparent Vmax was 0·4 µM min−1 (mg protein)−1. Azo1 was also able to metabolize Orange II, Amaranth, Ponceau BS and Ponceau S azo dyes. Azo1 represents the first azoreductase to be identified and characterized from human skin microflora. PMID:15870453

  6. Increased immunogenicity and protective efficacy of influenza M2e fused to a tetramerizing protein.

    Directory of Open Access Journals (Sweden)

    Anne-Marie Carola Andersson

    Full Text Available The ectodomain of the matrix 2 protein (M2e of influenza A virus represents an attractive target for developing a universal influenza A vaccine, with its sequence being highly conserved amongst human variants of this virus. With the aim of targeting conformational epitopes presumably shared by diverse influenza A viruses, a vaccine (M2e-NSP4 was constructed linking M2e (in its consensus sequence to the rotavirus fragment NSP4(98-135; due to its coiled-coil region this fragment is known to form tetramers in aqueous solution and in this manner we hoped to mimick the natural configuration of M2e as presented in membranes. M2e-NSP4 was then evaluated side-by-side with synthetic M2e peptide for its immunogenicity and protective efficacy in a murine influenza challenge model. Here we demonstrate that M2e fused to the tetramerizing protein induces an accelerated, augmented and more broadly reactive antibody response than does M2e peptide as measured in two different assays. Most importantly, vaccination with M2e-NSP4 caused a significant decrease in lung virus load early after challenge with influenza A virus and maintained its efficacy against a lethal challenge even at very low vaccine doses. Based on the results presented in this study M2e-NSP4 merits further investigation as a candidate for or as a component of a universal influenza A vaccine.

  7. 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

  8. 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...

  9. 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)

  10. 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.

  11. Structural analysis of β-glucosidase mutants derived from a hyperthermophilic tetrameric structure

    International Nuclear Information System (INIS)

    Nakabayashi, Makoto; Kataoka, Misumi; Mishima, Yumiko; Maeno, Yuka; Ishikawa, Kazuhiko

    2014-01-01

    Substitutive mutations that convert a tetrameric β-glucosidase into a dimeric state lead to improvement of its crystal quality. β-Glucosidase from Pyrococcus furiosus (BGLPf) is a hyperthermophilic tetrameric enzyme which can degrade cellooligosaccharides to glucose under hyperthermophilic conditions and thus holds promise for the saccharification of lignocellulosic biomass at high temperature. Prior to the production of large amounts of this enzyme, detailed information regarding the oligomeric structure of the enzyme is required. Several crystals of BGLPf have been prepared over the past ten years, but its crystal structure had not been solved until recently. In 2011, the first crystal structure of BGLPf was solved and a model was constructed at somewhat low resolution (2.35 Å). In order to obtain more detailed structural data on BGLPf, the relationship between its tetrameric structure and the quality of the crystal was re-examined. A dimeric form of BGLPf was constructed and its crystal structure was solved at a resolution of 1.70 Å using protein-engineering methods. Furthermore, using the high-resolution crystal structural data for the dimeric form, a monomeric form of BGLPf was constructed which retained the intrinsic activity of the tetrameric form. The thermostability of BGLPf is affected by its oligomeric structure. Here, the biophysical and biochemical properties of engineered dimeric and monomeric BGLPfs are reported, which are promising prototype models to apply to the saccharification reaction. Furthermore, details regarding the oligomeric structures of BGLPf and the reasons why the mutations yielded improved crystal structures are discussed

  12. 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.

  13. 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

  14. 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.

  15. 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

  16. 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...

  17. Biodegradable nanoparticles loaded with tetrameric melittin: preparation and membrane disruption evaluation.

    Science.gov (United States)

    Gonzalez-Horta, Azucena; Matamoros-Acosta, Arely; Chavez-Montes, Abelardo; Castro-Rios, Rocio; Lara-Arias, Jorge

    2017-10-01

    Melittin is the main component of bee venom consisting of 26 amino acids that has multiple effects, including antibacterial, antiviral and anti-inflammatory in various cell types. This peptide forms pores in biological membranes and triggers cell death. Therefore it has potential as an anti-cancer therapy. However, the therapeutic application of melittin is limited due to its main side effect, hemolysis, which is especially pronounced following intravenous administration. In the present study, we formulated tetrameric melittin-carrying poly-D,L-lactic-co-glycolic acid nanoparticles (PLGA-NPs) and analyzed the lytic activity of this system on liposomes that resembles breast cancer cells. Tetrameric melittin binds avidly to PLGA-NPs with an encapsulation efficiency of 97% and retains its lytic activity demonstrating the effectiveness of PLGA-NPs as nanocarriers for this cytolytic peptide.

  18. Insight on an arginine synthesis metabolon from the tetrameric structure of yeast acetylglutamate kinase.

    Science.gov (United States)

    de Cima, Sergio; Gil-Ortiz, Fernando; Crabeel, Marjolaine; Fita, Ignacio; Rubio, Vicente

    2012-01-01

    N-acetyl-L-glutamate kinase (NAGK) catalyzes the second, generally controlling, step of arginine biosynthesis. In yeasts, NAGK exists either alone or forming a metabolon with N-acetyl-L-glutamate synthase (NAGS), which catalyzes the first step and exists only within the metabolon. Yeast NAGK (yNAGK) has, in addition to the amino acid kinase (AAK) domain found in other NAGKs, a ~150-residue C-terminal domain of unclear significance belonging to the DUF619 domain family. We deleted this domain, proving that it stabilizes yNAGK, slows catalysis and modulates feed-back inhibition by arginine. We determined the crystal structures of both the DUF619 domain-lacking yNAGK, ligand-free as well as complexed with acetylglutamate or acetylglutamate and arginine, and of complete mature yNAGK. While all other known arginine-inhibitable NAGKs are doughnut-like hexameric trimers of dimers of AAK domains, yNAGK has as central structure a flat tetramer formed by two dimers of AAK domains. These dimers differ from canonical AAK dimers in the -110° rotation of one subunit with respect to the other. In the hexameric enzymes, an N-terminal extension, found in all arginine-inhibitable NAGKs, forms a protruding helix that interlaces the dimers. In yNAGK, however, it conforms a two-helix platform that mediates interdimeric interactions. Arginine appears to freeze an open inactive AAK domain conformation. In the complete yNAGK structure, two pairs of DUF619 domains flank the AAK domain tetramer, providing a mechanism for the DUF619 domain modulatory functions. The DUF619 domain exhibits the histone acetyltransferase fold, resembling the catalytic domain of bacterial NAGS. However, the putative acetyl CoA site is blocked, explaining the lack of NAGS activity of yNAGK. We conclude that the tetrameric architecture is an adaptation to metabolon formation and propose an organization for this metabolon, suggesting that yNAGK may be a good model also for yeast and human NAGSs.

  19. Insight on an arginine synthesis metabolon from the tetrameric structure of yeast acetylglutamate kinase.

    Directory of Open Access Journals (Sweden)

    Sergio de Cima

    Full Text Available N-acetyl-L-glutamate kinase (NAGK catalyzes the second, generally controlling, step of arginine biosynthesis. In yeasts, NAGK exists either alone or forming a metabolon with N-acetyl-L-glutamate synthase (NAGS, which catalyzes the first step and exists only within the metabolon. Yeast NAGK (yNAGK has, in addition to the amino acid kinase (AAK domain found in other NAGKs, a ~150-residue C-terminal domain of unclear significance belonging to the DUF619 domain family. We deleted this domain, proving that it stabilizes yNAGK, slows catalysis and modulates feed-back inhibition by arginine. We determined the crystal structures of both the DUF619 domain-lacking yNAGK, ligand-free as well as complexed with acetylglutamate or acetylglutamate and arginine, and of complete mature yNAGK. While all other known arginine-inhibitable NAGKs are doughnut-like hexameric trimers of dimers of AAK domains, yNAGK has as central structure a flat tetramer formed by two dimers of AAK domains. These dimers differ from canonical AAK dimers in the -110° rotation of one subunit with respect to the other. In the hexameric enzymes, an N-terminal extension, found in all arginine-inhibitable NAGKs, forms a protruding helix that interlaces the dimers. In yNAGK, however, it conforms a two-helix platform that mediates interdimeric interactions. Arginine appears to freeze an open inactive AAK domain conformation. In the complete yNAGK structure, two pairs of DUF619 domains flank the AAK domain tetramer, providing a mechanism for the DUF619 domain modulatory functions. The DUF619 domain exhibits the histone acetyltransferase fold, resembling the catalytic domain of bacterial NAGS. However, the putative acetyl CoA site is blocked, explaining the lack of NAGS activity of yNAGK. We conclude that the tetrameric architecture is an adaptation to metabolon formation and propose an organization for this metabolon, suggesting that yNAGK may be a good model also for yeast and human NAGSs.

  20. [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

  1. 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...

  2. 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...

  3. 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)].

  4. 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

  5. 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...

  6. 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.

  7. 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)

  8. The lysosomal potassium channel TMEM175 adopts a novel tetrameric architecture

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Changkeun; Guo, Jiangtao; Zeng, Weizhong; Kim, Sunghoon; She, Ji; Cang, Chunlei; Ren, Dejian; Jiang , Youxing (UPENN); (UTSMC); (HHMI)

    2017-07-19

    TMEM175 is a lysosomal K+ channel that is important for maintaining the membrane potential and pH stability in lysosomes1. It contains two homologous copies of a six-transmembrane-helix (6-TM) domain, which has no sequence homology to the canonical tetrameric K+ channels and lacks the TVGYG selectivity filter motif found in these channels2, 3, 4. The prokaryotic TMEM175 channel, which is present in a subset of bacteria and archaea, contains only a single 6-TM domain and functions as a tetramer. Here, we present the crystal structure of a prokaryotic TMEM175 channel from Chamaesiphon minutus, CmTMEM175, the architecture of which represents a completely different fold from that of canonical K+ channels. All six transmembrane helices of CmTMEM175 are tightly packed within each subunit without undergoing domain swapping. The highly conserved TM1 helix acts as the pore-lining inner helix, creating an hourglass-shaped ion permeation pathway in the channel tetramer. Three layers of hydrophobic residues on the carboxy-terminal half of the TM1 helices form a bottleneck along the ion conduction pathway and serve as the selectivity filter of the channel. Mutagenesis analysis suggests that the first layer of the highly conserved isoleucine residues in the filter is primarily responsible for channel selectivity. Thus, the structure of CmTMEM175 represents a novel architecture of a tetrameric cation channel whose ion selectivity mechanism appears to be distinct from that of the classical K+ channel family.

  9. New tetrameric forms of the rotavirus NSP4 with antiparallel helices.

    Science.gov (United States)

    Kumar, Sushant; Ramappa, Raghavendra; Pamidimukkala, Kiranmayee; Rao, C D; Suguna, K

    2018-06-01

    Rotavirus nonstructural protein 4, the first viral enterotoxin to be identified, is a multidomain, multifunctional glycoprotein. Earlier, we reported a Ca 2+ -bound coiled-coil tetrameric structure of the diarrhea-inducing region of NSP4 from the rotavirus strains SA11 and I321 and a Ca 2+ -free pentameric structure from the rotavirus strain ST3, all with a parallel arrangement of α-helices. pH was found to determine the oligomeric state: a basic pH favoured a tetramer, whereas an acidic pH favoured a pentamer. Here, we report two novel forms of the coiled-coil region of NSP4 from the bovine rotavirus strains MF66 and NCDV. These crystallized at acidic pH, forming antiparallel coiled-coil tetrameric structures without any bound Ca 2+ ion. Structural and mutational studies of the coiled-coil regions of NSP4 revealed that the nature of the residue at position 131 (Tyr/His) plays an important role in the observed structural diversity.

  10. 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)

  11. 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

  12. Secretory production of tetrameric native full-length streptavidin with thermostability using Streptomyces lividans as a host.

    Science.gov (United States)

    Noda, Shuhei; Matsumoto, Takuya; Tanaka, Tsutomu; Kondo, Akihiko

    2015-01-13

    Streptavidin is a tetrameric protein derived from Streptomyces avidinii, and has tight and specific biotin binding affinity. Applications of the streptavidin-biotin system have been widely studied. Streptavidin is generally produced using protein expression in Escherichia coli. In the present study, the secretory production of streptavidin was carried out using Streptomyces lividans as a host. In this study, we used the gene encoding native full-length streptavidin, whereas the core region is generally used for streptavidin production in E. coli. Tetrameric streptavidin composed of native full-length streptavidin monomers was successfully secreted in the culture supernatant of S. lividans transformants, and had specific biotin binding affinity as strong as streptavidin produced by E. coli. The amount of Sav using S. lividans was about 9 times higher than using E. coli. Surprisingly, streptavidin produced by S. lividans exhibited affinity to biotin after boiling, despite the fact that tetrameric streptavidin is known to lose its biotin binding ability after brief boiling. We successfully produced a large amount of tetrameric streptavidin as a secretory-form protein with unique thermotolerance.

  13. PET imaging of alphavbeta integrin expression in tumours with Ga-labelled mono-, di- and tetrameric RGD peptides

    NARCIS (Netherlands)

    Dijkgraaf, I.; Yim, C.B.; Franssen, G.M.; Schuit, R.C.; Luurtsema, G.; Liu, S.; Oyen, W.J.G.; Boerman, O.C.

    2011-01-01

    PURPOSE: Due to the restricted expression of alpha(v)beta(3) in tumours, alpha(v)beta(3) is considered a suitable receptor for tumour targeting. In this study the alpha(v)beta(3)-binding characteristics of (68)Ga-labelled monomeric, dimeric and tetrameric RGD peptides were determined and compared

  14. 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.

  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. 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. 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.

  18. 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-...

  19. 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

  20. Overexpression, purification and crystallization of the tetrameric form of SorC sorbitol operon regulator

    International Nuclear Information System (INIS)

    Sanctis, Daniele de; Rêgo, Ana T.; Marçal, David; McVey, Colin E.; Carrondo, Maria A.; Enguita, Francisco J.

    2007-01-01

    The sorbitol operon regulator from K. pneumoniae has been overexpressed in E. coli, purified and crystallized. Diffraction data were collected to 3.2 Å. The sorbitol operon regulator (SorC) regulates the metabolism of l-sorbose in Klebsiella pneumonia. SorC was overexpressed in Escherichia coli and purified, and crystals were obtained of a tetrameric form. A single crystal showed X-ray diffraction to 3.20 Å. The crystal belongs to space group P2 1 2 1 2 1 , with unit-cell parameters a = 91.6, b = 113.3, c = 184.1 Å. Analysis of the molecular-replacement solution indicates the presence of four SorC molecules in the asymmetric unit

  1. Tetrameric structure of the flagellar cap protein FliD from Serratia marcescens.

    Science.gov (United States)

    Cho, So Yeon; Song, Wan Seok; Hong, Ho Jeong; Lee, Geun-Shik; Kang, Seung Goo; Ko, Hyun-Jeong; Kim, Pyeung-Hyeun; Yoon, Sung-Il

    2017-07-15

    Bacterial motility is provided by the flagellum. FliD is located at the distal end of the flagellum and plays a key role in the insertion of each flagellin protein at the growing tip of the flagellar filament. Because FliD functions as an oligomer, the determination of the oligomeric state of FliD is critical to understanding the molecular mechanism of FliD-mediated flagellar growth. FliD has been shown to adopt a pentameric or a hexameric structure depending on the bacterial species. Here, we report another distinct oligomeric form of FliD based on structural and biochemical studies. The crystal structures of the D2 and D3 domains of Serratia marcescens FliD (smFliD) were determined in two crystal forms and together revealed that smFliD assembles into a tetrameric architecture that resembles a four-pointed star plate. smFliD tetramerization was also confirmed in solution by cross-linking experiments. Although smFliD oligomerizes in a head-to-tail orientation using a common primary binding interface between the D2 and D3' domains (the prime denotes the second subunit in the oligomer) similarly to other FliD orthologs, the smFliD tetramer diverges to present a unique secondary D2-D2' binding interface. Our structure-based comparative analysis of FliD suggests that bacteria have developed diverse species-specific oligomeric forms of FliD that range from tetramers to hexamers for flagellar growth. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Natural monomeric form of fetal bovine serum acetylcholinesterase lacks the C-terminal tetramerization domain.

    Science.gov (United States)

    Saxena, Ashima; Hur, Regina S; Luo, Chunyuan; Doctor, Bhupendra P

    2003-12-30

    Acetylcholinesterase isolated from fetal bovine serum (FBS AChE) was previously characterized as a globular tetrameric form. Analysis of purified preparations of FBS AChE by gel permeation chromatography revealed the presence of a stable, catalytically active, monomeric form of this enzyme. The two forms could be distinguished from each other based on their molecular weight, hydrodynamic properties, kinetic properties, thermal stability, and the type of glycans they carry. No differences between the two forms were observed for the binding of classical inhibitors such as edrophonium and propidium or inhibitors that are current or potential drugs for the treatment of Alzheimer's disease such as (-) huperzine A and E2020; tacrine inhibited the monomeric form 2-3-fold more potently than the tetrameric form. Sequencing of peptides obtained from an in-gel tryptic digest of the monomer and tetramer by tandem mass spectrometry indicated that the tetramer consists of 583 amino acid residues corresponding to the mature form of the enzyme, whereas the monomer consists of 543-547 amino acid residues. The subunit molecular weight of the protein component of the monomer (major species) was determined to be 59 414 Da and that of the tetramer as 64 239 Da. The N-terminal of the monomer and the tetramer was Glu, suggesting that the monomer is not a result of truncation at the N-terminal. The only differences detected were at the C-terminus. The tetramer yielded the expected C-terminus, CSDL, whereas the C-terminus of the monomer yielded a mixture of peptides, of which LLSATDTLD was the most abundant. These results suggest that monomeric FBS AChE is trimmed at the C-terminus, and the results are consistent with the involvement of C-terminal amino acids in the assembly of monomers into tetramers.

  3. Synthesis of Symmetrical Tetrameric Conjugates of the Radiolanthanide Chelator DOTPI for Application in Endoradiotherapy by Means of Click Chemistry

    Directory of Open Access Journals (Sweden)

    Alexander Wurzer

    2018-04-01

    Full Text Available Due to its 4 carbonic acid groups being available for bioconjugation, the cyclen tetraphosphinate chelator DOTPI, 1,4,7,10-tetraazacyclododecane-1,4,7, 10-tetrakis[methylene(2-carboxyethylphosphinic acid], represents an ideal scaffold for synthesis of tetrameric bioconjugates for labeling with radiolanthanides, to be applied as endoradiotherapeuticals. We optimized a protocol for bio-orthogonal DOTPI conjugation via Cu(I-catalyzed Huisgen-cycloaddition of terminal azides and alkynes (CuAAC, based on the building block DOTPI(azide4. A detailed investigation of kinetic properties of Cu(II-DOTPI complexes aimed at optimization of removal of DOTPI-bound copper by transchelation. Protonation and equilibrium properties of Ca(II-, Zn(II, and Cu(II-complexes of DOTPI and its tetra-cyclohexylamide DOTPI(Chx4 (a model for DOTPI conjugates as well as kinetic inertness (transchelation challenge in the presence of 20 to 40-fold excess of EDTA were investigated by pH-potentiometry and spectrophotometry. Similar stability constants of CaII-, ZnII, and CuII-complexes of DOTPI (logK(CaL = 8.65, logK(ZnL = 15.40, logK(CuL = 20.30 and DOTPI(Chx4 (logK(CaL = 8.99, logK(ZnL = 15.13, logK(CuL = 20.42 were found. Transchelation of Cu(II-complexes occurs via proton-assisted dissociation, whereafter released Cu(II is scavenged by EDTA. The corresponding dissociation rates [kd = 25 × 10−7 and 5 × 10−7 s−1 for Cu(DOTPI and Cu(DOTPI(Chx4, respectively, at pH 4 and 298 K] indicate that conjugation increases the kinetic inertness by a factor of 5. However, demetallation is completed within 4.5 and 7.2 h at pH 2 and 25°C, respectively, indicating that Cu(II removal after formation of CuAAC can be achieved in an uncomplicated manner by addition of excess H4EDTA. For proof-of-principle, tetrameric DOTPI conjugates of the prostate-specific membrane antigen (PSMA targeting motif Lys-urea-Glu (KuE were synthesized via CuAAC as well as dibenzo-azacyclooctine (DBCO based

  4. Synthesis of Symmetrical Tetrameric Conjugates of the Radiolanthanide Chelator DOTPI for Application in Endoradiotherapy by Means of Click Chemistry

    Science.gov (United States)

    Wurzer, Alexander; Vágner, Adrienn; Horváth, Dávid; Fellegi, Flóra; Wester, Hans-Jürgen; Kálmán, Ferenc K.; Notni, Johannes

    2018-01-01

    Due to its 4 carbonic acid groups being available for bioconjugation, the cyclen tetraphosphinate chelator DOTPI, 1,4,7,10-tetraazacyclododecane-1,4,7, 10-tetrakis[methylene(2-carboxyethylphosphinic acid)], represents an ideal scaffold for synthesis of tetrameric bioconjugates for labeling with radiolanthanides, to be applied as endoradiotherapeuticals. We optimized a protocol for bio-orthogonal DOTPI conjugation via Cu(I)-catalyzed Huisgen-cycloaddition of terminal azides and alkynes (CuAAC), based on the building block DOTPI(azide)4. A detailed investigation of kinetic properties of Cu(II)-DOTPI complexes aimed at optimization of removal of DOTPI-bound copper by transchelation. Protonation and equilibrium properties of Ca(II)-, Zn(II), and Cu(II)-complexes of DOTPI and its tetra-cyclohexylamide DOTPI(Chx)4 (a model for DOTPI conjugates) as well as kinetic inertness (transchelation challenge in the presence of 20 to 40-fold excess of EDTA) were investigated by pH-potentiometry and spectrophotometry. Similar stability constants of CaII-, ZnII, and CuII-complexes of DOTPI (logK(CaL) = 8.65, logK(ZnL = 15.40, logK(CuL) = 20.30) and DOTPI(Chx)4 (logK(CaL) = 8.99, logK(ZnL) = 15.13, logK(CuL) = 20.42) were found. Transchelation of Cu(II)-complexes occurs via proton-assisted dissociation, whereafter released Cu(II) is scavenged by EDTA. The corresponding dissociation rates [kd = 25 × 10−7 and 5 × 10−7 s−1 for Cu(DOTPI) and Cu(DOTPI(Chx)4), respectively, at pH 4 and 298 K] indicate that conjugation increases the kinetic inertness by a factor of 5. However, demetallation is completed within 4.5 and 7.2 h at pH 2 and 25°C, respectively, indicating that Cu(II) removal after formation of CuAAC can be achieved in an uncomplicated manner by addition of excess H4EDTA. For proof-of-principle, tetrameric DOTPI conjugates of the prostate-specific membrane antigen (PSMA) targeting motif Lys-urea-Glu (KuE) were synthesized via CuAAC as well as dibenzo-azacyclooctine (DBCO

  5. Synthesis of symmetrical tetrameric conjugates of the radiolanthanide chelator DOTPI for application in endoradiotherapy by means of click chemistry

    Science.gov (United States)

    Wurzer, Alexander; Vágner, Adrienn; Horváth, Dávid; Fellegi, Flóra; Wester, Hans-Jürgen; Kálmán, Ferenc K.; Notni, Johannes

    2018-04-01

    Due to its 4 carbonic acid groups being available for bioconjugation, the cyclen tetraphosphinate chelator DOTPI, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis[methylene(2-carboxyethylphosphinic acid)], represents an ideal scaffold for synthesis of tetrameric bioconjugates for labeling with radiolanthanides, to be applied as endoradiotherapeuticals. We optimized a protocol for bio-orthogonal DOTPI conjugation via Cu(I)-catalyzed Huisgen-cycloaddition of terminal azides and alkynes (CuAAC), based on the building block DOTPI(azide)4. A detailed investigation of kinetic properties of Cu(II)-DOTPI complexes aimed at optimization of removal of DOTPI-bound copper by transchelation. Protonation and equilibrium properties of Ca(II)-, Zn(II) and Cu(II)-complexes of DOTPI and its tetra-cyclohexylamide DOTPI(Chx)4 (a model for DOTPI conjugates) as well as kinetic inertness (transchelation challenge in the presence of 20 to 40-fold excess of EDTA) were investigated by pH-potentiometry and spectrophotometry. Similar stability constants of CaII-, ZnII and CuII-complexes of DOTPI (logK(CaL)=8.65, logK(ZnL=15.40, logK(CuL)=20.30) and DOTPI(Chx)4 (logK(CaL)=8.99, logK(ZnL)=15.13, logK(CuL)=20.42) were found. Transchelation of CuII-complexes occurs via proton-assisted dissociation, whereafter released Cu(II) is scavenged by EDTA. The corresponding dissociation rates (kd=25×10‑7 and 5×10‑7 s‑1 for Cu(DOTPI) and Cu(DOTPI(Chx)4), respectively, at pH 4 and 298 K) indicate that conjugation increases the kinetic inertness by a factor of 5. However demetallation is completed within 4.5 and 7.2 hours at pH 2 and 25 °C, respectively, indicating that CuII removal after formation of CuAAC can be achieved in an uncomplicated manner by addition of excess H4EDTA. For proof-of-principle, tetrameric DOTPI conjugates of the prostate-specific membrane antigen (PSMA) targeting motif Lys-urea-Glu (KuE) were synthesized via CuAAC as well as dibenzo-cyclooctine (DBCO) based, strain

  6. 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

  7. 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

  8. 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.

  9. 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)

  10. 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

  11. Preparation and characterization of dimeric and tetrameric clusters of molybdenum and tungsten

    Energy Technology Data Exchange (ETDEWEB)

    Ryan, T.R.

    1981-10-01

    The cyclo-addition of two Mo/sub 2/Cl/sub 4/(P(C/sub 6/H/sub 5/)/sub 3/)/sub 2/(CH/sub 3/OH)/sub 2/ molecules has produced a new type of tetrameric molybdenum cluster, Mo/sub 4/Cl/sub 8/L/sub 4/. Structural characterization of this dimer revealed weak molybdenum-methanol bonding which was consistent with the observed reactivity of the compound. New synthetic methods were devised for the preparation of Mo/sub 4/X/sub 8/L/sub 4/ clusters where X = Cl, Br, I and L = PR/sub 3/, Po/sub 3/, RCN, CH/sub 3/OH. A scheme for the metal-metal bonding in these clusters was presented which was in agreement with the known structural features of Mo/sub 4/Cl/sub 8/(PR/sub 3/)/sub 4/, R = C/sub 2/H/sub 5/, n-C/sub 4/H/sub 9/. The preparation of the analogous W/sub 4/Cl/sub 8/(PR/sub 3/)/sub 4/ cluster from WCl/sub 4/ was accomplished by application of techniques used in the molybdenum syntheses. The single crystal x-ray structure revealed slight differences from the molybdenum analog which were rationalized in terms of the known behavior in dimeric tungsten and molybdenum species. The attempted preparation of a tetrameric tungsten cluster from W/sub 2/(mhp)/sub 4/ was unsuccessful (mhp = anion of 2-methyl-6-hydroxypyridine). Instead, the new tungsten dimer, W/sub 2/Cl/sub 2/(mhp)/sub 3/, was isolated which possessed a metal-metal bond order of 3.5. The x-ray crystal structure of the dimer revealed that the chlorine atoms were situated cis, one bound to each tungsten. Cyclic voltammetry showed that the compound could be reversibly reduced, presumably to a W/sub 2//sup 4 +/ dimer containing a quadruple metal-metal bond.

  12. A Tetrameric Peptide Derived from Bovine Lactoferricin Exhibits Specific Cytotoxic Effects against Oral Squamous-Cell Carcinoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Víctor A. Solarte

    2015-01-01

    Full Text Available Several short linear peptides derived from cyclic bovine lactoferricin were synthesized and tested for their cytotoxic effect against the oral cavity squamous-cell carcinoma (OSCC cell lines CAL27 and SCC15. As a control, an immortalized and nontumorigenic cell line, Het-1A, was used. Linear peptides based on the RRWQWR core sequence showed a moderate cytotoxic effect and specificity towards tumorigenic cells. A tetrameric peptide, LfcinB(20–254, containing the RRWQWR motif, exhibited greater cytotoxic activity (>90% in both OSCC cell lines compared to the linear lactoferricin peptide or the lactoferrin protein. Additionally, this tetrameric peptide showed the highest specificity towards tumorigenic cells among the tested peptides. Interestingly, this effect was very fast, with cell shrinkage, severe damage to cell membrane permeability, and lysis within one hour of treatment. Our results are consistent with a necrotic effect rather than an apoptotic one and suggest that this tetrameric peptide could be considered as a new candidate for the therapeutic treatment of OSCC.

  13. A Tetrameric Peptide Derived from Bovine Lactoferricin Exhibits Specific Cytotoxic Effects against Oral Squamous-Cell Carcinoma Cell Lines.

    Science.gov (United States)

    Solarte, Víctor A; Rosas, Jaiver E; Rivera, Zuly J; Arango-Rodríguez, Martha L; García, Javier E; Vernot, Jean-Paul

    2015-01-01

    Several short linear peptides derived from cyclic bovine lactoferricin were synthesized and tested for their cytotoxic effect against the oral cavity squamous-cell carcinoma (OSCC) cell lines CAL27 and SCC15. As a control, an immortalized and nontumorigenic cell line, Het-1A, was used. Linear peptides based on the RRWQWR core sequence showed a moderate cytotoxic effect and specificity towards tumorigenic cells. A tetrameric peptide, LfcinB(20-25)4, containing the RRWQWR motif, exhibited greater cytotoxic activity (>90%) in both OSCC cell lines compared to the linear lactoferricin peptide or the lactoferrin protein. Additionally, this tetrameric peptide showed the highest specificity towards tumorigenic cells among the tested peptides. Interestingly, this effect was very fast, with cell shrinkage, severe damage to cell membrane permeability, and lysis within one hour of treatment. Our results are consistent with a necrotic effect rather than an apoptotic one and suggest that this tetrameric peptide could be considered as a new candidate for the therapeutic treatment of OSCC.

  14. Detection of anion-linked polymerization of the tetrameric hemoglobin from Scapharca inaequivalvis by 35Cl NMR spectroscopy

    International Nuclear Information System (INIS)

    Chiancone, E.; Univ. 'La Sapienza', Rome; Drakenberg, T.; Forsen, S.

    1988-01-01

    Ion binding to the hemoglobin components of Scaphara inaequivalvis has been measured directly in quadrupole relaxation experiments of 23 Na and 35 Cl. The dimeric and tetrameric hemoglobins interact weakly with sodium ions, but differ in their interaction with chloride ions. The dimeric hemoglobin binds chloride ions with low affinity, whereas the tetrameric protein has high affinity chloride binding sites. Binding of chloride ions to these high affinity sites brings about an oxygen-linked polymerization which manifests itself in an unusual dependence of the 35 Cl excess linewidth on the concentration of the anion. Polymerization is more pronounced in the deoxygenated than in the oxygenated derivative: in the former, it has been observed previously in sedimentation velocity experiments. The sensitivity of the 35 Cl excess linewidth on polymer formation indicates that the residence time of the transiently bound chloride on the tetrameric hemoglobin is not shorter than the correlation time of the molecule (2 X 10 -8 s -1 ). 17 refs.; 2 figs

  15. Identification of a tetramerization domain in the C terminus of the vanilloid receptor.

    Science.gov (United States)

    García-Sanz, Nuria; Fernández-Carvajal, Asia; Morenilla-Palao, Cruz; Planells-Cases, Rosa; Fajardo-Sánchez, Emmanuel; Fernández-Ballester, Gregorio; Ferrer-Montiel, Antonio

    2004-06-09

    TRPV1 (transient receptor potential vanilloid receptor subtype 1) is a member of the TRP channel family gated by vanilloids, protons, and heat. Structurally, TRPV1 appears to be a tetramer formed by the assembly of four identical subunits around a central aqueous pore. The molecular determinants that govern its subunit oligomerization remain elusive. Here, we report the identification of a segment comprising 684Glu-721Arg (referred to as the TRP-like domain) in the C terminus of TRPV1 as an association domain (AD) of the protein. Purified recombinant C terminus of TRPV1 (TRPV1-C) formed discrete and stable multimers in vitro. Yeast two-hybrid and pull-down assays showed that self-association of the TRPV1-C is blocked when segment 684Glu-721Arg is deleted. Biochemical and immunological analysis indicate that removal of the AD from full-length TRPV1 monomers blocks the formation of stable heteromeric assemblies with wild-type TRPV1 subunits. Deletion of the AD in a poreless TRPV1 subunit suppressed its robust dominant-negative phenotype. Together, these findings are consistent with the tenet that the TRP-like domain in TRPV1 is a molecular determinant of the tetramerization of receptor subunits into functional channels. Our observations suggest that the homologous TRP domain in the TRP protein family may function as a general, evolutionary conserved AD involved in subunit multimerization.

  16. Molecular determinants of tetramerization in the KcsA cytoplasmic domain.

    Science.gov (United States)

    Kamnesky, Guy; Hirschhorn, Orel; Shaked, Hadassa; Chen, Jingfei; Yao, Lishan; Chill, Jordan H

    2014-10-01

    The cytoplasmic C-terminal domain (CTD) of KcsA, a bacterial homotetrameric potassium channel, is an amphiphilic domain that forms a helical bundle with four-fold symmetry mediated by hydrophobic and electrostatic interactions. Previously we have established that a CTD-derived 34-residue peptide associates into a tetramer in a pH-dependent manner (Kamnesky et al., JMB 2012;418:237-247). Here we further investigate the molecular determinants of tetramer formation in the CTD by characterizing the kinetics of monomer-tetramer equilibrium for 10 alanine mutants using NMR, sedimentation equilibrium (SE) and molecular dynamics simulation. NMR and SE concur in finding single-residue contributions to tetramer stability to be in the 0.5 to 3.5 kcal/mol range. Hydrophobic interactions between residues lining the tetramer core generally contributed more to formation of tetramer than electrostatic interactions between residues R147, D149 and E152. In particular, alanine replacement of residue R147, a key contributor to inter-subunit salt bridges, resulted in only a minor effect on tetramer dissociation. Mutations outside of the inter-subunit interface also influenced tetramer stability by affecting the tetramerization on-rate, possibly by changing the inherent helical propensity of the peptide. These findings are interpreted in the context of established paradigms of protein-protein interactions and protein folding, and lay the groundwork for further studies of the CTD in full-length KcsA channels. © 2014 The Protein Society.

  17. 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

  18. 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)

  19. 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

  20. 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

  1. An H5N1-based matrix protein 2 ectodomain tetrameric peptide vaccine provides cross-protection against lethal infection with H7N9 influenza virus.

    Science.gov (United States)

    Leung, Ho-Chuen; Chan, Chris Chung-Sing; Poon, Vincent Kwok-Man; Zhao, Han-Jun; Cheung, Chung-Yan; Ng, Fai; Huang, Jian-Dong; Zheng, Bo-Jian

    2015-04-01

    In March 2013, a patient infected with a novel avian influenza A H7N9 virus was reported in China. Since then, there have been 458 confirmed infection cases and 177 deaths. The virus contains several human-adapted markers, indicating that H7N9 has pandemic potential. The outbreak of this new influenza virus highlighted the need for the development of universal influenza vaccines. Previously, we demonstrated that a tetrameric peptide vaccine based on the matrix protein 2 ectodomain (M2e) of the H5N1 virus (H5N1-M2e) could protect mice from lethal infection with different clades of H5N1 and 2009 pandemic H1N1 influenza viruses. In this study, we investigated the cross-protection of H5N1-M2e against lethal infection with the new H7N9 virus. Although five amino acid differences existed at positions 13, 14, 18, 20, and 21 between M2e of H5N1 and H7N9, H5N1-M2e vaccination with either Freund's adjuvant or the Sigma adjuvant system (SAS) induced a high level of anti-M2e antibody, which cross-reacted with H7N9-M2e peptide. A mouse-adapted H7N9 strain, A/Anhui/01/2013m, was used for lethal challenge in animal experiments. H5N1-M2e vaccination provided potent cross-protection against lethal challenge of the H7N9 virus. Reduced viral replication and histopathological damage of mouse lungs were also observed in the vaccinated mice. Our results suggest that the tetrameric H5N1-M2e peptide vaccine could protect against different subtypes of influenza virus infections. Therefore, this vaccine may be an ideal candidate for developing a universal vaccine to prevent the reemergence of avian influenza A H7N9 virus and the emergence of potential novel reassortants of influenza virus.

  2. 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

  3. 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

  4. 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

  5. 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.)

  6. 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.

  7. 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.

  8. 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

  9. Crystal structures of two tetrameric β-carbonic anhydrases from the filamentous ascomycete Sordaria macrospora.

    Science.gov (United States)

    Lehneck, Ronny; Neumann, Piotr; Vullo, Daniela; Elleuche, Skander; Supuran, Claudiu T; Ficner, Ralf; Pöggeler, Stefanie

    2014-04-01

    Carbonic anhydrases (CAs) are metalloenzymes catalyzing the reversible hydration of carbon dioxide to bicarbonate (hydrogen carbonate) and protons. CAs have been identified in archaea, bacteria and eukaryotes and can be classified into five groups (α, β, γ, δ, ζ) that are unrelated in sequence and structure. The fungal β-class has only recently attracted attention. In the present study, we investigated the structure and function of the plant-like β-CA proteins CAS1 and CAS2 from the filamentous ascomycete Sordaria macrospora. We demonstrated that both proteins can substitute for the Saccharomyces cerevisiae β-CA Nce103 and exhibit an in vitro CO2 hydration activity (kcat /Km of CAS1: 1.30 × 10(6) m(-1) ·s(-1) ; CAS2: 1.21 × 10(6 ) m(-1) ·s(-1) ). To further investigate the structural properties of CAS1 and CAS2, we determined their crystal structures to a resolution of 2.7 Å and 1.8 Å, respectively. The oligomeric state of both proteins is tetrameric. With the exception of the active site composition, no further major differences have been found. In both enzymes, the Zn(2) (+) -ion is tetrahedrally coordinated; in CAS1 by Cys45, His101 and Cys104 and a water molecule and in CAS2 by the side chains of four residues (Cys56, His112, Cys115 and Asp58). Both CAs are only weakly inhibited by anions, making them good candidates for industrial applications. CAS1 and CAS2 bind by x-ray crystallography (View interaction) Structural data have been deposited in the Protein Data Bank database under accession numbers 4O1J for CAS1 and 4O1K for CAS2. © 2014 FEBS.

  10. 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)

  11. Genetic variation observed at three tetrameric short tandem repeat loci HumTHO1, TPOX, and CSF1PO--in five ethnic population groups of northeastern India.

    Science.gov (United States)

    Ranjan, D; Kashyap, V K

    2001-01-01

    This paper portrays the genetic variation observed at three tetrameric short tandem repeat (STR) loci HumTHO1, TPOX, and CSF1PO in five ethnic population groups from northeastern India. The study also specifies the suitability of use of these markers for forensic testing. The populations studied included three tribal groups (Kuki, Naga and Hmar), one Mongoloid caste group (Meitei), and a religious caste group (Manipuri Muslims). The loci were highly polymorphic in the populations, and all loci met Hardy-Weinberg expectations. No evidence for association of alleles among the loci was detected. The probability of match for the three loci of the most frequent genotype in the five population groups ranged between 2.6 x 10(-4) and 6.6 x 10(-5). The average heterozygosity among the population groups was approximately 70% with the overall extent of gene differentiation among the five groups being high (Gst = 0.046). Genetic affinity among the populations reveal very close association between the Kuki, Hmar, Naga, and Meitei. The Manipuri Muslims, despite being found in the same region, have had no admixture with these populations and maintain a substantial distance with the other groups. The genetic polymorphism data suggest that the studied systems can be used for human identity testing to estimate the frequency of a multiple locus STR DNA profile in population groups of northeastern India.

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. Model compounds for heavy crude oil components and tetrameric acids: Characterization and interfacial behaviour

    Energy Technology Data Exchange (ETDEWEB)

    Nordgaard, Erland Loeken

    2009-07-01

    The tendency during the past decades in the quality of oil reserves shows that conventional crude oil is gradually being depleted and the demand being replaced by heavy crude oils. These oils contain more of a class high-molecular weight components termed asphaltenes. This class is mainly responsible for stable water-in-crude oil emulsions. Both heavy and lighter crude oils in addition contain substantial amounts of naphthenic acids creating naphthenate deposits in topside facilities. The asphaltene class is defined by solubility and consists of several thousand different structures which may behave differently in oil-water systems. The nature of possible sub fractions of the asphaltene has been received more attention lately, but still the properties and composition of such is not completely understood. In this work, the problem has been addressed by synthesizing model compounds for the asphaltenes, on the basis that an acidic function incorporated could be crucial. Such acidic, poly aromatic surfactants turned out to be highly inter facially active as studied by the pendant drop technique. Langmuir monolayer compressions combined with fluorescence of deposited films indicated that the interfacial activity was a result of an efficient packing of the aromatic cores in the molecules, giving stabilizing interactions at the o/w interface. Droplet size distributions of emulsions studied by PFG NMR and adsorption onto hydrophilic silica particles demonstrated the high affinity to o/w interfaces and that the efficient packing gave higher emulsion stability. Comparing to a model compound lacking the acidic group, it was obvious that sub fractions of asphaltenes that contain an acidic, or maybe similar hydrogen bonding functions, could be responsible for stable w/o emulsions. Indigenous tetrameric acids are the main constituent of calcium naphthenate deposits. Several synthetic model tetra acids have been prepared and their properties have been compared to the indigenous

  18. Isolation of dimeric, trimeric, tetrameric and pentameric procyanidins from unroasted cocoa beans (Theobroma cacao L.) using countercurrent chromatography.

    Science.gov (United States)

    Esatbeyoglu, Tuba; Wray, Victor; Winterhalter, Peter

    2015-07-15

    The main procyanidins, including dimeric B2 and B5, trimeric C1, tetrameric and pentameric procyanidins, were isolated from unroasted cocoa beans (Theobroma cacao L.) using various techniques of countercurrent chromatography, such as high-speed countercurrent chromatography (HSCCC), low-speed rotary countercurrent chromatography (LSRCCC) and spiral-coil LSRCCC. Furthermore, dimeric procyanidins B1 and B7, which are not present naturally in the analysed cocoa beans, were obtained after semisynthesis of cocoa bean polymers with (+)-catechin as nucleophile and separated by countercurrent chromatography. In this way, the isolation of dimeric procyanidin B1 in considerable amounts (500mg, purity>97%) was possible in a single run. This is the first report concerning the isolation and semisynthesis of dimeric to pentameric procyanidins from T. cacao by countercurrent chromatography. Additionally, the chemical structures of tetrameric (cinnamtannin A2) and pentameric procyanidins (cinnamtannin A3) were elucidated on the basis of (1)H NMR spectroscopy. Interflavanoid linkage was determined by NOE-correlations, for the first time. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Identification of a tetrameric assembly domain in the C terminus of heat-activated TRPV1 channels.

    Science.gov (United States)

    Zhang, Feng; Liu, Shuang; Yang, Fan; Zheng, Jie; Wang, KeWei

    2011-04-29

    Transient receptor potential (TRP) channels as cellular sensors are thought to function as tetramers. Yet, the molecular determinants governing channel multimerization remain largely elusive. Here we report the identification of a segment comprising 21 amino acids (residues 752-772 of mouse TRPV1) after the known TRP-like domain in the channel C terminus that functions as a tetrameric assembly domain (TAD). Purified recombinant C-terminal proteins of TRPV1-4, but not the N terminus, mediated the protein-protein interaction in an in vitro pulldown assay. Western blot analysis combined with electrophysiology and calcium imaging demonstrated that TAD exerted a robust dominant-negative effect on wild-type TRPV1. When fused with the membrane-tethered peptide Gap43, the TAD blocked the formation of stable homomultimers. Calcium imaging and current recordings showed that deletion of the TAD in a poreless TRPV1 mutant subunit suppressed its dominant-negative phenotype, confirming the involvement of the TAD in assembly of functional channels. Our findings suggest that the C-terminal TAD in TRPV1 channels functions as a domain that is conserved among TRPV1-4 and mediates a direct subunit-subunit interaction for tetrameric assembly.

  20. 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

  1. 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)

  2. 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

  3. 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)

  4. 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.

  5. PET imaging of alpha(v)beta(3) integrin expression in tumours with Ga-68-labelled mono-, di- and tetrameric RGD peptides

    NARCIS (Netherlands)

    Dijkgraaf, Ingrid; Yim, Cheng-Bin; Franssen, Gerben M.; Schuit, Robert C.; Luurtsema, Gert; Liu, Shuang; Oyen, Wim J. G.; Boerman, Otto C.

    Due to the restricted expression of alpha(v)beta(3) in tumours, alpha(v)beta(3) is considered a suitable receptor for tumour targeting. In this study the alpha(v)beta(3)-binding characteristics of Ga-68-labelled monomeric, dimeric and tetrameric RGD peptides were determined and compared with their

  6. 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

  7. 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...

  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. 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

  11. 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...

  12. 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...

  13. 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

  14. 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

  15. 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.

  16. 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

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

    Directory of Open Access Journals (Sweden)

    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.

  18. 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

  19. 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.

  20. 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...

  1. Ipomoelin, a Jacalin-Related Lectin with a Compact Tetrameric Association and Versatile Carbohydrate Binding Properties Regulated by Its N Terminus

    Science.gov (United States)

    Chang, Wei-Chieh; Liu, Kai-Lun; Hsu, Fang-Ciao; Jeng, Shih-Tong; Cheng, Yi-Sheng

    2012-01-01

    Many proteins are induced in the plant defense response to biotic stress or mechanical wounding. One group is lectins. Ipomoelin (IPO) is one of the wound-inducible proteins of sweet potato (Ipomoea batatas cv. Tainung 57) and is a Jacalin-related lectin (JRL). In this study, we resolved the crystal structures of IPO in its apo form and in complex with carbohydrates such as methyl α-D-mannopyranoside (Me-Man), methyl α-D-glucopyranoside (Me-Glc), and methyl α-D-galactopyranoside (Me-Gal) in different space groups. The packing diagrams indicated that IPO might represent a compact tetrameric association in the JRL family. The protomer of IPO showed a canonical β-prism fold with 12 strands of β-sheets but with 2 additional short β-strands at the N terminus. A truncated IPO (ΔN10IPO) by removing the 2 short β-strands of the N terminus was used to reveal its role in a tetrameric association. Gel filtration chromatography confirmed IPO as a tetrameric form in solution. Isothermal titration calorimetry determined the binding constants (KA) of IPO and ΔN10IPO against various carbohydrates. IPO could bind to Me-Man, Me-Glc, and Me-Gal with similar binding constants. In contrast, ΔN10IPO showed high binding ability to Me-Man and Me-Glc but could not bind to Me-Gal. Our structural and functional analysis of IPO revealed that its compact tetrameric association and carbohydrate binding polyspecificity could be regulated by the 2 additional N-terminal β-strands. The versatile carbohydrate binding properties of IPO might play a role in plant defense. PMID:22808208

  2. 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

  3. 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...

  4. [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

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. Direct spectroscopic evidence for competition between thermal molecular agitation and magnetic field in a tetrameric protein in aqueous solution

    Science.gov (United States)

    Calabrò, Emanuele; Magazù, Salvatore

    2018-05-01

    Samples of a typical tetrameric protein, the hemoglobin, at the concentration of 150 mg/ml in bidistilled water solution, were exposed to a uniform magnetic field at 200 mT at different temperatures of 15∘C, 40∘C and 65∘C. Fourier Transform Infrared Spectroscopy was used to analyze the response of the secondary structure of the protein to both stress agents, heating and static magnetic field. The most relevant result which was observed was the significant increasing in intensity of the Amide I band after exposure to the uniform magnetic field at the room temperature of 15∘C. This result can be explained assuming that protein's α-helices aligned along the direction of the applied magnetic field due to their large dipole moment, inducing the alignment of the entire protein. Increasing of temperature up to 40∘C and 65∘C induced a significant reduction of the increasing in intensity of the Amide I band. This effect may be easily explained assuming that Brownian motion of the protein in water solution caused by thermal molecular agitation increased with increasing of temperature, contrasting the effect of the torque of the magnetic field applied to the protein in water solution.

  10. The Vip3Ag4 Insecticidal Protoxin from Bacillus thuringiensis Adopts A Tetrameric Configuration That Is Maintained on Proteolysis

    Directory of Open Access Journals (Sweden)

    Leopoldo Palma

    2017-05-01

    Full Text Available The Vip3 proteins produced during vegetative growth by strains of the bacterium Bacillus thuringiensis show insecticidal activity against lepidopteran insects with a mechanism of action that may involve pore formation and apoptosis. These proteins are promising supplements to our arsenal of insecticidal proteins, but the molecular details of their activity are not understood. As a first step in the structural characterisation of these proteins, we have analysed their secondary structure and resolved the surface topology of a tetrameric complex of the Vip3Ag4 protein by transmission electron microscopy. Sites sensitive to proteolysis by trypsin are identified and the trypsin-cleaved protein appears to retain a similar structure as an octomeric complex comprising four copies each of the ~65 kDa and ~21 kDa products of proteolysis. This processed form of the toxin may represent the active toxin. The quality and monodispersity of the protein produced in this study make Vip3Ag4 a candidate for more detailed structural analysis using cryo-electron microscopy.

  11. PET imaging of {alpha}{sub v}{beta}{sub 3} integrin expression in tumours with {sup 68}Ga-labelled mono-, di- and tetrameric RGD peptides

    Energy Technology Data Exchange (ETDEWEB)

    Dijkgraaf, Ingrid; Franssen, Gerben M.; Oyen, Wim J.G.; Boerman, Otto C. [Radboud University Nijmegen Medical Centre, Department of Nuclear Medicine, P.O. Box 9101, Nijmegen (Netherlands); Yim, Cheng-Bin [Radboud University Nijmegen Medical Centre, Department of Nuclear Medicine, P.O. Box 9101, Nijmegen (Netherlands); Utrecht University, Department of Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Utrecht (Netherlands); Schuit, Robert C. [VU University Medical Centre, Department of Nuclear Medicine and PET Research, P.O. Box 7057, Amsterdam (Netherlands); Luurtsema, Gert [University Medical Center Groningen, Department of Nuclear Medicine and Molecular Imaging, Hanzeplein 1, P.O. Box 30.001, Groningen (Netherlands); Liu, Shuang [Purdue University, School of Health Sciences, West Lafayette, IN (United States)

    2011-01-15

    Due to the restricted expression of {alpha}{sub v}{beta}{sub 3} in tumours, {alpha}{sub v}{beta}{sub 3} is considered a suitable receptor for tumour targeting. In this study the {alpha}{sub v}{beta}{sub 3}-binding characteristics of {sup 68}Ga-labelled monomeric, dimeric and tetrameric RGD peptides were determined and compared with their {sup 111}In-labelled counterparts. A monomeric (E-c(RGDfK)), a dimeric (E-[c(RGDfK)]{sub 2}) and a tetrameric (E{l_brace}E[c(RGDfK)]{sub 2}{r_brace}{sub 2}) RGD peptide were synthesised, conjugated with DOTA and radiolabelled with {sup 68}Ga. In vitro {alpha}{sub v}{beta}{sub 3}-binding characteristics were determined in a competitive binding assay. In vivo {alpha}{sub v}{beta}{sub 3}-targeting characteristics of the compounds were assessed in mice with subcutaneously growing SK-RC-52 xenografts. In addition, microPET images were acquired using a microPET/CT scanner. The IC{sub 50} values for the Ga(III)-labelled DOTA-E-c(RGDfK), DOTA-E-[c(RGDfK)]{sub 2} and DOTA-E{l_brace}E[c(RGDfK)]{sub 2}{r_brace}{sub 2} were 23.9 {+-} 1.22, 8.99 {+-} 1.20 and 1.74 {+-} 1.18 nM, respectively, and were similar to those of the In(III)-labelled mono-, di- and tetrameric RGD peptides (26.6 {+-} 1.15, 3.34 {+-} 1.16 and 1.80 {+-} 1.37 nM, respectively). At 2 h post-injection, tumour uptake of the {sup 68}Ga-labelled mono-, di- and tetrameric RGD peptides (3.30 {+-} 0.30, 5.24 {+-} 0.27 and 7.11 {+-} 0.67%ID/g, respectively) was comparable to that of their {sup 111}In-labelled counterparts (2.70 {+-} 0.29, 5.61 {+-} 0.85 and 7.32 {+-} 2.45%ID/g, respectively). PET scans were in line with the biodistribution data. On all PET scans, the tumour could be clearly visualised. The integrin affinity and the tumour uptake followed the order of DOTA-tetramer > DOTA-dimer > DOTA-monomer. The {sup 68}Ga-labelled tetrameric RGD peptide has excellent characteristics for imaging of {alpha}{sub v} {beta}{sub 3} expression with PET. (orig.)

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. Structure and Mechanism of Proton Transport Through the Transmembrane Tetrameric M2 Protein Bundle of the Influenza A Virus

    Energy Technology Data Exchange (ETDEWEB)

    R Acharya; V Carnevale; G Fiorin; B Levine; A Polishchuk; V Balannick; I Samish; R Lamb; L Pinto; et al.

    2011-12-31

    The M2 proton channel from influenza A virus is an essential protein that mediates transport of protons across the viral envelope. This protein has a single transmembrane helix, which tetramerizes into the active channel. At the heart of the conduction mechanism is the exchange of protons between the His37 imidazole moieties of M2 and waters confined to the M2 bundle interior. Protons are conducted as the total charge of the four His37 side chains passes through 2{sup +} and 3{sup +} with a pK{sub a} near 6. A 1.65 {angstrom} resolution X-ray structure of the transmembrane protein (residues 25-46), crystallized at pH 6.5, reveals a pore that is lined by alternating layers of sidechains and well-ordered water clusters, which offer a pathway for proton conduction. The His37 residues form a box-like structure, bounded on either side by water clusters with well-ordered oxygen atoms at close distance. The conformation of the protein, which is intermediate between structures previously solved at higher and lower pH, suggests a mechanism by which conformational changes might facilitate asymmetric diffusion through the channel in the presence of a proton gradient. Moreover, protons diffusing through the channel need not be localized to a single His37 imidazole, but instead may be delocalized over the entire His-box and associated water clusters. Thus, the new crystal structure provides a possible unification of the discrete site versus continuum conduction models.

  20. 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...

  1. 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

  2. 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

  3. 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.)

  4. 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.)

  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. 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

  7. 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)

  8. 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

  9. 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.

  10. 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)

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

    Directory of Open Access Journals (Sweden)

    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.

  13. 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.

  14. 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.

  15. 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)

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. A tetrameric peptide derived from bovine lactoferricin as a potential therapeutic tool for oral squamous cell carcinoma: A preclinical model.

    Science.gov (United States)

    Solarte, Víctor Alfonso; Conget, Paulette; Vernot, Jean-Paul; Rosas, Jaiver Eduardo; Rivera, Zuly Jenny; García, Javier Eduardo; Arango-Rodríguez, Martha Ligia

    2017-01-01

    Oral squamous cell carcinoma is the fifth most common epithelial cancer in the world, and its current clinical treatment has both low efficiency and poor selectivity. Cationic amphipathic peptides have been proposed as new drugs for the treatment of different types of cancer. The main goal of the present work was to determine the potential of LfcinB(20-25)4, a tetrameric peptide based on the core sequence RRWQWR of bovine lactoferricin LfcinB(20-25), for the treatment of OSCC. In brief, OSCC was induced in the buccal pouch of hamsters by applying 7,12-Dimethylbenz(a)anthracene, and tumors were treated with one of the following peptides: LfcinB(20-25)4, LfcinB(20-25), or vehicle (control). Lesions were macroscopically evaluated every two days and both histological and serum IgG assessments were conducted after 5 weeks. The size of the tumors treated with LfcinB(20-25)4 and LfcinB(20-25) was smaller than that of the control group (46.16±4.41 and 33.92±2.74 mm3 versus 88.77±10.61 mm3, respectively). Also, LfcinB(20-25)4 caused acellularity in the parenchymal tumor compared with LfcinB(20-25) and vehicle treatments. Furthermore, our results demonstrated that both LfcinB(20-25)4 and LfcinB(20-25) induced higher degree of apoptosis relative to the untreated tumors (75-86% vs 8%, respectively). Moreover, although the lowest inflammatory response was achieved when LfcinB(20-25)4 was used, this peptide appeared to induce higher levels of IgG antibodies relative to the vehicle and LfcinB(20-25). In addition the cellular damage and selectivity of the LfcinB(20-25)4 peptide was evaluated in vitro. These assays showed that LfcinB(20-25)4 triggers a selective necrotic effect in the carcinoma cell line. Cumulatively, these data indicate that LfcinB(20-25)4 could be considered as a new therapeutic agent for the treatment of OSCC.

  3. A tetrameric peptide derived from bovine lactoferricin as a potential therapeutic tool for oral squamous cell carcinoma: A preclinical model.

    Directory of Open Access Journals (Sweden)

    Víctor Alfonso Solarte

    Full Text Available Oral squamous cell carcinoma is the fifth most common epithelial cancer in the world, and its current clinical treatment has both low efficiency and poor selectivity. Cationic amphipathic peptides have been proposed as new drugs for the treatment of different types of cancer. The main goal of the present work was to determine the potential of LfcinB(20-254, a tetrameric peptide based on the core sequence RRWQWR of bovine lactoferricin LfcinB(20-25, for the treatment of OSCC. In brief, OSCC was induced in the buccal pouch of hamsters by applying 7,12-Dimethylbenz(aanthracene, and tumors were treated with one of the following peptides: LfcinB(20-254, LfcinB(20-25, or vehicle (control. Lesions were macroscopically evaluated every two days and both histological and serum IgG assessments were conducted after 5 weeks. The size of the tumors treated with LfcinB(20-254 and LfcinB(20-25 was smaller than that of the control group (46.16±4.41 and 33.92±2.74 mm3 versus 88.77±10.61 mm3, respectively. Also, LfcinB(20-254 caused acellularity in the parenchymal tumor compared with LfcinB(20-25 and vehicle treatments. Furthermore, our results demonstrated that both LfcinB(20-254 and LfcinB(20-25 induced higher degree of apoptosis relative to the untreated tumors (75-86% vs 8%, respectively. Moreover, although the lowest inflammatory response was achieved when LfcinB(20-254 was used, this peptide appeared to induce higher levels of IgG antibodies relative to the vehicle and LfcinB(20-25. In addition the cellular damage and selectivity of the LfcinB(20-254 peptide was evaluated in vitro. These assays showed that LfcinB(20-254 triggers a selective necrotic effect in the carcinoma cell line. Cumulatively, these data indicate that LfcinB(20-254 could be considered as a new therapeutic agent for the treatment of OSCC.

  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. 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

  6. 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

  7. 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

  8. A mannose-specific tetrameric lectin with mitogenic and antibacterial activities from the ovary of a teleost, the cobia (Rachycentron canadum).

    Science.gov (United States)

    Ngai, Patrick H K; Ng, T B

    2007-02-01

    A tetrameric lectin, with hemagglutinating activity toward rabbit erythrocytes and with specificity toward D-mannosamine and D(+)-mannose, was isolated from the ovaries of a teleost, the cobia Rachycentron canadum. The isolation protocol comprised ion exchange chromatography on CM-cellulose and Q-Sepharose, ion exchange chromatography by fast protein liquid chromatography (FPLC) on Mono Q, and finally gel filtration by FPLC on Superose 12. The lectin was adsorbed on all ion exchangers used. It exhibited a molecular mass of 180 kDa in gel filtration on Superose 12 and a single 45-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that it is a tetrameric protein. The hemagglutinating activity of the lectin was stable up to 40 degrees C and between pH 4 and pH 10. All hemagglutinating activity disappeared at 60 degrees C and at pH 1 and pH 13. The hemagglutinating activity was doubled in the presence of 0.1 microM FeCl3. The lectin exerted antibacterial activity against Escherichia coli with 50% inhibition at 250 microg. There was no antifungal activity toward Coprinus comatus, Fusarium oxysporum, Mycosphaerella arachidicola, and Rhizoctonia solani at a dose of 300 microg. The lectin exhibited maximal mitogenic response from mouse splenocytes at a concentration of 14 microM.

  9. 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.

  10. 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.

  11. 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...

  12. 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

  13. 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

  14. 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

  15. 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...

  16. 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.

  17. 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.

  18. 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.)

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

    DEFF Research Database (Denmark)

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

    1989-01-01

    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...

  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. Inhibition by nucleosides of glucose-transport activity in human erythrocytes.

    OpenAIRE

    Jarvis, S M

    1988-01-01

    The interaction of nucleosides with the glucose carrier of human erythrocytes was examined by studying the effect of nucleosides on reversible cytochalasin B-binding activity and glucose transport. Adenosine, inosine and thymidine were more potent inhibitors of cytochalasin B binding to human erythrocyte membranes than was D-glucose [IC50 (concentration causing 50% inhibition) values of 10, 24, 28 and 38 mM respectively]. Moreover, low concentrations of thymidine and adenosine inhibited D-glu...

  2. 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.

  3. 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.

  4. 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.

  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. Studies on the decomposition of ethyl diazoacetate and its reaction with coal. Formation of a new tetrameric product and reagent access within the coal

    Energy Technology Data Exchange (ETDEWEB)

    Pomerantz, M.; Rooney, P.

    A new tetrameric pyrazoline, 10, has been observed in the thermal and Fe/sub 2/O/sub 3/-catalyzed decomposition of ethyl diazoacetate (2) as well as when several coal samples were treated thermally with 2 under various conditions. Identification of 10 was based on spectral properties and an independent synthesis. A comparison of the amounts of diethyl fumarate (3), diethyl maleate (4), the trimeric pyrazoline 5, triethyl trans-cyclopropane-1,2,3-tricarboxylate (8), and the tetrameric pyrazoline 10 formed in the coal reactions with the relative quantities produced in the thermal and Fe/sub 2/O/sub 3/-catalyzed reactions of 2, both neat and diluted with p-xylene, showed that there were several successive and competing reactions occurring, one of which was independent of the concentration of 2. Further, on the basis of the observation that the product distribution of 3-5, 8, and 10 in the Fe/sub 2/O/sub 3/-catalyzed decomposition of 2 in relatively dilute solution is similar to that observed in the coal reactions, with cyclopropane 8 being the major product in both cases, and that 2 is reacting mainly with the coal, it is concluded that 2 is fairly well dispersed within the coal. In addition, it is clear that swelling of the coal with dioxane did very little to facilitate access of 2 into the coal. Instead the dioxane merely acted to allow for more complete extraction of the products after 2 had reacted with the coal, presumably by keeping the matrix structure more open, than when the dioxane was not used. 26 refs., 2 tabs.

  7. Development of a tetrameric streptavidin mutein with reversible biotin binding capability: engineering a mobile loop as an exit door for biotin.

    Directory of Open Access Journals (Sweden)

    Valerie J O'Sullivan

    Full Text Available A novel form of tetrameric streptavidin has been engineered to have reversible biotin binding capability. In wild-type streptavidin, loop(3-4 functions as a lid for the entry and exit of biotin. When biotin is bound, interactions between biotin and key residues in loop(3-4 keep this lid in the closed state. In the engineered mutein, a second biotin exit door is created by changing the amino acid sequence of loop(7-8. This door is mobile even in the presence of the bound biotin and can facilitate the release of biotin from the mutein. Since loop(7-8 is involved in subunit interactions, alteration of this loop in the engineered mutein results in an 11° rotation between the two dimers in reference to wild-type streptavidin. The tetrameric state of the engineered mutein is stabilized by a H127C mutation, which leads to the formation of inter-subunit disulfide bonds. The biotin binding kinetic parameters (k(off of 4.28×10(-4 s(-1 and K(d of 1.9×10(-8 M make this engineered mutein a superb affinity agent for the purification of biotinylated biomolecules. Affinity matrices can be regenerated using gentle procedures, and regenerated matrices can be reused at least ten times without any observable reduction in binding capacity. With the combination of both the engineered mutein and wild-type streptavidin, biotinylated biomolecules can easily be affinity purified to high purity and immobilized to desirable platforms without any leakage concerns. Other potential biotechnological applications, such as development of an automated high-throughput protein purification system, are feasible.

  8. Development of a Tetrameric Streptavidin Mutein with Reversible Biotin Binding Capability: Engineering a Mobile Loop as an Exit Door for Biotin

    Science.gov (United States)

    O'Sullivan, Valerie J.; Barrette-Ng, Isabelle; Hommema, Eric; Hermanson, Greg T.; Schofield, Mark; Wu, Sau-Ching; Honetschlaeger, Claudia; Ng, Kenneth K.-S.; Wong, Sui-Lam

    2012-01-01

    A novel form of tetrameric streptavidin has been engineered to have reversible biotin binding capability. In wild-type streptavidin, loop3–4 functions as a lid for the entry and exit of biotin. When biotin is bound, interactions between biotin and key residues in loop3–4 keep this lid in the closed state. In the engineered mutein, a second biotin exit door is created by changing the amino acid sequence of loop7–8. This door is mobile even in the presence of the bound biotin and can facilitate the release of biotin from the mutein. Since loop7–8 is involved in subunit interactions, alteration of this loop in the engineered mutein results in an 11° rotation between the two dimers in reference to wild-type streptavidin. The tetrameric state of the engineered mutein is stabilized by a H127C mutation, which leads to the formation of inter-subunit disulfide bonds. The biotin binding kinetic parameters (koff of 4.28×10−4 s−1 and Kd of 1.9×10−8 M) make this engineered mutein a superb affinity agent for the purification of biotinylated biomolecules. Affinity matrices can be regenerated using gentle procedures, and regenerated matrices can be reused at least ten times without any observable reduction in binding capacity. With the combination of both the engineered mutein and wild-type streptavidin, biotinylated biomolecules can easily be affinity purified to high purity and immobilized to desirable platforms without any leakage concerns. Other potential biotechnological applications, such as development of an automated high-throughput protein purification system, are feasible. PMID:22536357

  9. 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

  10. 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

  11. 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)

  12. 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...

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

    International Nuclear Information System (INIS)

    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)

  14. 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)

  15. 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 ...

  16. 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...

  17. 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

  18. 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

  19. 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

  20. 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.

  1. 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...

  2. 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)

  3. 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.

  4. 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.).

  5. 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.

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. 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.)

  11. 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.

  12. 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)

  13. 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)

  14. Assay for mutagenesis in heterozygous diploid human lymphoblasts

    Science.gov (United States)

    Skopek, Thomas R.; Liber, Howard L.; Penman, Bruce W.; Thilly, William G.; Hoppe, IV, Henry

    1981-01-01

    An assay is disclosed for determining mutagenic damage caused by the administration of a known or suspected mutagen to diploid human lymphoblastoid cell lines. The gene locus employed for this assay is the gene for thymidine kinase, uridine kinase, or cytidine deaminase. Since human lymphoblastoid cells contain two genes for these enzymes, heterozygotes of human lymphoblastoid cells are used in this assay.

  15. 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

  16. Nematodes from Swainson's spurfowl Pternistis swainsonii and an Orange River francolin Scleroptila levaillantoides in Free State Province, South Africa, with a description of Tetrameres swainsonii n. sp. (Nematoda: Tetrameridae).

    Science.gov (United States)

    Junker, K; Davies, O R; Jansen, R; Crowe, T M; Boomker, J

    2008-12-01

    Five Swainson's spurfowl collected in Free State Province, South Africa, were examined for helminth parasites, and the nematodes Acuaria gruveli, Cyrnea parroti, Gongylonema congolense, Subulura dentigera, Subulura suctoria and a new Tetrameres species were recovered. Their respective prevalence was 100, 20, 80, 20, 20 and 20%. These nematodes are all new parasite records for Swainson's spurfowl, and Acuaria gruveli constitutes a new geographical record as well. A single specimen of Cyrnea eurycerca was found in an Orange River francolin, representing a new host and geographical record for this parasite. The new species, for which the name Tetrameres swainsonii is proposed, can be differentiated from its congeners by a combination of the following characters of males: two rows of body spines, a single spicule which is 1152-1392 microm long, and eight pairs of caudal spines arranged in two ventral and two lateral rows of four spines each. The single female has the globular shape typical of the genus.

  17. 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.

  18. 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.

  19. Comparative analysis of the heme iron electronic structure and stereochemistry in tetrameric rabbit hemoglobin and monomeric soybean leghemoglobin a using Mössbauer spectroscopy with a high velocity resolution

    Science.gov (United States)

    Alenkina, I. V.; Kumar, A.; Berkovsky, A. L.; Oshtrakh, M. I.

    2018-02-01

    A comparative study of tetrameric rabbit hemoglobin and monomeric soybean leghemoglobin a in the oxy- and deoxy-forms was carried out using 57Fe Mössbauer spectroscopy with a high velocity resolution in order to analyze the heme iron electronic structure and stereochemistry in relation to the Mössbauer hyperfine parameters. The Mössbauer spectra of tetrameric rabbit hemoglobin in both forms were fitted using two quadrupole doublets related to the 57Fe in ɑ- and β-subunits. In contrast, the Mössbauer spectra of monomeric soybean leghemoglobin a were fitted using: (i) two quadrupole doublets for the oxy-form related to two conformational states of the distal His E7 imidazole ring and different hydrogen bonding of oxygen molecule in the oxy-form and (ii) using three quadrupole doublets for deoxy-form related to three conformational states of the proximal His F8 imidazole ring. Small variations of Mössbauer hyperfine parameters related to small differences in the heme iron electronic structure and stereochemistry in tetrameric rabbit hemoglobin and monomeric soybean leghemoglobin a are discussed.

  20. Molecular dynamics characterization of the SAMHD1 Aicardi-Goutières Arg145Gln mutant: structural determinants for the impaired tetramerization

    Science.gov (United States)

    Cardamone, Francesca; Falconi, Mattia; Desideri, Alessandro

    2018-05-01

    Aicardi-Goutières syndrome, a rare genetic disorder characterized by calcification of basal ganglia, results in psychomotor delays and epilepsy states from the early months of children life. This disease is caused by mutations in seven different genes encoding proteins implicated in the metabolism of nucleic acids, including SAMHD1. Twenty SAMHD1 gene variants have been discovered and in this work, a structural characterization of the SAMHD1 Aicardi-Goutières Arg145Gln mutant is reported by classical molecular dynamics simulation. Four simulations have been carried out and compared. Two concerning the wild-type SAMHD1 form in presence and absence of cofactors, in order to explain the role of cofactors in the SAMHD1 assembly/disassembly process and, two concerning the Arg145Gln mutant, also in presence and absence of cofactors, in order to have an accurate comparison with the corresponding native forms. Results show the importance of native residue Arg145 in maintaining the tetramer, interacting with GTP cofactor inside allosteric sites. Replacement of arginine in glutamine gives rise to a loosening of GTP-protein interactions, when cofactors are present in allosteric sites, whilst in absence of cofactors, the occurrence of intra and inter-chain interactions is observed in the mutant, not seen in the native enzyme, making energetically unfavourable the tetramerization process.

  1. 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

  2. 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.

  3. 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.)

  4. 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.

  5. 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

  6. 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.

  7. 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)

  8. Isolation of human simple repeat loci by hybridization selection.

    Science.gov (United States)

    Armour, J A; Neumann, R; Gobert, S; Jeffreys, A J

    1994-04-01

    We have isolated short tandem repeat arrays from the human genome, using a rapid method involving filter hybridization to enrich for tri- or tetranucleotide tandem repeats. About 30% of clones from the enriched library cross-hybridize with probes containing trimeric or tetrameric tandem arrays, facilitating the rapid isolation of large numbers of clones. In an initial analysis of 54 clones, 46 different tandem arrays were identified. Analysis of these tandem repeat loci by PCR showed that 24 were polymorphic in length; substantially higher levels of polymorphism were displayed by the tetrameric repeat loci isolated than by the trimeric repeats. Primary mapping of these loci by linkage analysis showed that they derive from 17 chromosomes, including the X chromosome. We anticipate the use of this strategy for the efficient isolation of tandem repeats from other sources of genomic DNA, including DNA from flow-sorted chromosomes, and from other species.

  9. 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.

  10. 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.

  11. 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)

  12. The monomeric, tetrameric, and fibrillar organization of Fib: the dynamic building block of the bacterial linear motor of Spiroplasma melliferum BC3.

    Science.gov (United States)

    Cohen-Krausz, Sara; Cabahug, Pamela C; Trachtenberg, Shlomo

    2011-07-08

    Spiroplasmas belong to the class Mollicutes, representing the minimal, free-living, and self-replicating forms of life. Spiroplasmas are helical wall-less bacteria and the only ones known to swim by means of a linear motor (rather than the near-universal rotary bacterial motor). The linear motor follows the shortest path along the cell's helical membranal tube. The motor is composed of a flat monolayered ribbon of seven parallel fibrils and is believed to function in controlling cell helicity and motility through dynamic, coordinated, differential length changes in the fibrils. The latter cause local perturbations of helical symmetry, which are essential for net directional displacement in environments with a low Reynolds number. The underlying fibrils' core building block is a circular tetramer of the 59-kDa protein Fib. The fibrils' differential length changes are believed to be driven by molecular switching of Fib, leading consequently to axial ratio and length changes in tetrameric rings. Using cryo electron microscopy, diffractometry, single-particle analysis of isolated ribbons, and sequence analyses of Fib, we determined the overall molecular organization of the Fib monomer, tetramer, fibril, and linear motor of Spiroplasma melliferum BC3 that underlies cell geometry and motility. Fib appears to be a bidomained molecule, of which the N-terminal half is apparently a globular phosphorylase. By a combination of reversible rotation and diagonal shift of Fib monomers, the tetramer adopts either a cross-like nonhanded conformation or a ring-like handed conformation. The sense of Fib rotation may determine the handedness of the linear motor and, eventually, of the cell. A further change in the axial ratio of the ring-like tetramers controls fibril lengths and the consequent helical geometry. Analysis of tetramer quadrants from adjacent fibrils clearly demonstrates local differential fibril lengths. Copyright © 2011 Elsevier Ltd. All rights reserved.

  13. 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.

  14. 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

  15. 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.

  16. 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...

  17. Role of α-globin H helix in the building of tetrameric human hemoglobin: interaction with α-hemoglobin stabilizing protein (AHSP) and heme molecule.

    Science.gov (United States)

    Domingues-Hamdi, Elisa; Vasseur, Corinne; Fournier, Jean-Baptiste; Marden, Michael C; Wajcman, Henri; Baudin-Creuza, Véronique

    2014-01-01

    Alpha-Hemoglobin Stabilizing Protein (AHSP) binds to α-hemoglobin (α-Hb) or α-globin and maintains it in a soluble state until its association with the β-Hb chain partner to form Hb tetramers. AHSP specifically recognizes the G and H helices of α-Hb. To investigate the degree of interaction of the various regions of the α-globin H helix with AHSP, this interface was studied by stepwise elimination of regions of the α-globin H helix: five truncated α-Hbs α-Hb1-138, α-Hb1-134, α-Hb1-126, α-Hb1-123, α-Hb1-117 were co-expressed with AHSP as two glutathione-S-transferase (GST) fusion proteins. SDS-PAGE and Western Blot analysis revealed that the level of expression of each truncated α-Hb was similar to that of the wild type α-Hb except the shortest protein α-Hb1-117 which displayed a decreased expression. While truncated GST-α-Hb1-138 and GST-α-Hb1-134 were normally soluble; the shorter globins GST-α-Hb1-126 and GST-α-Hb1-117 were obtained in very low quantities, and the truncated GST-α-Hb1-123 provided the least material. Absorbance and fluorescence studies of complexes showed that the truncated α-Hb1-134 and shorter forms led to modified absorption spectra together with an increased fluorescence emission. This attests that shortening the H helix leads to a lower affinity of the α-globin for the heme. Upon addition of β-Hb, the increase in fluorescence indicates the replacement of AHSP by β-Hb. The CO binding kinetics of different truncated AHSPWT/α-Hb complexes showed that these Hbs were not functionally normal in terms of the allosteric transition. The N-terminal part of the H helix is primordial for interaction with AHSP and C-terminal part for interaction with heme, both features being required for stability of α-globin chain.

  18. 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...

  19. 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...

  20. 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)

  1. 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

  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. 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

  5. Spontaneous unscheduled DNA synthesis in human lymphocytes

    International Nuclear Information System (INIS)

    Forell, B.; Myers, L.S. Jr.; Norman, A.

    1979-01-01

    The rate of spontaneous unscheduled DNA synthesis in human lymphocytes was estimated from measurements of tritiated thymidine incorporation into double-stranded DNA (ds-DNA) during incubation of cells in vitro. The contribution of scheduled DNA synthesis to the observed incorporation was reduced by inhibiting replication with hydroxyurea and by separating freshly replicated single-stranded DNA (ss-DNA) from repaired ds-DNA by column chromatography. The residual contribution of scheduled DNA synthesis was estimated by observing effects on thymidine incorporation of: (a) increasing the rate of production of apurinic sites, and alternatively, (b) increasing the number of cells in S-phase. Corrections based on estimates of endogenous pool size were also made. The rate of spontaneous unscheduled DNA synthesis is estimated to be 490 +- 120 thymidine molecules incorporated per cell per hour. These results compare favorably with estimates made from rates of depurination and depyrimidination of DNA, measured in molecular systems if we assume thymidine is incorporated by a short patch mechanism which incorporates an average of four bases per lesion

  6. 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

  7. 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)

  8. 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....

  9. 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

  10. 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.

  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. 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.

  13. 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

  14. 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

  15. 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)

  16. 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)

  17. 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.

  18. 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.)

  19. 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.

  20. 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.

  1. 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.)

  2. 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)

  3. 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.

  4. 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

  5. A repeated injection of polyethyleneglycol-conjugated recombinant human butyrylcholinesterase elicits immune response in mice

    International Nuclear Information System (INIS)

    Chilukuri, Nageswararao; Sun Wei; Parikh, Kalpana; Naik, Ramachandra S.; Tang Lin; Doctor, Bhupendra P.; Saxena, Ashima

    2008-01-01

    Human serum butyrylcholinesterase (Hu BChE) serves as an efficacious bioscavenger of highly toxic organophosphorus (OP) compounds. Since there is a concern that the supply of native Hu BChE may be limited, monomeric and tetrameric forms of recombinant Hu BChE (rHu BChE) were evaluated as replacements and found that they lacked sufficient stability in vivo. However, their in vivo stability could be significantly prolonged by conjugation with polyethyleneglycol-20K (PEG) suggesting that monomeric and tetrameric PEG-rHu BChE could function as bioscavengers. Here, the immunogenicity of PEG-rHu BChE was evaluated in mice following two injections given four weeks apart. In addition to pharmacokinetic parameters, such as mean residence time, maximal concentration, time to reach the maximal concentration, elimination half-life and area under the plasma concentration-time curve extrapolated to infinity, the presence of circulating anti-rHu BChE antibodies was also determined. Although the pharmacokinetic parameters were significantly improved for the first injection of monomeric and tetrameric PEG-rHu BChEs, they were much lower for the second injection. Anti-rHu BChE antibodies were detected in the blood of mice following the first and second enzyme injections and their levels were approximately higher by 5-fold and 2-fold in mice injected with monomeric and tetrameric PEG-rHu BChEs as compared to mice injected with unconjugated enzymes. The findings that the rapid clearance of a repeat injection of PEG-rHu BChEs in mice which coincides with the presence of circulating anti-rHu BChE antibodies suggest that PEG conjugation prolonged the circulatory stability of rHu BChE but failed to eliminate its immunogenicity in mice

  6. 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

  7. 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.

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

    Directory of Open Access Journals (Sweden)

    Joan Villarroya

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

  9. 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.

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

    International Nuclear Information System (INIS)

    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)

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

    Directory of Open Access Journals (Sweden)

    Gilad Oren

    2005-03-01

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

  12. 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)

  13. 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

  14. 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.)

  15. PHA-induced cytotoxicity of human lymphocytes against adherent hela-cells

    NARCIS (Netherlands)

    Huges-Law, G.; de Gast, G. C.; The, T. Hauw

    The conditions for a phytohaemagglutinin(PHA)-induced cytotoxicity test of human peripheral blood lymphocytes were investigated. [3H]thymidine prelabelled HeLa cells were used as target cells. Stimulation with 10 μl PHA/ml during 24 h gave the best measure of lymphocyte cytotoxic capacity.

  16. 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

  17. 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

  18. In vitro exposure to X-radiation of stimulated and non-stimulated human B lymphocytes and T lymphocytes. In vitro Roentgenbestrahlung stimulierter und unstimulierter menschlicher B- und T-Lymphozyten

    Energy Technology Data Exchange (ETDEWEB)

    Krystossek, H.

    1986-09-25

    The sensitivity of human type B and type T lymphocytes to 130 kV X-radiation was investigated in vitro. The degree to which 3H thymidine was incorporated into the DNA of these cells was taken as a measure of cellular viability. The results led to the conclusion that the in vitro reactions to X-rays following stimulation and radiation are considerably more pronounced in human B lymphocytes than in human T lymphocytes. The rapid radiation-induced lessening of thymidine incorporation into stimulated B lymphocytes was interpreted as a sign that cellular decay occurred during the interphase. The relative increases in the thymidine incorporation rates seen following radiation of T cells in the presence of hydroxyurea or caffeinemust, however, not be mistaken for an augmentation of resistance that was brought about by these inhibitors. The latter effect is believed to be rather due to an overreaction of the repair mechanisms of DNA which is characterised by short chains.

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. 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...

  7. 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.

  8. Antiproliferative effect of UTP on human arterial and venous smooth muscle cells.

    Science.gov (United States)

    White, P J; Kumari, R; Porter, K E; London, N J; Ng, L L; Boarder, M R

    2000-12-01

    We have investigated the hypothesis that responses associated with proliferation are regulated by extracellular nucleotides such as ATP and UTP in cultured human vascular smooth muscle cells (VSMC) derived from internal mammary artery (IMA) and saphenous vein (SV). Platelet-derived growth factor (PDGF), ATP, and UTP each generated an increase in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) in both IMA- and SV-derived cells in the absence of detectable inositol 1,4,5-trisphosphate production. ATP alone had no effect on [(3)H]thymidine incorporation into DNA, but with a submaximal concentration of PDGF it raised [(3)H]thymidine incorporation in SV- but not IMA-derived cells. UTP alone also was without effect on [(3)H]thymidine incorporation or cell number. However, in both SV- and IMA-derived cells, UTP reduced the PDGF-stimulated [(3)H]thymidine response and PDGF-stimulated cell proliferation. This cannot be explained by an inhibitory effect on the p42/p44 mitogen-activated protein kinase (MAPK) cascade, since this response to PDGF was not attenuated by UTP. We conclude that, in human VSMC of both arterial and venous origin, UTP acts as an anti-proliferative regulator.

  9. Effect of pyrimethamine and sulphadoxine on human lymphocyte proliferation

    DEFF Research Database (Denmark)

    Bygbjerg, I C; Odum, Niels; Theander, T G

    1986-01-01

    The in vitro effect of pyrimethamine (PYR) on human blood mononuclear cells stimulated with phytohaemagglutinin (PHA), pokeweed mitogen (PWM) and purified protein derivative of tuberculin (PPD) was studied by 14C-thymidine incorporation, by cell counting and by total DNA estimation. PYR in concen......The in vitro effect of pyrimethamine (PYR) on human blood mononuclear cells stimulated with phytohaemagglutinin (PHA), pokeweed mitogen (PWM) and purified protein derivative of tuberculin (PPD) was studied by 14C-thymidine incorporation, by cell counting and by total DNA estimation. PYR...... in concentrations 10 times higher than serum values obtained in clinical practice inhibited lymphocyte proliferation irreversibly. PYR in concentrations corresponding to clinical practice quickly and irreversibly suppressed the proliferation of PWM-stimulated cells, and more slowly the proliferation of PPD...

  10. Low-resolution structure of the tetrameric phenylalanyl-tRNA synthetase from Escherichia coli. A neutron small-angle scattering study of hybrids composed of protonated and deuterated protomers

    International Nuclear Information System (INIS)

    Dessen, P.; Ducruix, A.; May, R.P.; Blanquet, S.

    1990-01-01

    Escherichia coli phenylalanyl-tRNA synthetase is a tetrameric protein composed of two types of protomers. In order to resolve the subunit organization, neutron small-angle scattering experiments have been performed in different contrasts with all types of isotope hybrids that could be obtained by reconstituting the alpha 2 beta 2 enzyme from the protonated and deuterated forms of the alpha and beta subunits. Experiments have been also made with the isolated alpha promoter. A model for the alpha 2 beta 2 tetramer is deduced where the two alpha promoters are elongated ellipsoids (45 x 45 x 160 A3) lying side by side with an angle of about 40 degrees between their long axes and where the two beta subunits are also elongated ellipsoids (31 x 31 x 130 A3) with an angle of 30 degrees between their axes. This model was obtained by assuming that the two pairs of subunits are in contact in an orthogonal manner and by taking advantage of the measured distance between the centers of mass of the alpha 2 and beta 2 pairs (d = 23 +/- 2 A)

  11. Purification of human platelet-derived growth factor

    International Nuclear Information System (INIS)

    Raines, E.W.; Ross, R.

    1985-01-01

    The paper describes a method for purification of human platelet-derived growth factor (PDGF) from outdated platelet-rich plasma (PRP) using commonly available laboratory reagents and yielding a mitogen purified 800,000-fold over the starting material. [ 3 H]thymidine incorporation into DNA of cultured cells responsive to PDGF represents the most readily available method to follow its purification and define the biological activity of a purified preparation. Other assays to quantitate PDGF include radioreceptor assay and radioimmunoassay

  12. 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.

  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. 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

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. Site-specific semisynthetic variant of human hemoglobin

    International Nuclear Information System (INIS)

    Hefta, S.A.; Lyle, S.B.; Busch, M.R.; Harris, D.E.; Matthew, J.B.; Gurd, F.R.N.

    1988-01-01

    A single round of Edman degradation was employed to remove the NH 2 -terminal valine from isolated α chains of human hemoglobin. Reconstitution of normal β chains with truncated or substituted α chains was used to form truncated (des-Val 1 -α1) and substituted ([[1- 13 C]Gly 1 ]α1) tetrameric hemoglobin analogs. Structural homology of the analogs with untreated native hemoglobin was established by using several spectroscopic and physical methods. Functional studies indicate that the reconstituted tetrameric protein containing des-Val 1 -α chains has a higher affinity for oxygen, is less influenced by chloride ions or 2,3-biphosphoglycerate, and shows lower cooperativity than native hemoglobin. These results confirm the key functional role of the α-chain NH 2 terminus in mediating cooperative oxygen binding across the dimer interface. The NH 2 -terminal pK/sub 1/2/ value was determined for the [ 13 C]glycine-substituted analog to be 7.46 +/- 0.09 at 15 0 C in the carbon monoxide-liganded form. This value, measured directly by 13 C NMR, agrees with the determination made by the less-direct 13 CO 2 method and confirms the role of this residue as a contributor to the alkaline Bohr effect; however, it is consistent with the presence of an NH 2 -terminal salt bridge to the carboxylate of Arg-141 of the α chain in the liganded form

  6. Kinetics of Human Haematopoiesis

    Energy Technology Data Exchange (ETDEWEB)

    Cronkite, E. P. [Forschungsgruppe Freiburg, Institut fuer Haematologie der Gesellschaft fuer Strahlenforschung Assoziation mit EURATOM, Freiburg/Breisgau, Federal Republic of Germany (Germany); Medical Research Center, Brookhaven National Laboratory, Upton, Long Island, NY (United States)

    1967-07-15

    An understanding of the parameters of normal cell proliferation is a prerequisite for the understanding of the disturbances induced by radiation. The disturbances induced by radiation on cell renewal systems has been presented in detail by Bond, Fliedner and Archambeau. In this paper a method of studying the concepts and actual observations on human haemopoiesis will be summarized. Details of the procedures, methods and concepts are published. The method employed is the serial.autoradiographic study of haemopoietic tissues after labelling the DNA of proliferating compartments by intravenous injection of tritiated thymidine (3HTDR) a specific DNA precursor. One can then observe the flow of labelled cells through mitosis, the transit of labelled cells from a proliferating compartment to a non proliferating compartment and the migration of cells from tissues to blood.

  7. Ex vivo analysis of human memory CD4 T cells specific for hepatitis C virus using MHC class II tetramers

    OpenAIRE

    Day, Cheryl L.; Seth, Nilufer P.; Lucas, Michaela; Appel, Heiner; Gauthier, Laurent; Lauer, Georg M.; Robbins, Gregory K.; Szczepiorkowski, Zbigniew M.; Casson, Deborah R.; Chung, Raymond T.; Bell, Shannon; Harcourt, Gillian; Walker, Bruce D.; Klenerman, Paul; Wucherpfennig, Kai W.

    2003-01-01

    Containment of hepatitis C virus (HCV) and other chronic human viral infections is associated with persistence of virus-specific CD4 T cells, but ex vivo characterization of circulating CD4 T cells has not been achieved. To further define the phenotype and function of these cells, we developed a novel approach for the generation of tetrameric forms of MHC class II/peptide complexes that is based on the cellular peptide-exchange mechanism. HLA-DR molecules were expressed as precursors with a c...

  8. 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

  9. Characterization of Staphylococcus aureus Primosomal DnaD Protein: Highly Conserved C-Terminal Region Is Crucial for ssDNA and PriA Helicase Binding but Not for DnaA Protein-Binding and Self-Tetramerization.

    Directory of Open Access Journals (Sweden)

    Yen-Hua Huang

    Full Text Available The role of DnaD in the recruitment of replicative helicase has been identified. However, knowledge of the DNA, PriA, and DnaA binding mechanism of this protein for the DnaA- and PriA-directed replication primosome assemblies is limited. We characterized the DNA-binding properties of DnaD from Staphylococcus aureus (SaDnaD and analyzed its interactions with SaPriA and SaDnaA. The gel filtration chromatography analysis of purified SaDnaD and its deletion mutant proteins (SaDnaD1-195, SaDnaD1-200 and SaDnaD1-204 showed a stable tetramer in solution. This finding indicates that the C-terminal region aa 196-228 is not crucial for SaDnaD oligomerization. SaDnaD forms distinct complexes with ssDNA of different lengths. In fluorescence titrations, SaDnaD bound to ssDNA with a binding-site size of approximately 32 nt. A stable complex of SaDnaD1-195, SaDnaD1-200, and SaDnaD1-204 with ssDNA dT40 was undetectable, indicating that the C-terminal region of SaDnaD (particularly aa 205-228 is crucial for ssDNA binding. The SPR results revealed that SaDnaD1-195 can interact with SaDnaA but not with SaPriA, which may indicate that DnaD has different binding sites for PriA and DnaA. Both SaDnaD and SaDnaDY176A mutant proteins, but not SaDnaD1-195, can significantly stimulate the ATPase activity of SaPriA. Hence, the stimulation effect mainly resulted from direct contact within the protein-protein interaction, not via the DNA-protein interaction. Kinetic studies revealed that the SaDnaD-SaPriA interaction increases the Vmax of the SaPriA ATPase fivefold without significantly affecting the Km. These results indicate that the conserved C-terminal region is crucial for ssDNA and PriA helicase binding, but not for DnaA protein-binding and self-tetramerization.

  10. 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

  11. 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.

  12. 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

  13. Kinetics of growth and differentiation of cultured human epidermal keratinocytes

    International Nuclear Information System (INIS)

    Albers, K.M.

    1985-01-01

    A study was made of the interrelationship between replication and differentiation in cultures of human epidermal keratinocytes. Measures of both parameters were made using newly developed methods to quantify the rate at which keratinocytes replicate and the rate at which they withdraw from the cell cycle. Keratinocyte replication was measured by determining the cell doubling time, labeling index, and cell cycle duration. Cell cycle length was measured using a double label assay that determines the length of time between two successive phases of DNA synthesis. The first DNA synthesis phase was marked by labeling keratinocytes with 14 C-thymidine. At the next round of DNA synthesis, cells were labeled with bromodeoxyuridine, a heavy analog of thymidine. The cell cycle length is given by the time required for the 14 C-labeled DNA to become double labeled. To measure keratinocyte differentiation, the rate at which cells withdraw from the cell cycle was determined. To measure withdrawal, the percentage of cells labeled by a pulse of 14 C-thymidine that failed to undergo a second cycle of DNA synthesis, as measured by bromodeoxyuridine incorporation, was determined. Cells which failed to undergo a second cycle of synthesis were considered to have differentiated and withdrawn from the cell cycle

  14. Hapten-specific lymphocyte transformation in humans sensitized with NDMA or DNCB.

    Science.gov (United States)

    SoebergB; Andersen, V

    1976-01-01

    The primary immune response to a contact sensitizing dose of para-N-dimethylnitrosaniline (NDMA) and dinitrochlorobenzene (DNCB) was obtained in humans and measured in vitro by increased thymidine incorporation into sensitized lymphocytes. No cross-reaction was found between these two haptens, and it is thus possible on two separate occasions to quantify and follow the primary cellular immune response in man. PMID:963911

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 2-Methoxypyridine as a Thymidine Mimic in Watson-Crick Base Pairs of DNA and PNA: Synthesis, Thermal Stability, and NMR Structural Studies.

    Science.gov (United States)

    Novosjolova, Irina; Kennedy, Scott D; Rozners, Eriks

    2017-11-02

    The development of nucleic acid base-pair analogues that use new modes of molecular recognition is important both for fundamental research and practical applications. The goal of this study was to evaluate 2-methoxypyridine as a cationic thymidine mimic in the A-T base pair. The hypothesis was that including protonation in the Watson-Crick base pairing scheme would enhance the thermal stability of the DNA double helix without compromising the sequence selectivity. DNA and peptide nucleic acid (PNA) sequences containing the new 2-methoxypyridine nucleobase (P) were synthesized and studied by using UV thermal melting and NMR spectroscopy. Introduction of P nucleobase caused a loss of thermal stability of ≈10 °C in DNA-DNA duplexes and ≈20 °C in PNA-DNA duplexes over a range of mildly acidic to neutral pH. Despite the decrease in thermal stability, the NMR structural studies showed that P-A formed the expected protonated base pair at pH 4.3. Our study demonstrates the feasibility of cationic unnatural base pairs; however, future optimization of such analogues will be required. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Reduction of fatal graft-versus-host disease by 3H--thymidine suicide of donor cells cultured with host cells

    International Nuclear Information System (INIS)

    Cheever, M.A.; Einstein, A.B. Jr.; Kempf, R.A.; Fefer, A.

    1977-01-01

    The effect of the tritiated thymidine ( 3 H-TdR) suicide technique on the ability of donor cells to induce fatal graft-versus-host disease (GVHD) was studied. C57BL/6 (H-2/sup b/) spleen cells were stimulated in vitro with irradiated BALB/c (H-2/sup d/) Moloney lymphoma cells in mixed culture and 3 H-TdR of high-specific activity added to eliminate proliferating cells. The ability of such cells to induce fatal GVHD was assayed by injecting them i.v. into adult BALB/c mice immunosuppressed with cyclophosphamide (180 mg/kg). These cells induced fatal GVHD in fewer mice (52 percent) than did C57BL/6 cells cultured with BALB/c lymphoma cells but without 3 H-TdR (87 percent) and C57BL/6 cells cultured with irradiated C57BL/6 cells with (95 percent) or without 3 H-TdR (86 percent). Thus, the 3 H-TdR suicide technique greatly diminished the ability of cells to induce lethal GVHD

  2. 3H-thymidine autoradiographic study of cell proliferation and the influence of isoproterenol and kallikrein in various cell populations of the gastrointestinal tract in animal experiments

    International Nuclear Information System (INIS)

    Albrecht, S.

    1981-01-01

    Morphological studies were carried out with the aim of studying cell proliferation in various tissues (stable and epithelial tissues) of the gastrointestinal tract. The cells were studied by 3 H thymidine autoradiography in the normal age cycle and under the influence of kallikrein or isoproterenol. Epithelial cells and smooth muscle cells of the forestomach, stomach, small intestine and colon were investigated and, in the case of the stomach, also connective-tissue cells of the mucotic stroma. Counts across the total epithelial thickness yielded similar results for the oesophagus and the forestomach. A count of 1000 cells per animal was found to yield representative information on a defined type of cell. Kallikrein was not found to have a constant mitosis-promoting effect. Isoproterenol caused the labelling index to increase or to decrease; in higher concentrations, it increased the proliferation rate in all cell types. In male animals, the labelling indices of connective-tissue cells of the gastric mucosa were significantly higher than in female animals. (orig./MG) [de

  3. Thymidine phosphorylase and hypoxia-inducible factor 1-α expression in clinical stage II/III rectal cancer: association with response to neoadjuvant chemoradiation therapy and prognosis.

    Science.gov (United States)

    Lin, Shuhan; Lai, Hao; Qin, Yuzhou; Chen, Jiansi; Lin, Yuan

    2015-01-01

    The aim of this study was to determine whether pretreatment status of thymidine phosphorylase (TP), and hypoxia-inducible factor alpha (HIF-1α) could predict pathologic response to neoadjuvant chemoradiation therapy with oxaliplatin and capecitabine (XELOXART) and outcomes for clinical stage II/III rectal cancer patients. A total of 180 patients diagnosed with clinical stage II/III rectal cancer received XELOXART. The status of TP, and HIF-1α were determined in pretreatment biopsies by immunohistochemistry (IHC). Tumor response was assessed in resected regimens using the tumor regression grade system and TNM staging system. 5-year disease free survival (DFS) and 5-year overall survival (OS) were evaluated with the Kaplan-Meier method and were compared by the log-rank test. Over expression of TP and low expression of HIF-1α were associated with pathologic response to XELOXART and better outcomes (DFS and OS) in clinical stage II/III rectal cancer patients (P rectal cancer received XELOXART. Additional well-designed, large sample, multicenter, prospective studies are needed to confirm the result of this study.

  4. 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.

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

    Directory of Open Access Journals (Sweden)

    Jianyong Xiao

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

  6. 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.

  7. Distribution of tritiated compounds (tritiated thymidine and tritiated water) in the mother-fetus system and its consequences for the radiotoxic effect of tritium

    Energy Technology Data Exchange (ETDEWEB)

    Schreml, W; Fliedner, T M [Ulm Univ. (Germany, F.R.). Abt. Klinische Physiologie

    1978-01-01

    The incorporation and distribution of tritiated thymidine (/sup 3/H-TdR) and tritiated water (HTO) have been measured in newborn rats exposed to various levels of tritium by continuous infusion into pregnant rats from day 9 until term. In the animals exposed to HTO, the tritium activity was homogeneously distributed while /sup 3/H-TdR led to accumulation of DNA-bound and homogeneously distributed tritium. The incorporated activity and the specific activity of DNA from ovaries which showed a reduction of total oocyte number by approximately 50% were used to estimate the dose absorbed by the ovarian cell nuclei in both systems. From the absorbed dose a factor of 3.7 was calculated for the 'internal relative biological effectiveness' of DNA-bound tritium as compared to homogeneously distributed /sup 3/H under the restrictive assumption that the static description of the system at birth reflects the situation during the time of dynamic development of the ovaries when the toxic effect occurs. The influence of these dynamic factors of changing nuclear size and tritium incorporation during the sensitive period is weighed against the possibility that the continuous /sup 3/H-TdR infusion during pregnancy might represent a model in which DNA-bound tritium shows a higher effectiveness than homogeneously distributed tritium.

  8. In vitro and in vivo evaluations of a radioiodinated thymidine phosphorylase inhibitor as a tumor diagnostic agent for angiogenic enzyme imaging

    International Nuclear Information System (INIS)

    Akizawa, Hiromichi; Zhao, Songji; Takahashi, Masayuki; Nishijima, Ken-ichi; Kuge, Yuji; Tamaki, Nagara; Seki, Koh-ichi; Ohkura, Kazue

    2010-01-01

    Introduction: The expression of thymidine phosphorylase (TP) is closely associated with angiogenesis, tumor invasiveness and activation of antitumor agents. We evaluated radioiodinated 5-iodo-6-[(2-iminoimidazolidinyl)methyl]uracil ([ 125 I]IIMU) having high TP-inhibitory potency as the new radiotracer for SPECT targeting of TP expression in tumors. Methods: The characteristics of the radioiodinated TP inhibitor IIMU were determined by evaluating the uptake by tumor cells in vitro and by biodistribution studies in vivo. The distribution of the radiotracer and the extent of TP-specific uptake by tumors were evaluated by a counting method in tumor-bearing mice. Results: The in vitro uptake of radiolabeled IIMU by A431 cells along with high TP expressions was attributed to the binding of the radiotracer to its target enzyme, i.e., TP. In vivo distribution of the radiotracer in A431 tumor-bearing mice revealed tumor/blood and tumor/muscle activity uptake ratios of 36 and 106, respectively, at 3 h after the radiotracer injection. On using low TP-expressing tumors and TP blocking studies as controls, minor TP-specific accumulation of the radiotracer was detected in these studies. Conclusion: According to the binding of radioiodinated IIMU to the angiogenic enzyme TP, it can be concluded that radioiodinated IIMU might be suitable as a SPECT tracer for tumor imaging.

  9. Enhancement in irradiated mononuclear cells in culture of mitogen-induced incorporation of [3H]thymidine by homologous conditioned medium

    International Nuclear Information System (INIS)

    Sandru, G.; Greiner, R.

    1994-01-01

    Incorporation of [ 3 H]thymidine in irradiated peripheral blood mononuclear cell cultures irradiated in vitro was stimulated significantly by either concanavalin A or phytohemagglutinin only in the presence of homologous conditioned medium. Production of this activity by mononuclear cells was enhanced by irradiation and/or pulsed exposure to puromycin but was abolished by actinomycin D. Addition of anti-interleukin 1 or anti-interleukin 2 monoclonal antibodies to the conditioned medium before assay did not influence the stimulatory action. A similar significant stimulation of mononuclear cell cultures irradiated with 6 Gy by concanavalin A was obtained when purified preparations of homologous conditioned medium were used in the assay. Purification was done by ultrafiltration and concentration, heparin agarose chromatography, ammonium sulfate precipitation, concanavalin A agarose chromatography, DEAE-ion exchange chromatography and HPLC gel filtration chromatography. With SDS-PAGE and silver staining, the active HPLC fraction gave one band of 50 kDa, suggesting that this protein is responsible for the co-stimulatory effect of homologous conditioned medium for both mitogen-induced irradiated and nonirradiated mononuclear cell cultures. 42 refs., 9 figs., 3 tabs

  10. Effect of D-valine and cytosine arabinoside on [3H]thymidine incorporation in rat and rabbit epididymal epithelial cell cultures

    International Nuclear Information System (INIS)

    Orgebin-Crist, M.C.; Jonas-Davies, J.; Storey, P.; Olson, G.E.

    1984-01-01

    Epithelial cell enriched primary cultures were established from the rat and the rabbit epididymis. Epithelial cell aggregates, obtained after pronase digestion of minced epididymis, attached to the culture dish and after 72 h in vitro spread out to form discrete patches of cells. These cells have an epithelioid morphology and form a monolayer of closely apposed polygonal cells where DNA synthesis, as judged by [ 3 H]thymidine uptake, is very low. In L-valine medium the nonepithelial cell contamination was no more than 10% in rat and rabbit epididymal primary cultures. The labeling index of rat epididymal cells cultured in D-valine medium was significantly lower than that of cells cultured in L-valine medium. In contrast, the labeling index of rabbit epididymal cells cultured in D-valine medium was significantly higher than that of cells cultured in L-valine medium. Cytosine arabinoside decreased the number of labeled cells in both L-valine and D-valine cultures. From these results, it appears that D-valine is a selective agent for rat epididymal epithelial cells, but not for rabbit epithelial cells, and that cytosine arabinoside is a simple and effective means to control the proliferation of fibroblast-like cells in both rat and rabbit epididymal cell cultures

  11. The Use of Tritium-Labelled Thymidine in Studies on the Synthesis of Deoxyribonucleic Acids; Emploi de la Thymidine Tritiee Dans L'etude de la Synthese de l'Acide Desoxyribonucleique; 0418 0441 043f 043e 0414 ; Empleo de Timidina Tritiada para Estudiar la Sintesis de los Acidos Desoxirtibonucleicos

    Energy Technology Data Exchange (ETDEWEB)

    Bianchi, P. A.; Crathorn, A. R.; Shooter, K. V. [Chester Beatty Research Institute, Institute of Cancer Research, London (United Kingdom)

    1962-02-15

    In the course of studies on the biosynthesis of DNA some experiments were performed using Ehrlich ascites cells, examining the uptake and incorporation of H{sup 3}-thymidine. After in-vitro incubation of the cells with this compound, autoradiographs of the cells were made and DNA was also isolated and assayed for H{sup 3} activity using a flow-counter. A comparison of the two methods of assay showed a marked discrepancy; the H{sup 3} activity per cell, calculated from the autoradiographs always appeared to be greater than the activity of the isolated DNA. Subsequent flow-counter assay of the activity of washed, homogenized whole cells gave figures which agreed with the autoradiographic assay. It thus appeared that, in this system, the use of autoradiography as a measure for DNA synthesis was open to criticism. Analysis of the bound, non-DNA activity has been made and similar studies of total cellular H{sup 3} activity and the activity of isolated DNA have been undertaken on other cell types; these have also shown similar effects. From this information it has been possible to divide the synthetic process into various stages: (1) The initial incorporation of thymidine into the cell; (2) Subsequent phosphorylation in at least two steps; (3) Polymerization of the phosphorylated thymidine into DNA. Thus, although the assumption that the incorporation of thymidine into the cell gives a measure of DNA synthesis is an oversimplification, it would seem that considerable information about the preliminary stages in the process can be obtained by use of this tracer. (author) [French] Au cours d'etudes sur la biosynthese de l'ADN, l'auteur a fait certaines experiences, a l'aide de cellules eosinophiles ascitiques, en vue de mesurer l'absorption et l'incorporation de la thymidine tritiee. Apres incubation in vitro des cellules avec ce compose, on a fait des autoradiographies; on a, d'autre part, isole l'ADN et determine son activite due au tritium a l'aide d'un compteur a

  12. Autoradiographic Study of Incorporation of Tritiated Thymidine in the Rat; Etude Autoradiographique de l'Incorporation de Thymidine Tritiee chez le Rat; 0420 0430 0434 0438 043e 0430 0432 0442 043e 0414 ; Estudio Autorradiografico de la Incorporacion de Timidina Tritiada en la Rata

    Energy Technology Data Exchange (ETDEWEB)

    Maldague, P.; Hong-Que, Pham; Maisin, J. [Laboratoire de Radiobiologie, Institut du Cancer, Louvain (Belgium)

    1962-02-15

    The authors used tritiated thymidine to evaluate desoxyribonucleic acid synthesis in various rat organs. They show by autoradiographs that this synthesis takes place mainly in tissue having intensive mitotic activity (bone marrow, seminiferous tubules of the testis, mucous membrane of the intestine, oesophagus and tongue). The authors also studied the regeneration of the convoluted renal tubules during the months following local irradiation of the kidney at various doses. (author) [French] Les auteurs ont utilise de la thymidine tritiee pour evaluer la synthese de l'acide desoxyribonucleique au niveau de differents organes chez le rat. Ils montrent par autoradiographies que cette synthese s'effectue principalement au niveau des tissus presentant une activite mitotique intense (moelle osseuse, tubes seminiferes du testicule, muqueuse intestinale, oesophagienne et de la lingue). Les auteurs ont en outre etudie la regeneration des tubes contournes du rein apres irradiation locale de cet organe, a differentes doses et dans les mois qui suivent cette irradiation. (author) [Spanish] Los autores han utilizado timidina tritiada para evaluar la sintesis del acido desoxirribonucleico en distintos organos de la rata. Demuestran mediante autorradiografias que esta sintesis se produce principalmente en los tejidos de intensa actividad mitotica (medula osea, tubos seminiferos de los testiculos, mucosas intestinal, del esofago y de la lengua). Finalmente, los autores han estudiado la regeneracion de los tubos y convolvulados del rinon en los meses que siguen a la irradiacion local de este organo con distintas dosis. (author) [Russian] Avtory pol'zovalis' mechennym tritiem timidinom dlja ocenki sin- teza dezoksiribonukleinovoj kisloty v razlichnyh organah krysy. Pri po- moshhi radioavtografii avtory pokazyvajut, chto jetot sintez proishodit glavnym obrazom v tkanjah s intensivnoj mitoticheskoj dejatel'nost'ju (kostnyj mozg, semenenosnye kanaly testikula, slizistaja obolochka

  13. 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...

  14. Characterization of the growth of murine fibroblasts that express human insulin receptors. I. The effect of insulin in the absence of other growth factors

    International Nuclear Information System (INIS)

    Randazzo, P.A.; Morey, V.A.; Polishook, A.K.; Jarett, L.

    1990-01-01

    The effect of insulin on the growth of murine fibroblasts transfected with an expression vector containing human insulin receptor cDNA (NIH 3T3/HIR) and the parental cells (NIH/3T3) was characterized. Insulin in the absence of other mitogens increased the rate of incorporation of thymidine into NIH 3T3/HIR cells with a half-maximal response occurring at an insulin concentration of 35 ng/ml and a maximal response that was equivalent to that elicited by 10% fetal calf serum. The thymidine incorporation rate was increased by 12 h, was maximal at approximately 16 h, and returned to basal rates at 24 h after the addition of insulin. Insulin induced a maximum of 65% of cells to incorporate thymidine. The increased DNA synthesis was accompanied by net growth. Addition of insulin to the NIH 3T3/HIR cells resulted in increased DNA content with a half-maximal response occurring at approximately 30 ng/ml insulin and a maximal response equivalent to that elicited by serum. An increase in cell number detected after the addition of insulin to the NIH 3T3/HIR suggests that the cells had progressed through mitosis. Insulin did not increase the rate of thymidine incorporation, DNA content, or number of the parental NIH 3T3 cells. These data show that insulin, in the absence of a second mitogen, is able to induce NIH 3T3/HIR fibroblasts to traverse the cell cycle

  15. Inhibitory effect of calcium channel blockers on proliferation of human glioma cells in vitro

    International Nuclear Information System (INIS)

    Kunert-Radek, J.; Stepien, H.; Lyson, K.; Pawlikowski, M.; Radek, A.

    1989-01-01

    The effects of 2 specific calcium channel blockers, verapamil and nimodipine, on the proliferation of human glioma tumour cells were investigated in vitro. Tumour tissues for primary cell cultures were obtained bioptically from 3 patients with the histopathological diagnosis of glioblastoma. The [ 3 H]-thymidine incorporation into glioma tumour cells DNA was used as a sensitive index of the cell proliferation. It was found that varapamil (10 4 -10 5 M) and nimodipine (10 4 -10 6 M) significantly inhibited the [ 3 H]-thymidine uptake in a dose-related manner. The inhibitory effect of both calcium channel antagonists was reversed by stimultancous addition of calcium chloride (5x10 3 M). These results indicate that verapamil and nimodipine may exert an antiproliferative effect on glioma cells growth acting through a blokade of specific voltage-dependent calcium channels. (author)

  16. Effect of chloroquine on human lymphocyte proliferation

    DEFF Research Database (Denmark)

    Bygbjerg, Ib Christian; Flachs, H

    1986-01-01

    The effect of chloroquine on human blood mononuclear cells was studied. High concentrations of chloroquine in vitro profoundly suppressed the proliferation of mitogen- and antigen-stimulated cells, as indicated by decreased 14C-thymidine incorporation. Lower concentrations of chloroquine increase...... to large particulate antigens; the response to small antigens was not affected. The mode of action of chloroquine and the possible consequences of the findings for dosage of chloroquine when used for malaria prophylaxis is discussed.......The effect of chloroquine on human blood mononuclear cells was studied. High concentrations of chloroquine in vitro profoundly suppressed the proliferation of mitogen- and antigen-stimulated cells, as indicated by decreased 14C-thymidine incorporation. Lower concentrations of chloroquine increased...... the response to pokeweed mitogen. The response to concanavalin A and to various antigens was suppressed, especially the response to large particulate antigens. Oral intake of 300 mg of chloroquine base/week did not affect the lymphocyte proliferative responses. 600 mg of base/week decreased the response...

  17. The 3H-thymidine incorporation into the DNA of different tissues of the guinea pip after BCG-vaccination and the radiation insultus

    International Nuclear Information System (INIS)

    Langbein, U.

    1976-01-01

    The radioprotective effects of BCG vaccines have been examined. The 3 H-thymidine incorporation into the DNA of different tissues of the guinea-pig after solitary whole-body irradiation by the doses of 160, 400, and 700 R have been used as a parameter for radiation injuries and radioprotection. The specific activity of DNA has been detected by means of liquid scintillation counting and by indirect photomeric determination of the amount at 7 h p.r. It has been revealed that independent of the chosen irradiation dose, there was no significant difference in the rate of DNA synthesis in the duodenal, testicular, bone marrow, liver, and lymphatic ganglion tissues of animals vaccinated 30 days before irradiation insultus and the rate of DNA synthesis in normal animals. Based on medical evidence, effect principles which can be observed on other antigenous radioprotective substances can be excluded this time. The dose effect curve has qualitatively the same features as the curves of cell cultures and synchronized cell systems in mammals. Furthermore, the process of DNA synthesis was observed for 56 days. During this observation period there was no significant difference to be seen in the rate of duodenal, testicular, bone marrow, and liver tissues in vaccinated and in normal animals. Only in lymphatic tissues the synthesis rate of vaccinated animals has shown a significantly more decreasing tendency than that of normal animals. A relation concerning radioprotective substances containing SH-groups and 'short-term' protectors (endotoxines) could be excluded because of medical evidence. It is suggested to carry out further tests with parameters affecting the RES in order to comprehend radioprotection after BCG vaccination. (orig./MG) [de

  18. Studies on the cytodifferentiation of the neuroblasts and visual cells in the chick embryo retina, using the electron-microscopic autoradiography of 3H-thymidine

    International Nuclear Information System (INIS)

    Mishima, H.; Fujita, H.

    1978-01-01

    Studies on the histogenetic analysis of cytodifferentiation of the neuroblast and visual cell in the chick embryo retina were made using the autoradiography of 3 H-thymidine. The posterior pole region of the eyeball was observed in all the animals used. The retina in a 4-day-old chick embryo consists exclusively of matrix cells forming the matrix layer. In a 5-day-old chick embryo retina, neuroblasts first differentiated from the matrix cells migrate into the outer part of the matrix layer, forming the mantle layer. The matrix cell is a homogeneous epithelial cell containing abundant free ribosomes and a poorly developed cytoplasmic membrane system in the cytoplasm. The characteristic sign of differentiation of the neuroblast is an appearance of elements of rough-surfaced endoplasmic reticulum and an indentation of the nucleus. The primitive visual cell having just lost its ability to synthesize DNA appears just beneath the pigment epithelium in a 7-day-old chick embryo, and all the cells lying beneath the pigment epithelium lose the ability to synthesize DNA at 10 days of incubation. The cytoplasmic process of the matrix cell is in contact with the adjacent one, making an apicolateral junction. When the matrix cell loses its ability to synthesize DNA, a big tentlike process extending over the level of the apicolateral junction appears. This phenomenon is considered to be a sign of differentiation from matrix cell to primitive visual cell, and this big tentlike process containing 2 centrioles is a primordium of the inner segment of the visual cell. (orig.) [de

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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.)

  4. 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.

  5. Cell turnover in the odontogenic organ of the rat incisor as visualized by graphic reconstructions following a single injection of 3H-thymidine.

    Science.gov (United States)

    Smith, C E

    1980-07-01

    Turnover of cells within the odontogenic organ was studied in three dimensions by preparing serial sections of incisors from young male rats killed at various times following a single intraperitoneal injection of 1 muCi/g body weight of 3H-thymidine. Radioautographs showed that at 1 hour after injection labeled cells were present in all cell layers throughout the entire depth of the odontogenic organ. They were encountered frequently within the inner dental epithelium and stratum intermedium but appeared less abundant within the stellate reticulum and outer dental epithelium. With time, the frequency of labeled cells in each layer declined progressively, and more rapidly at the anterior and labial side of the odontogenic organ than toward its posterior and lingual side. Hence labeled cells were observed over the longest time interval in regions where cell layers were in closest proximity to the opening of the apical foramen, that is, near the apical and cervical loops. By 32 days after injection, numerous labeled cells could still be identified within the outer dental epithelium and stellate reticulum near the apical loop (bulbous part of the odontogenic organ) and the outer dental epithelium near the cervical loops ("U"-shaped part of the odontogenic organ). These findings support the hypothesis that cells originate within the bulbous part of the odontogenic organ and migrate anteriorly through the "U"-shaped and root sheath parts of the odontogenic organ during renewal of the incisor. It appears that individual stem cell compartments may be maintained for surface (outer/inner dental epithelium) and intermediate layers (stellate reticulum/stratum intermedium) in the odontogenic epithelium.

  6. 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.

  7. Dynamics of neuroepithelial body (NEB) formation in developing hamster lung: Light microscopic autoradiography after 3H-thymidine labeling in vivo

    International Nuclear Information System (INIS)

    Hoyt, R.F. Jr.; McNelly, N.A.; Sorokin, S.P.

    1990-01-01

    Autoradiographs were prepared from lungs of a newborn Syrian golden hamster exposed continuously to 3H-thymidine for the final 4.5 days of a normal 16 day gestation. Silver grains were counted over nuclei of 1,298 small-granule endocrine cells in 165 neuroepithelial bodies (NEBs) in the right upper lobe and along the left axial bronchus, where nodal NEBs occurred at branch points and internodal NEBs in the airway between them. Nuclei of 1,005 nonendocrine airway epithelial cells were counted next to the NEBs. Label was distributed differently in the two populations: All nonendocrine cells were labeled, whereas many endocrine cells were not. In NEBs of the right upper lobe, total label (net grains/nuclear profile) averaged only 23% of that in nonendocrine cells. Along the left axial bronchus, mean label in nonendocrine cells and internodal NEBs rose 10-fold between the hilum and the periphery. Increases for both populations were linear and parallel, but total label in the NEBs was consistently lower than that in the surrounding epithelium by 15 grains/nuclear profile. Nodal NEBs were more lightly labeled than those of the internodes, consistent with their earlier formation. A few very heavily labeled small-granule cells (0.9%) occurred singly in the periphery of large, otherwise lightly labeled NEBs. In contrast to NEBs, neurons in 10 bronchial ganglia of the right lung were virtually unlabeled. These arise from vagal neural crest and seem to comprise an entirely distinct population. We conclude that NEBs belong intrinsically to pulmonary endoderm, not neural crest. During fetal life each develops from a cell or cells programmed to stop dividing well ahead of other elements in the epithelium. Their formation is linked closely to early proliferation of the bronchial tree and is an integral part of growth and differentiation of the airway lining

  8. The influence of elevated levels of platelet-derived endothelial cell growth factor/thymidine phosphorylase on tumourigenicity, tumour growth, and oxygenation

    International Nuclear Information System (INIS)

    Griffiths, L.; Stratford, I.J.

    1998-01-01

    Purpose: Investigation of the effect of platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) on various aspects of tumour growth in a xenograft model, including growth rate, tumourigenicity and oxygenation levels. Methods and Materials: MDA 231 breast cancer cells overexpressing PD-ECGF/TP protein were made by retroviral transduction. These cells were grown in vitro and in vivo as xenografts. Direct measurement of tumours was used to record growth parameters, while the comet assay with the bioreductive drug RSU 1069 was used to assess tumour cell oxygenation. Results: We report that MDA 231 breast tumour cell lines expressing an increased range of levels of PD-ECGF/TP have increased tumourigenicity positively related to the level of PD-ECGF/TP when implanted in nude mice. As previously reported, tumours grown from these overexpressing cell lines grew faster than the parental line. These tumours expressed higher levels of TP activity and showed increased immunocytochemical staining for PD-ECGF. In addition, the rate of growth was found to be positively related to the level of PD-ECGF/TP expressed by the tumour cells. When the comet assay was used to compare the oxygenation status of cells between the parental and PD-ECGF/TP overexpressing tumours, the latter were found to have a larger proportion of well oxygenated cells. This is consistent with these tumours having an increased and functionally competent vascular supply in response to the expression of PD-ECGF/TP. Conclusion: PD-ECGF/TP appears to be capable of influencing tumourigenicity, angiogenesis and tumour growth in a proportional manner and can directly influence tumour oxygenation levels via its role in formation of functional vasculature

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

    Directory of Open Access Journals (Sweden)

    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.

  10. Effect of varying the exposure and 3H-thymidine labeling period upon the outcome of the primary hepatocyte DNA repair assay

    International Nuclear Information System (INIS)

    Barfknecht, T.R.; Mecca, D.J.; Naismith, R.W.

    1988-01-01

    The results presented in this report demonstrate that an 18-20 hour exposure/ 3 H-thymidine DNA labeling period is superior to a 4 hour incubation interval for general genotoxicity screening studies in the rat primary hepatocyte DNA repair assay. When DNA damaging agents which give rise to bulky-type DNA base adducts such as 2-acetylaminofluorene, aflatoxin B1 and benzidine were evaluated, little or no difference was observed between the 4 hour or an 18-20-hour exposure/labeling period. Similar results were also noted for the DNA ethylating agent diethylnitrosamine. However, when DNA damaging chemicals which produce a broader spectrum of DNA lesions were studied, differences in the amount of DNA repair as determined by autoradiographic analysis did occur. Methyl methanesulfonate and dimethylnitrosamine induced repairable DNA damage that was detected at lower dose levels with the 18-20 hour exposure/labeling period. Similar results were also observed for the DNA cross-linking agents, mitomycin C and nitrogen mustard. Ethyl methanesulfonate produced only a marginal amount of DNA repair in primary hepatocytes up to a dose level of 10(-3) M during the 4 hour incubation period, whereas a substantial amount of DNA repair was detectable at a dose level of 2.5 X 10(-4) M when the 18-20 hour exposure/labeling period was employed. The DNA alkylating agent 4-nitroquinoline-1-oxide, which creates DNA base adducts that are slowly removed from mammalian cell DNA, induced no detectable DNA repair in hepatocytes up to a toxic dose level of 2 X 10(-5) M with the 4 hour exposure period, whereas a marked DNA repair response was observed at 10(-5) M when the 18-20 hour exposure/labeling period was used

  11. Radiation-Induced Thymidine Phosphorylase Upregulation in Rectal Cancer Is Mediated by Tumor-Associated Macrophages by Monocyte Chemoattractant Protein-1 From Cancer Cells

    International Nuclear Information System (INIS)

    Kim, Tae-Dong; Li Ge; Song, Kyoung-Sub; Kim, Jin-Man; Kim, Jun-Sang; Kim, Jong-Seok; Yun, Eun-Jin; Park, Jong-Il; Park, Hae-Duck; Hwang, Byung-Doo; Lim, Kyu; Yoon, Wan-Hee

    2009-01-01

    Purpose: The mechanisms of thymidine phosphorylase (TP) regulation induced by radiation therapy (XRT) in various tumors are poorly understood. We investigated the effect and mechanisms of preoperative XRT on TP expression in rectal cancer tissues. Methods and Materials: TP expression and CD68 and monocyte chemoattractant protein-1 (MCP-1) levels in rectal cancer tissues and cancer cell lines were evaluated before and after XRT in Western blotting, immunohistochemistry, enzyme-linked immunoassay, and reverse transcription-polymerase chain reaction studies. Isolated peripheral blood monocytes were used in the study of chemotaxis under the influence of MCP-1 released by irradiated colon cancer cells. Results: Expression of TP was significantly elevated by 9 Gy of XRT in most rectal cancer tissues but not by higher doses of XRT. In keeping with the close correlation of the increase in both TP expression and the number of tumor-associated macrophages (TAMs), anti-TP immunoreactivity was found in the CD68-positive TAMs and not the neoplastic cells. Expression of MCP-1 was increased in most cases after XRT, and this increase was strongly correlated with TP expression. However, this increase in MCP-1 expression occurred in tumor cells and not stromal cells. The XRT upregulated MCP-1 mRNA and also triggered the release of MCP-1 protein from cultured colon cancer cells. The supernatant of irradiated colon cancer cells showed strong chemotactic activity for monocyte migration, but this activity was completely abolished by neutralizing antibody. Conclusions: Use of XRT induces MCP-1 expression in cancer cells, which causes circulating monocytes to be recruited into TAMs, which then upregulate TP expression in rectal cancer tissues

  12. Generation patterns of four groups of cholinergic neurons in rat cervical spinal cord: a combined tritiated thymidine autoradiographic and choline acetyltransferase immunocytochemical study

    International Nuclear Information System (INIS)

    Phelps, P.E.; Barber, R.P.; Vaughn, J.E.

    1988-01-01

    This report examines the generation of cholinergic neurons in the spinal cord in order to determine whether the transmitter phenotype of neurons is associated with specific patterns of neurogenesis. Previous immunocytochemical studies identified four groups of choline acetyltransferase (ChAT)-positive neurons in the cervical enlargement of the rat spinal cord. These cell groups vary in both somatic size and location along the previously described ventrodorsal neurogenic gradient of the spinal cord. Thus, large (and small) motoneurons are located in the ventral horn, medium-sized partition cells are found in the intermediate gray matter, small central canal cluster cells are situated within lamina X, and small dorsal horn neurons are scattered predominantly through laminae III-V. The relationships among the birthdays of these four subsets of cholinergic neurons have been examined by combining 3H-thymidine autoradiography and ChAT immunocytochemistry. Embryonic day 11 was the earliest time that neurons were generated within the cervical enlargement. Large and small ChAT-positive motoneurons were produced on E11 and 12, with 70% of both groups being born on E11. ChAT-positive partition cells were produced between E11 and 13, with their peak generation occurring on E12. Approximately 70% of the cholinergic central canal cluster and dorsal horn cells were born on E13, and the remainder of each of these groups was generated on E14. Other investigators have shown that all neurons within the rat cervical spinal cord are produced in a ventrodorsal sequence between E11 and E16. In contrast, ChAT-positive neurons are born only from E11 to E14 and are among the earliest cells generated in the ventral, intermediate, and dorsal subdivisions of the spinal cord

  13. 5-fluorouracil Toxicity Mechanism Determination in Human Keratinocytes: in vitro Study on HaCaT

    Directory of Open Access Journals (Sweden)

    Jan Hartinger

    2018-01-01

    Full Text Available 5-fluorouracil (5-FU and capecitabine therapy is often accompanied by palmar-plantar erythrodysesthesia (PPE which is manifestation of 5-FU toxicity in keratinocytes. The main mechanisms of 5-FU action are thymidylate synthase (TS inhibition which can be abrogated by thymidine and strengthened by calciumfolinate (CF and incorporation of fluorouridinetriphosphate into RNA which can be abrogated by uridine. For proper PPE treatment 5-FU mechanism of action in keratinocytes needs to be elucidated. We used the 5-FU toxicity modulators uridine, thymidine and CF to discover the mechanism of 5-FU action in human keratinocyte cell line HaCaT. To measure the cellular viability, we used MTT test and RTCA test. CF did not augment 5-FU toxicity and 5-FU toxicity was weakened by uridine. Therefore, the primary mechanism of 5-FU toxicity in keratinocytes is 5-FU incorporation into RNA. The uridine protective effect cannot fully develop in the presence of CF. Thymidine addition to 5-FU and uridine treated cells not only prevents the toxicity-augmenting CF effect but it also prolongs the 5-FU treated cells survival in comparison to uridine only. Therefore, it can be assumed that in the presence of uridine the 5-FU toxicity mechanism is switched from RNA incorporation to TS inhibition. Although particular 5-FU toxicity mechanisms were previously described in various cell types, this is the first time when various combinations of pyrimidine nucleosides and CF were used for 5-FU toxicity mechanism elucidation in human keratinocytes. We suggest that for PPE treatment ointment containing uridine and thymidine should be further clinically tested.

  14. The Use of Direct Tritium Assay Techniques in Studies with Tritiated Thymidine; Application des Methodes de Dosage Direct du Tritium aux Etudes avec la Thymidine Tritiee; 041f 0440 0438 043c 0435 043d 0435 0414 ; Aplicacion de Tecnicas de Medicion Directa del Tritio a Estudios con Timidina Tritiada

    Energy Technology Data Exchange (ETDEWEB)

    Gordon Steel, G. [Physics Department, Institute of Cancer Research, Royal Cancer Hospital, Clifton Avenue, Belmont, Surrey (United Kingdom)

    1962-02-15

    Results are described of investigations into the catabolism of tritiated thymidine in the rat, during the period of its initial localization in tissues, and into the retention of tritium-labelled cells in intestine and bone marrow up to 16 d after labelling. During the first hour after the injection of tritiated thymidine, whilst the concentration of nonvolatile tritium in most tissues rose to a saturation level, the concentration in liver fell rapidly from an initially high level. Using a device which enabled a sample of tissue water to be extracted from a cold tissue specimen, measurements were made of the specific activity of tritiated water from various organs. By one hour after injection, the tritium concentration was almost constant throughout the body water, but during the period of establishment of this equilibrium, considerable gradients of tissue water specific activity could be detected. In liver, the gradient indicated a flow of tritiated water from the liver into the blood; in spleen, testis and muscle the flow was from the blood into the tissue. Measurements of tritium retention in intestine and bone marrow after giving tritiated thymidine, indicated that in both tissues there was an initial plateau and that the subsequent decay had two prominent exponentials. In bone marrow the observation of a plateau conflicts with other published work; the plateau is not observed in animals which have received protracted irradiation. (author) [French] L'auteur decrit les resultats de recherches sur le catabolisme de la thymidine tritiee dans le rat, au cours de la periode de fixation initiale dans les tissus, et sur la retention de cellules marquees au tritium par l'intestin et la moelle osseuse dans un delai allant jusqu'a 16 jours apres le marquage. Pendant la premiere heure suivant l'injection de thymidine tritiee, alors que la concentration du tritium non volatil dans la plupart des tissus atteignait rapidement un degre de saturation, la concentration dans le

  15. Mitogenic Activity of a Water-Soluble Adjuvant (Bu-WSA) Obtained from Bacterionema matruchotii: IV. Synergistic Effects of Bu-WSA on Concanavalin A-Induced Proliferative Response of Human Peripheral Blood Lymphocytes.

    Science.gov (United States)

    Nitta, Toshimasa; Okumura, Seiichi; Tsushi, Masao; Nakano, Masayasu

    1982-07-01

    Butanol-extracted water-soluble adjuvant (Bu-WSA) obtained from Bacterionema matruchotii was cultured with peripheral blood mononuclear cells (PBM) in the presence of sub- and/or supra-optimal mitogenic concentrations of concanavalin A (Con A). The addition of Bu-WSA resulted in increased tritiated thymidine incorporation above that produced by Con A alone. Bu-WSA by itself is not mitogenic for PBM and in fact produced a decrease in thymidine uptake compared to the control. We investigated the response of subpopulation(s) of PBM to Bu-WSA, Con A and a mixture of Bu-WSA and Con A. Separation of PBM into purified T cells, B cells and macrophages showed that cell-cell cooperation of T cells with B cells or macrophages is necessary for the observed synergistic effect of Bu-WSA with Con A. A marked increase in thymidine incorporation by the mixture of T and B cell populations occurred, while only a small amount of thymidine was incorporated when the B cell population was absent. Mitomycin treatment revealed that the response could be ascribed to the T-cell response with a B-cell helper effect. Moreover, Con A and Bu-WSA appeared to act on the same T cell population. This model may provide unique information about the activation of human peripheral blood T cells compared with the activation of these cells by other mitogens. © owned by Center for Academic Publications Japan (Publisher).

  16. Mitogenic activity of a water-soluble adjuvant (Bu-WSA) obtained from Bacterionema matruchotii. IV. Synergistic effects of Bu-WSA on Concanavalin A-induced proliferative response of human peripheral blood lymphocytes.

    Science.gov (United States)

    Nitta, T; Okumura, S; Tsushi, M; Nakano, M

    1982-01-01

    Butanol-extracted water-soluble adjuvant (Bu-WSA) obtained from Bacterionema matruchotii was cultured with peripheral blood mononuclear cells (PBM) in the presence of sub- and/or supra-optimal mitogenic concentrations of concanavalin A (Con A). The addition of Bu-WSA resulted in increased tritiated thymidine incorporation above that produced by Con A alone. Bu-WSA by itself is not mitogenic for PBM and in fact produced a decrease in thymidine uptake compared to the control. We investigated the response of subpopulation(s) of PBM to Bu-WSA, Con A and a mixture of Bu-WSA and Con A. Separation of PBM into purified T cells, B cells and macrophages showed that cell-cell cooperation of T cells with B cells or macrophages is necessary for the observed synergistic effect of Bu-WSA with Con A. A marked increase in thymidine incorporation by the mixture of T and B cell populations occurred, while only a small amount of thymidine was incorporated when the B cell population was absent. Mitomycin treatment revealed that the response could be ascribed to the T-cell response with a B-cell helper effect. Moreover, Con A and Bu-WSA appeared to act on the same T cell population. This model may provide unique information about the activation of human peripheral blood T cells compared with the activation of these cells by other mitogens.

  17. 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.

  18. MCF-7 human mammary adenocarcinoma cells exhibit augmented responses to human insulin on a collagen IV surface

    DEFF Research Database (Denmark)

    Listov-Saabye, Nicolai; Jensen, Marianne Blirup; Kiehr, Benedicte

    2009-01-01

    Human mammary cell lines are extensively used for preclinical safety assessment of insulin analogs. However, it is essentially unknown how mitogenic responses can be optimized in mammary cell-based systems. We developed an insulin mitogenicity assay in MCF-7 human mammary adenocarcinoma cells......, under low serum (0.1% FCS) and phenol red-free conditions, with 3H thymidine incorporation as endpoint. Based on EC50 values determined from 10-fold dilution series, beta-estradiol was the most potent mitogen, followed by human IGF-1, human AspB10 insulin and native human insulin. AspB10 insulin...... was significantly more mitogenic than native insulin, validating the ability of the assay to identify hypermitogenic human insulin analogs. With MCF-7 cells on a collagen IV surface, the ranking of mitogens was maintained, but fold mitogenic responses and dynamic range and steepness of dose-response curves were...

  19. Effect of oral proguanil on human lymphocyte proliferation

    DEFF Research Database (Denmark)

    Bygbjerg, Ib Christian; Flachs, H

    1986-01-01

    In vitro studies have indicated that the antifolates pyrimethamine [4, 6] and cycloguanil (the active metabolite of proguanil) suppress the proliferation of stimulated human lymphocytes; proguanil has no effect [2]. During the early growth phase of the cells, 14C-thymidine (14C-TdR) incorporation...... is increased by pyrimethamine and cycloguanil, reflecting blockage of endogenous TdR synthesis [3]. Proguanil (Paludrine) is increasingly being used for malaria prophylaxis. It is considered the most innocuous of the antimalarials currently employed. Since nothing is known about the effect of oral proguanil...

  20. 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)

  1. Auger electron emitter against multiple myeloma - targeted endo-radio-therapy with 125I-labeled thymidine analogue 5-iodo-4'-thio-2'-deoxyuridine

    International Nuclear Information System (INIS)

    Morgenroth, Agnieszka; Dinger, Cornelia; Zlatopolskiy, Boris D.; Al-Momani, Ehab; Glatting, Gerhard; Mottaghy, Felix M.; Reske, Sven N.

    2011-01-01

    Introduction: Multiple myeloma (MM) is a plasma cell malignancy characterized by accumulation of malignant, terminally differentiated B cells in the bone marrow. Despite advances in therapy, MM remains an incurable disease. Novel therapeutic approaches are, therefore, urgently needed. Auger electron-emitting radiopharmaceuticals are attractive for targeted nano-irradiation therapy, given that DNA of malignant cells is selectively addressed. Here we evaluated the antimyeloma potential of the Auger electron-emitting thymidine analogue 125 I-labeled 5-iodo-4'-thio-2'-deoxyuridine ([ 125 I]ITdU). Methods: Cellular uptake and DNA incorporation of [ 125 I]ITdU were determined in fluorodeoxyuridine-pretreated KMS12BM, U266, dexamethasone-sensitive MM1.S and -resistant MM1.R cell lines. The effect of stimulation with interleukin 6 (IL6) or insulin-like growth factor 1 (IGF1) on the intracellular incorporation of [ 125 I]ITdU was investigated in cytokine-sensitive MM1.S and MM1.R cell lines. Apoptotic cells were identified using Annexin V. Cleavage of caspase 3 and PARP was visualized by Western blot. DNA fragmentation was investigated using laddering assay. Therapeutic efficiency of [ 125 I]ITdU was proven by clonogenic assay. Results: [ 125 I]ITdU was shown to be efficiently incorporated into DNA of malignant cells, providing a promising mechanism for delivering highly toxic Auger radiation emitters into tumor DNA. [ 125 I]ITdU had a potent antimyeloma effect in cell lines representing distinct disease stages and, importantly, in cell lines sensitive or resistant to the conventional therapeutic agent, but was not toxic for normal plasma and bone marrow stromal cells. Furthermore, [ 125 I]ITdU abrogated the protective actions of IL6 and IGF1 on MM cells. [ 125 I]ITdU induced massive damage in the DNA of malignant plasma cells, which resulted in efficient inhibition of clonogenic growth. Conclusion: These studies may provide a novel treatment strategy for overcoming

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. Increased levels of unscheduled DNA synthesis in UV-irradiated human fibroblasts pretreated with sodium butyrate

    International Nuclear Information System (INIS)

    Williams, J.I.; Friedberg, E.C.

    1982-01-01

    Pretreatment of growing normal and xeroderma pigmentosum (XP) human fibroblasts with sodium butyrate at concentrations of 5-20 mM results in increased levels of DNA repair synthesis measured by autoradiography after exposure of the cells to 254 nm UV radiation in the fluence range 0-25 J/m 2 . The phenomenon manifests as an increased extent and an increased initial rate of unscheduled DNA synthesis (UDS). This experimental result is not due to an artifact of autoradiography related to cell size. Xeroderma pigmentosum cells from complementation groups A, C, D and E and XP variant cells all exhibit increases in the levels of UV-induced UDS in response to sodium butyrate proportional to those observed with normal cells. These UDS increases associated with butyrate pretreatment correlate with demonstrable changes in intracellular thymidine pool size and suggest that sodium butyrate enhances uptake of exogenous radiolabeled thymidine during UV-induced repair synthesis by reducing endogenous levels of thymidine. (author)

  7. Vasoactive intestinal polypeptide and peptide histidine methionine. Presence in human follicular fluid and effects on DNA synthesis and steroid secretion in cultured human granulosa/lutein cells

    DEFF Research Database (Denmark)

    Gräs, S; Ovesen, P; Andersen, A N

    1994-01-01

    Vasoactive intestinal polypeptide (VIP) and peptide histidine methionine (PHM) originate from the same precursor molecule, prepro VIP. In the present study we examined the concentrations of VIP and PHM in human follicular fluid and their effects on cultured human granulosa/lutein cells. Follicular....../l, respectively. VIP at a concentration of 10 nmol/l caused a significant increase in [3H]thymidine incorporation, and at 1000 nmol/l a significant increase in oestradiol secretion was observed. VIP had no effect on progesterone secretion. PHM at the concentrations tested did not influence any of the activities...

  8. Effects of tritium-labeled pyrimidine nucleosides on epithelial cell proliferation in the mouse. I. Cytodynamic perturbations in normal circadian rhythms after a single injection of 3H-thymidine

    International Nuclear Information System (INIS)

    Olsson, L.

    1976-01-01

    In a combined stathmokinetic, autoradiographic, and microspectrophotometric study on the cytodynamics in mouse epidermis, cirdardian rhythms were observed in the number of cells within the various phases (except G 2 ) of the mitotic cell cycle, and in mitotic rate. S-Phase influx and efflux were computed and were found to vary during a 24-hr period. No diurnal rhythm was observed in mitotic rate in the epithelium of small intestine. 3 H-Thymidine (1 μCi/g; sp act 5 Ci/mmol) induced mitotic delay in labeled cells 2 to 8 hr after administration. A supranormal mitotic activity in unlabeled cells was observed 12 to 24 hr after injection of 3 H-TdR. ''Cold'' thymidine had no effects on epidermal cell proliferation. S-Phase duration and average cell cycle time were estimated by traditional cell kinetic methods. The results suggest a 3 H-TdR-induced synchrony in cells entering the S phase 0 to 15 hr after injection of the precursor. A mitotic delay was seen in small intestinal epithelium 2 to 6 hr after injection of the precursor

  9. Effect of periodontal dressings on human gingiva fibroblasts in vitro

    International Nuclear Information System (INIS)

    Eber, R.M.; Shuler, C.F.; Buchanan, W.; Beck, F.M.; Horton, J.E.

    1989-01-01

    In vitro cytotoxicity studies of periodontal dressings have not generally produced a result consistent with in vivo observations. These prior in vitro studies have not used human intraoral cell lines. We tested the effects of two eugenol containing and two non-eugenol periodontal dressings on cultured human gingival fibroblasts (HGF) (ATCC No. 1292). Replicate HGF cultures grown in microtiter plates were exposed to stock, 1:4 and 1:16 dilutions of extracts made from each of the four periodontal dressings. The HGF cultures were pulse labelled with tritiated thymidine (3HTdR) after 24, 48, and 72 hours. Incorporations of the labelled thymidine were measured using liquid scintillation counting and expressed as counts per minute. The results showed that undiluted extracts from all four periodontal dressings totally inhibited 3HTdR uptake (P less than 0.05). The 1:4 dilution of eugenol dressings inhibited 3HTdR uptake significantly more than non-eugenol dressings (P less than 0.05). Interestingly, at 72 hours the 1:16 dilution of the non-eugenol dressings caused significantly increased 3HTdR uptake which was not observed with the eugenol dressings. The present results suggest that the use of a human fibroblastic cell line for testing the effects of periodontal dressings may provide information about the relative biological effects of these dressings. Using this cell line, we have found that eugenol dressings inhibit fibroblast proliferation to a greater extent than non-eugenol dressings

  10. Effects of mercury on the proliferation of human peripheral lymphocytes in vitro

    International Nuclear Information System (INIS)

    Piwecka, K.; Poniedzialek, B.; Rzymski, P.; Karczewski, J.; Zurawski, J.; Wiktorowicz, K.

    2011-01-01

    Our project aimed to investigate the effects of mercury on the proliferation of human peripheral lymphocytes in vitro. The lymphocytes were isolated from the blood collected from healthy donors at Regionalne Centrum Krwiodawstwa i Krwiolecznictwa in Poznan, Poland. For the purpose of cell culture, the lymphocyte suspension (25 · 10 4 cells/ml) in Eagle's medium supplemented with 10% fetal calf serum was prepared. Phytohaemagglutinin-L (PHA-L) was used in a concentration of 2.5 mg/ml to stimulate cell proliferation. Mercuric chloride (HgCl 2 ) in four different concentrations (1 μM, 10 μM, 50 μM, 100 μM) and [3H]-thymidine were added after 48 hours of incubation and the cell culture was continued for the next 24 hours. The rate of lymphocyte proliferation was measured by [3H]-thymidine incorporation method with a liquid scintillation counter. Results indicate that higher concentrations of mercury (50 μM, 100 μM) inhibit the [3H]-thymidine incorporation of human peripheral lymphocytes in vitro. The incorporation was lower than the control sample by 65% at a concentration of 50 μM, while at a concentration of 100 μM it fell to virtually zero. Moreover, the phase of lymphocyte proliferation cycle affected by mercuric chloride was also investigated. For this purpose HgCl 2 in 2 concentrations (10 μM, 50 μM) was added to the cell culture in 4 different time points: at the start of the cell culture and after 4, 24, and 48 hours of incubation. After 48 hours, [3H]-thymidine was added and the cell culture was continued for an additional 24 hours. The rate of cell proliferation was estimated by [3H]-thymidine incorporation using a liquid scintillation counter. The inhibition effect was observed in samples with metal added at the start of the cell culture and after 4 h of incubation, i.e. at the initial phase of the lymphocyte proliferation cycle. (authors)

  11. 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

  12. Monoclonal antibody to the type I insulin-like growth factor (IGF-I) receptor blocks IGF-I receptor-mediated DNA synthesis: clarification of the mitogenic mechanisms of IGF-I and insulin in human skin fibroblasts

    International Nuclear Information System (INIS)

    Flier, J.S.; Usher, P.; Moses, A.C.

    1986-01-01

    Insulin and insulin-like growth factor type I (IGF-I) stimulate an overlapping spectrum of biological responses in human skin fibroblasts. Although insulin and IGF-I are known to stimulate the incorporation of [ 3 H]thymidine into DNA in these cells, the identify of the receptor(s) that mediates this effect has not been fully clarified. The mouse anti-human IGF-I receptor antibody αIR-3 binds with specificity to IGF-I but not to insulin receptors in human placental membranes; it also specifically inhibits the binding of 125 I-labeled IGF-I but not 125 I-labeled insulin to suspensions of human skin fibroblasts in a dose-dependent manner. αIR-3 competitively inhibits IGF-I-mediated stimulation of [ 3 H]thymidine incorporation into DNA. This inhibition is dependent on the concentration of αIR-3 and in the presence of a fixed antibody concentration can be partially overcome by high concentrations of IGF-I. In contrast, at concentrations of 3 H]thymidine incorporation is not inhibited by αIR-3. However, the incremental effects of higher concentrations (> 1 μg/ml) of insulin on [ 3 H]thymidine incorporation are inhibited by αIR-3. αIR-3 is a highly specific antagonist of IGF-I receptor-mediated mitogenesis in human skin fibroblasts. By using this antibody, it is shown directly that insulin can act through the IGF-I receptor to stimulate DNA synthesis but can also activate this effect through the insulin receptor itself

  13. Effects of anticonvulsant drugs on the synthesis of DNA and protein by human bone marrow cells in vitro

    International Nuclear Information System (INIS)

    Wickramasinghe, S.N.; Saunders, J.; Williams, G.

    1976-01-01

    Suspensions of human bone marrow cells were incubated with various concentrations of phenobarbitone or phenytoin sodium for 2 h, and the effects of this incubation on the subsequent incorporation of 3 H-thymidine and 3 H-leucine into DNA and protein, respectively, were studied. Both drugs caused a depression of 3 H-thymidine incorporation and this phenomenon was not prevented by the addition of 100 μg of pteroylglutamic acid, folinic acid or 5-methyltetrahydrofolate per ml of marrow culture. The lowest concentration of drug which caused a statistically significant depression of 3 H-thymidine incorporation was 200μg per ml for phenobarbitone and 50 μg per ml for phenytoin sodium. Both phenobarbitone and phenytoin sodium also caused an increase in the incorporation of 3 H-leucine at concentrations of 50 and 20 μg per ml., respectively, suggesting the possibility that a stimulation of protein synthesis within erythropoietic cells may play an important role in the development of anticonvulsant-induced macrocytosis. (authod)

  14. Human cytomegalovirus replicates in gamma-irradiated fibroblasts

    International Nuclear Information System (INIS)

    Shanley, J.D.

    1986-01-01

    Because of the unique interdependence of human cytomegalovirus (HCMV) and the physiological state of the host cell, we evaluated the ability of human foreskin fibroblasts (HFF), exposed to gamma radiation, to support HCMV growth. Irradiation of HFF with 2,500 rADS prevented cellular proliferation and suppressed cellular DNA, but not RNA or protein synthesis. Treatment of HFF cells with 2,500 rADS 6 or 48 hours prior to infection did not alter the time course or virus yield during HCMV replication. Virus plaquing efficiency in irradiated cells was comparable to that of nonirradiated cells. As judged by thymidine incorporation and BUdR inhibition of virus replication, HCMV infection induced both thymidine kinase activity and host cell DNA synthesis in irradiated cells. In addition, virus could be recovered from HFF exposed to radiation 0-2 days after infection with HCMV. These studies indicate that the damage to cells by gamma irradiation does not alter the capacity of host cells to support HCMV replication

  15. Primary structure of human alpha 2-macroglobulin. V. The complete structure

    DEFF Research Database (Denmark)

    Sottrup-Jensen, Lars; Stepanik, Terrence M; Kristensen, Torsten

    1984-01-01

    The primary structure of the tetrameric plasma glycoprotein human alpha 2-macroglobulin has been determined. The identical subunits contain 1451 amino acid residues. Glucosamine-based oligosaccharide groups are attached to asparagine residues 32, 47, 224, 373, 387, 846, 968, and 1401. Eleven......-SH group of Cys-949 is thiol esterified to the gamma-carbonyl group of Glx-952, thus forming an activatable reactive site which can mediate covalent binding of nucleophiles. A putative transglutaminase cross-linking site is constituted by Gln-670 and Gln-671. The primary sites of proteolytic cleavage......-macroglobulin subunit is discussed. A comparison of stretches of sequences from alpha 2-macroglobulin with partial sequence data for complement components C3 and C4 indicates that these proteins are evolutionary related. The properties of alpha 2-macroglobulin are discussed within the context of proteolytically...

  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. Classification and risk assessment of individuals with familial polyposis, Gardner's syndrome, and familial non-polyposis colon cancer from [3H]thymidine labeling patterns in colonic epithelial cells

    International Nuclear Information System (INIS)

    Lipkin, M.; Blattner, W.A.; Gardner, E.J.; Burt, R.W.; Lynch, H.; Deschner, E.; Winawer, S.; Fraumeni, J.F. Jr.

    1984-01-01

    A probabilistic analysis has been developed to assist the binary classification and risk assessment of members of familial colon cancer kindreds. The analysis is based on the microautoradiographic observation of [ 3 H]thymidine-labeled epithelial cells in colonic mucosa of the kindred members. From biopsies of colonic mucosa which are labeled with [ 3 H]thymidine in vitro, the degree of similarity of each subject's cell-labeling pattern measured over entire crypts was automatically compared to the labeling patterns of high-risk and low-risk reference populations. Each individual was then presumptively classified and assigned to one of the reference populations, and a degree of risk for the classification was provided. In carrying out the analysis, a linear score was calculated for each individual relative to each of the reference populations, and the classification was based on the polarity of the score difference; the degree of risk was then quantitated from the magnitude of the score difference. When the method was applied to kindreds having either familial polyposis or familial non-polyposis colon cancer, it effectively segregated individuals affected with disease from others at low risk, with sensitivity and specificity ranging from 71 to 92%. Further application of the method to asymptomatic family members believed to be at 50% risk on the basis of pedigree evaluation revealed a biomodal distribution to nearly zero or full risk. The accuracy and simplicity of this approach and its capability of revealing early stages of abnormal colonic epithelial cell development indicate potential for preclinical screening of subjects at risk in cancer-prone kindreds and for assisting the analysis of modes of inheritance

  18. Human reporter genes: potential use in clinical studies

    Energy Technology Data Exchange (ETDEWEB)

    Serganova, Inna [Department of Neurology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021 (United States); Ponomarev, Vladimir [Department of Radiology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021 (United States); Blasberg, Ronald [Department of Neurology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021 (United States); Department of Radiology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021 (United States)], E-mail: blasberg@neuro1.mskcc.org

    2007-10-15

    The clinical application of positron-emission-tomography-based reporter gene imaging will expand over the next several years. The translation of reporter gene imaging technology into clinical applications is the focus of this review, with emphasis on the development and use of human reporter genes. Human reporter genes will play an increasingly more important role in this development, and it is likely that one or more reporter systems (human gene and complimentary radiopharmaceutical) will take leading roles. Three classes of human reporter genes are discussed and compared: receptors, transporters and enzymes. Examples of highly expressed cell membrane receptors include specific membrane somatostatin receptors (hSSTrs). The transporter group includes the sodium iodide symporter (hNIS) and the norepinephrine transporter (hNET). The endogenous enzyme classification includes human mitochondrial thymidine kinase 2 (hTK2). In addition, we also discuss the nonhuman dopamine 2 receptor and two viral reporter genes, the wild-type herpes simplex virus 1 thymidine kinase (HSV1-tk) gene and the HSV1-tk mutant (HSV1-sr39tk). Initial applications of reporter gene imaging in patients will be developed within two different clinical disciplines: (a) gene therapy and (b) adoptive cell-based therapies. These studies will benefit from the availability of efficient human reporter systems that can provide critical monitoring information for adenoviral-based, retroviral-based and lenteviral-based gene therapies, oncolytic bacterial and viral therapies, and adoptive cell-based therapies. Translational applications of noninvasive in vivo reporter gene imaging are likely to include: (a) quantitative monitoring of gene therapy vectors for targeting and transduction efficacy in clinical protocols by imaging the location, extent and duration of transgene expression; (b) monitoring of cell trafficking, targeting, replication and activation in adoptive T-cell and stem/progenitor cell therapies

  19. Human reporter genes: potential use in clinical studies

    International Nuclear Information System (INIS)

    Serganova, Inna; Ponomarev, Vladimir; Blasberg, Ronald

    2007-01-01

    The clinical application of positron-emission-tomography-based reporter gene imaging will expand over the next several years. The translation of reporter gene imaging technology into clinical applications is the focus of this review, with emphasis on the development and use of human reporter genes. Human reporter genes will play an increasingly more important role in this development, and it is likely that one or more reporter systems (human gene and complimentary radiopharmaceutical) will take leading roles. Three classes of human reporter genes are discussed and compared: receptors, transporters and enzymes. Examples of highly expressed cell membrane receptors include specific membrane somatostatin receptors (hSSTrs). The transporter group includes the sodium iodide symporter (hNIS) and the norepinephrine transporter (hNET). The endogenous enzyme classification includes human mitochondrial thymidine kinase 2 (hTK2). In addition, we also discuss the nonhuman dopamine 2 receptor and two viral reporter genes, the wild-type herpes simplex virus 1 thymidine kinase (HSV1-tk) gene and the HSV1-tk mutant (HSV1-sr39tk). Initial applications of reporter gene imaging in patients will be developed within two different clinical disciplines: (a) gene therapy and (b) adoptive cell-based therapies. These studies will benefit from the availability of efficient human reporter systems that can provide critical monitoring information for adenoviral-based, retroviral-based and lenteviral-based gene therapies, oncolytic bacterial and viral therapies, and adoptive cell-based therapies. Translational applications of noninvasive in vivo reporter gene imaging are likely to include: (a) quantitative monitoring of gene therapy vectors for targeting and transduction efficacy in clinical protocols by imaging the location, extent and duration of transgene expression; (b) monitoring of cell trafficking, targeting, replication and activation in adoptive T-cell and stem/progenitor cell therapies

  20. Scintillometric determination of DNA repair in human cell lines. A critical appraisal

    Energy Technology Data Exchange (ETDEWEB)

    Bianchi, V.; Zantedeschi, A.; Levis, A.G. (Padua Univ. (Italy). Ist. di Biologica Animale); Nuzzo, F.; Stefanini, M. (Consiglio Nazionale delle Ricerche, Pavia (Italy). Ist. di Genetica Biochimica ed Evoluzionistica); Abbondandolo, A.; Bonatti, S.; Fiorio, R.; Mazzaccaro, A. (Consiglio Nazionale delle Ricerche, Pisa (Italy). Ist. di Mutagenesi e Differenziamento); Capelli, E. (Pavia Univ. (Italy). Ist. di Genetica)

    1982-04-01

    The ability of a variety of chemical and physical agents to stimulate DNA repair synthesis in human cell cultures was tested by a simplified scintillometric procedure, with the use of hydroxyurea (HU) to suppress DNA replicative synthesis. After incubation with (/sup 3/H)thymidine, the radioactivity incorporated into DNA was determined in controls (C) and treated (T) cultures and in the corresponding HU series (Csub(HU), Tsub(HU)). The ratios Tsub(HU)/Csub(HU) and Tsub(HU)/T:Csub(HU)/C, indicating absolute and relative increases of DNA radioactivity, were calculated. When both ratios were significantly higher than 1, they were taken as indices of DNA repair stimulation.

  1. Monoclonal antibodies to antigens on human neutrophils, activated T lymphocytes, and acute leukemia blast cells

    International Nuclear Information System (INIS)

    Miterev, G.Yu.; Burova, G.F.; Puzhitskaya, M.S.; Danilevich, S.V.; Bulycheva, T.I.

    1987-01-01

    The authors describe the production of two mouse hybridomas secreting monoclonal antibodies to antigenic determinants of the surface membranes of human neutrophils, activated T lymphocytes, and acute leukemic blast cells. The degree of lymphocyte stimulation was estimated from incorporation of 3 H-thymidine with parallel microculture. Monoclonal antibodies of supernatants of hybridoma cultures shown here reacted in both immunofluorescence test and cytotoxicity test with surface membrane antigens on the majority of neutrophils and PHA-activated peripheral blood lymphocytes from healthy subjects, but did not give positive reactions with unactivated lymphocytes, adherent monocytes, erythrocytes, and alloantigen-stimulated lymphocytes

  2. Human mixed lymphocyte cultures. Evaluation of microculture technique utilizing the multiple automated sample harvester (MASH)

    Science.gov (United States)

    Thurman, G. B.; Strong, D. M.; Ahmed, A.; Green, S. S.; Sell, K. W.; Hartzman, R. J.; Bach, F. H.

    1973-01-01

    Use of lymphocyte cultures for in vitro studies such as pretransplant histocompatibility testing has established the need for standardization of this technique. A microculture technique has been developed that has facilitated the culturing of lymphocytes and increased the quantity of cultures feasible, while lowering the variation between replicate samples. Cultures were prepared for determination of tritiated thymidine incorporation using a Multiple Automated Sample Harvester (MASH). Using this system, the parameters that influence the in vitro responsiveness of human lymphocytes to allogeneic lymphocytes have been investigated. PMID:4271568

  3. Monoclonal antibodies to antigens on human neutrophils, activated T lymphocytes, and acute leukemia blast cells

    Energy Technology Data Exchange (ETDEWEB)

    Miterev, G.Yu.; Burova, G.F.; Puzhitskaya, M.S.; Danilevich, S.V.; Bulycheva, T.I.

    1987-11-01

    The authors describe the production of two mouse hybridomas secreting monoclonal antibodies to antigenic determinants of the surface membranes of human neutrophils, activated T lymphocytes, and acute leukemic blast cells. The degree of lymphocyte stimulation was estimated from incorporation of /sup 3/H-thymidine with parallel microculture. Monoclonal antibodies of supernatants of hybridoma cultures shown here reacted in both immunofluorescence test and cytotoxicity test with surface membrane antigens on the majority of neutrophils and PHA-activated peripheral blood lymphocytes from healthy subjects, but did not give positive reactions with unactivated lymphocytes, adherent monocytes, erythrocytes, and alloantigen-stimulated lymphocytes.

  4. Exposure to 60-Hz magnetic fields and proliferation of human astrocytoma cells in vitro.

    Science.gov (United States)

    Wei, M; Guizzetti, M; Yost, M; Costa, L G

    2000-02-01

    Epidemiological studies have suggested that exposure to electric and magnetic fields (EMF) may be associated with an increased incidence of brain tumors, most notably astrocytomas. However, potential cellular or molecular mechanisms involved in these effects of EMF are not known. In this study we investigated whether exposure to 60-Hz sinusoidal magnetic fields (0.3-1.2 G for 3-72 h) would cause proliferation of human astrocytoma cells. Sixty-Hertz magnetic fields (MF) caused a time- and dose-dependent increase in proliferation of astrocytoma cells, measured by (3)H-thymidine incorporation and by flow cytometry, and strongly potentiated the effect of two agonists (the muscarinic agonist carbachol and the phorbol ester PMA). However, MF had no effect on DNA synthesis of rat cortical astrocytes, i.e., of similar, nontransformed cells. To determine the amount of heating induced by MF, temperatures were also recorded in the medium. Both 1.2 G MF and a sham exposure caused a 0.7 degrees C temperature increase in the medium; however, (3)H-thymidine incorporation induced by sham exposure was significantly less than that caused by MF. GF 109203X, a rather specific protein kinase C (PKC) inhibitor, and down-regulation of PKC inhibited the effect of MF on basal and on agonist-stimulated (3)H-thymidine incorporation. These data indicate that MF can increase the proliferation of human astrocytoma cells and strongly potentiate the effects of two agonists. These findings may provide a biological basis for the observed epidemiological associations between MF exposure and brain tumors. Copyright 2000 Academic Press.

  5. Assembly of human C-terminal binding protein (CtBP) into tetramers.

    Science.gov (United States)

    Bellesis, Andrew G; Jecrois, Anne M; Hayes, Janelle A; Schiffer, Celia A; Royer, William E

    2018-06-08

    C-terminal binding protein 1 (CtBP1) and CtBP2 are transcriptional coregulators that repress numerous cellular processes, such as apoptosis, by binding transcription factors and recruiting chromatin-remodeling enzymes to gene promoters. The NAD(H)-linked oligomerization of human CtBP is coupled to its co-transcriptional activity, which is implicated in cancer progression. However, the biologically relevant level of CtBP assembly has not been firmly established; nor has the stereochemical arrangement of the subunits above that of a dimer. Here, multi-angle light scattering (MALS) data established the NAD + - and NADH-dependent assembly of CtBP1 and CtBP2 into tetramers. An examination of subunit interactions within CtBP1 and CtBP2 crystal lattices revealed that both share a very similar tetrameric arrangement resulting from assembly of two dimeric pairs, with specific interactions probably being sensitive to NAD(H) binding. Creating a series of mutants of both CtBP1 and CtBP2, we tested the hypothesis that the crystallographically observed interdimer pairing stabilizes the solution tetramer. MALS data confirmed that these mutants disrupt both CtBP1 and CtBP2 tetramers, with the dimer generally remaining intact, providing the first stereochemical models for tetrameric assemblies of CtBP1 and CtBP2. The crystal structure of a subtle destabilizing mutant suggested that small structural perturbations of the hinge region linking the substrate- and NAD-binding domains are sufficient to weaken the CtBP1 tetramer. These results strongly suggest that the tetramer is important in CtBP function, and the series of CtBP mutants reported here can be used to investigate the physiological role of the tetramer. © 2018 Bellesis et al.

  6. 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.

  7. Tritiated Thymidine as Tracer in DNA Metabolism and Cell Dynamics of Experimental Myeloid Leukaemia; Emploi de la Thymidine Tritiee comme Indicateur pour l'Etude du Metabolisme de l'ADN et de la Dynamique des Cellules dans la Leucemie Myeloide Experimentale; 0422 0440 0438 0442 0414 ; Empleo de la Timidina Tritiada como Indicador para Estudiar el Metabolismo del Acido Desoxirribonucleico y la Dinamica Celular en la Leucemia Mieloide Experimental

    Energy Technology Data Exchange (ETDEWEB)

    Zajicek, G.; Rosin, A.; Gross, J. [Department of Experimental Medicine and Cancer Research, Hebrew University, Hadassah Medical School, Jerusalem (Israel)

    1962-02-15

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

  8. Quantitative analysis of fluorodeoxyglucose-, fluoro-misonidazole- and fluoro-thymidine-PET images on five patients irradiated for a non-small-cell bronchial cancer; Analyse quantitative d'images de TEP au fluorodesoxyglucose, au fluoromisonidazole et a la fluorothymidine chez cinq patients irradies pour un cancer bronchique non a petites cellules

    Energy Technology Data Exchange (ETDEWEB)

    Thureau, S.; Dubray, B. [Departement de radiotherapie et physique medicale, centre Henri-Becquerel, Rouen (France); Litis EA 4108, QuantiF, universite de Rouen, Rouen (France); Modzelewski, R.; Lelandais, B.; Edet Sanson, A.; Gardin, I.; Vera, P. [Litis EA 4108, QuantiF, universite de Rouen, Rouen (France); Departement de medecine nucleaire, centre Henri-Becquerel, Rouen (France)

    2011-10-15

    As fluorodeoxyglucose-PET (positron emission tomography) is used for the planning of non-small-cell bronchial carcinoma radiotherapy in order to help to define the gross tumour volume (GTV), and as new tracers have been developed to define a biological target volume, the authors report the comparison of several thresholding methods by using fluorodeoxyglucose-, fluoro-misonidazole- and fluoro-thymidine-PET images. They notice important differences between the delineated volumes. Short communication

  9. Quantitative autoradiographic investigations on hairs, skin, and nails with the precursors 35S-cystine or 35S-methionine and 3H-thymidine respectively in animal experiments

    International Nuclear Information System (INIS)

    Schmiegelow, P.; Berndt, G.; Lindner, J.; Puschmann, M.

    1981-01-01

    By quantitative autoradiographic methods we tested the possible influence of the sulphurous amino acid L-cystine on the growth of hair, skin and nails. In the 3 H-thymidine autoradiographic method the tracing index (percentage of traced cell cores in relation to the total number of cell cores of a cell population) is calculated morphologically. In the 35 S-cystine and the 35 S-methionine autoradiography a quantification is carried out by applying the microscopic photometer for the investigation of defined hair root cross-sections. Measured values are indicated which depend on dosage and on the incorporation time. The investigation of other agents provoking negative or positive reactions of the hair growth by means of this method will have to be realized. Unlabelled L-cystine seems to promote the growth of hairs, epidermic basal cells and of germ cells of mouse nails, according to experiences made in animal experiments. The latter findings will have to be completed and confirmed by additional experiments. (orig.) [de

  10. Relative mRNA expression of prostate-derived E-twenty-six factor and E-twenty-six variant 4 transcription factors, and of uridine phosphorylase-1 and thymidine phosphorylase enzymes, in benign and malignant prostatic tissue

    Science.gov (United States)

    CAVAZZOLA, LUCIANE ROSTIROLA; CARVALHAL, GUSTAVO FRANCO; DEVES, CANDIDA; RENCK, DAIANA; ALMEIDA, RICARDO; SANTOS, DIóGENES SANTIAGO

    2015-01-01

    Prostate cancer is the most frequent urological tumor, and the second most common cancer diagnosed in men. Incidence and mortality are variable and appear to depend on behavioral factors and genetic predisposition. The prostate-derived E-twenty-six factor (PDEF) and E-twenty-six variant 4 (ETV4) transcription factors, and the thymidine phosphorylase (TP) and uridine phosphorylase-1 (UP-1) enzymes, are reported to be components of the pathways leading to tumorigenesis and/or metastasis in a number of tumors. The present study aimed to analyze the mRNA expression levels of these proteins in prostatic cancerous and benign tissue, and their association with clinical and pathological variables. Using quantitative reverse transcription polymerase chain reaction, the mRNA expression levels of PDEF, ETV4, TP and UP-1 were studied in 52 tissue samples (31 of benign prostatic hyperplasia and 21 of prostate adenocarcinomas) obtained from patients treated by transurethral resection of the prostate or by radical prostatectomy. Relative expression was assessed using the ∆-CT method. Data was analyzed using Spearman's tests for correlation. Pbenign and malignant prostatic tissues. Further studies are necessary to define the role of these proteins as therapeutic targets in prostate cancer. PMID:26137165

  11. Rate of accumulation of thymidine analogue mutations in patients continuing to receive virologically failing regimens containing zidovudine or stavudine: implications for antiretroviral therapy programs in resource-limited settings

    DEFF Research Database (Denmark)

    Cozzi-Lepri, Alessandro; Phillips, Andrew N; Martinez-Picado, Javier

    2009-01-01

    BACKGROUND: Because changes in antiretroviral therapy in resource-limited settings (RLSs) are delayed until patients experience immunological or clinical failure, it is important to be able to estimate the consequences in terms of accumulation of thymidine analogue (TA) mutations (TAMs). METHODS...... until the second GRT. RESULTS: At the time of the first GRT in a pair (t0), 1 year after virological failure, a median of 3 TAMs were detected, mutations 41L and 215Y in 65% of pairs and 67N in 52%. Overall, 126 TAMs were accumulated during 548 person-years of follow-up (PYFUs) (1/4.3 years; 95......% confidence interval, 3.7-5.0 years). Greater predicted activity of the TA at t0, TAM profile 2 (TAM2; vs TAM profile 1 [TAM1]) profiles at t0, use of a nonnucleoside reverse-transcriptase inhibitor (NNRTI) at t0 (vs combined NNRTI and protease inhibitor), and acquisition of HIV infection through heterosexual...

  12. Growth inhibitory effects of endotoxins from Bacteroides gingivalis and intermedius on human gingival fibroblasts in vitro

    International Nuclear Information System (INIS)

    Layman, D.L.; Diedrich, D.L.

    1987-01-01

    Purified endotoxin or lipopolysaccharide from Bacteroides gingivalis and Bacteroides intermedius caused a similar dose-dependent inhibition of growth of cultured human gingival fibroblasts as determined by 3 H-thymidine incorporation and direct cell count. Approximately 200 micrograms/ml endotoxin caused a 50% reduction in 3 H-thymidine uptake of logarithmically growing cells. Inhibition of growth was similar in cultures of fibroblasts derived from either healthy or diseased human gingiva. When examining the change in cell number with time of exposure in culture, the rate of proliferation was significantly suppressed during the logarithmic phase of growth. However, the cells recovered so that the rate of proliferation, although reduced, was sufficient to produce a cell density similar to the control cells with prolonged culture. The endotoxins were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The profiles of the Bacteroides endotoxins were different. B. gingivalis endotoxin showed a wide range of distinct bands indicating a heterogeneous distribution of molecular species. Endotoxin from B. intermedius exhibited a few discrete low molecular weight bands, but the majority of the lipopolysaccharides electrophoresed as a diffuse band of high molecular weight material. The apparent heterogeneity of the two Bacteroides endotoxins and the similarity in growth inhibitory capacity suggest that growth inhibitory effects of these substances cannot be attributed to any polysaccharide species of endotoxin

  13. Intracellular calcium mobilization in human lymphocytes in the presence of synthetic IgG Fc peptides

    International Nuclear Information System (INIS)

    Plummer, J.M.; Panahi, Y.P.; McClurg, M.R.; Hahn, G.S.; Naemura, J.R.

    1986-01-01

    Certain synthetic peptides derived from the Fc region of human IgG can suppress the mixed lymphocyte response. These peptides were tested for the ability to induce intracellular calcium mobilization in human lymphocytes using fura-2/calcium fluorescence. T cells were isolated by rosetting and were > 90% OKT3 positive. Lymphocytes were incubated with the acetoxymethyl ester of fura-2 (10 μM) for 60 minutes at 37 0 C. Fluorescence intensity changes at 505 nm were monitored at an excitation lambda of 340 nm. Fura-2 was not cytotoxic compared to quin-2 since fura-2 loaded mononuclear cells incorporated 3 H-thymidine when stimulated by PHA, succinyl Con A, PWM or LPS-STM whereas quin-2 loaded cells showed a dose dependent inhibition of proliferation. Those synthetic peptides (5 to 400 μg/ml) that suppressed the MLR induced a dose dependent increase in intracellular calcium in mononuclear cells, lymphocytes, non-T cells and T cells. The fura-2 calcium fluorescence time course response was similar for peptide, PHA and succinyl Con A. These results suggest that these immunoregulatory peptides suppress 3 H-thymidine incorporation at a point after intracellular calcium mobilization and that fura-2 has advantages over quin-2 in measuring intracellular calcium levels in lymphocytes

  14. Studies on chromosome aberrations induced in human lymphocytes by very low-dose exposure to tritium

    International Nuclear Information System (INIS)

    Hori, T.; Moriya, Junko; Nakai, Sayaka

    1978-01-01

    Assessment of potential hazard from environmental tritium to man becomes very important with increasing the development of nuclear-power industry. However, little data are available as to the determination on the genetic effect of tritium especially at the low levels. The object of the present study is to obtain quantitative data for chromosome aberrations in human lymphocytes, as an indicator for genetic risk estimation, induced by tritium at very low dose levels. Leukocyte cultures of human peripheral blood were chronically exposed for 48h to tritiated water and 3 H-thymidine using a wide range of tritium doses, and aberrations in lymphocyte chromosomes at the first metaphases were examined. In the experimental conditions, the types of aberrations induced by radiation emitted from both tritiated water and 3 H-thymidine were mostly chromatid types, such as chromatid gaps and deletions. The dose-response relations for chromatid breaks per cell exhibited unusual dose-dependency in both cases. It was demonstrated that at higher dose range the yields of chromatid breaks increased linearly with dose, while those at lower dose range were significantly higher than would be expected by a downward extraporation from the linear relation. Partial-hit or partial-target kinetics events appeared at very low dose exposure. (author)

  15. Optimal conditions in (/sup 3/H)-thymidine uptake studies to prevent radiation damage to cells. A scintimetric and cytofluorographic analysis

    Energy Technology Data Exchange (ETDEWEB)

    Ruiz-Arguelles, A; Llorente, L; Diaz-Jouanen, E; Alarcon-Segovia, D [Instituto Nacional de la Nutricion, Mexico City

    1981-12-01

    Cells subjected to nucleoside incorporation studies using radiolabelled materials may suffer radiation damage that can alter the results. Scintimetric and cytofluorographic analyses were performed to confirm this and to determine the optimal experimental doses of, and exposure times to, (/sup 3/H)-TdR, in order to prevent or minimize such radiation damage to cells. The results showed that cultures of human mononuclear cells should be pulsed with 0.125 ..mu..Ci for 14 hr when stimulated with phytohaemagglutinin, 0.125 ..mu..Ci for 18 hr when stimulated with pokeweed mitogen, 0.5 ..mu..Ci for 8 hr when stimulated with concanavalin A and 0.5 ..mu..Ci for 8 hr when subjected to allogeneic stimulus, in order to achieve optimal incorporation with minimal disturbances of the cell cycle.

  16. Effects of extracellular plaque components on the chlorhexidine sensitivity of strains of Streptococcus mutans and human dental plaque

    International Nuclear Information System (INIS)

    Wolinsky, L.E.; Hume, W.R.

    1985-01-01

    An in vitro study was undertaken to determine the effects of sucrose-derived extracellular plaque components on the sensitivity of selected oral bacteria to chlorhexidine (CX). Cultures of Streptococcus mutans HS-6, OMZ-176, Ingbritt C, 6715-wt13, and pooled human plaque were grown in trypticase soy media with or without 1% sucrose. The sensitivity to CX of bacteria grown in each medium was determined by fixed-time exposure to CX and subsequent measurement of 3 H-thymidine uptake. One-hour exposure to CX at concentrations of 10(-4) M (0.01% w/v) or greater substantially inhibited subsequent cellular division among all the S. mutans strains and human plaque samples tested. An IC50 (the CX concentration which depressed 3 H-thymidine incorporation to 50% of control level) of close to 10(-4) M was noted for S. mutans strains HS-6, OMZ-176, and 6715-wt13 when grown in the presence of sucrose. The same strains grown in cultures without added sucrose showed about a ten-fold greater sensitivity to CX (IC50 close to 10(-5) M). A three-fold difference was noted for S. mutans Ingbritt C. Only a slight increase in the IC50 was noted for the plaque samples cultured in sucrose-containing media, but their threshold for depression of 3 H-thymidine uptake by CX was lower than that for the sucrose-free plaque samples. The study showed that extracellular products confer some protection against CX to the bacteria examined, and provided an explanation for the disparity between clinically-recommended concentrations for plaque suppression and data on in vitro susceptibility

  17. Tyrosine phosphorylation of a 66KD soluble protein and augmentation of lectin induced mitogenesis by DMSO in human T lymphocytes

    International Nuclear Information System (INIS)

    Wedner, H.J.; Bass, G.

    1986-01-01

    The authors have demonstrated that induction of mitogenesis in human T lymphocytes is associated with the tyrosine phosphorylation of a 66KD soluble substrate-TPP 66. Since DMSO has been shown to be a non-specific stimulator of tyrosine protein kinases they have examined the effect of DMSO on both activation and tyrosine phosphorylation in human T cells. Human peripheral blood T lymphocytes were isolated by dextran sedimentation, Ficol/Paque centrifugation and nylon wool filtration. Phosphorylation was performed in cells incubated with [ 32 P] orthophosphate followed by DMSO for 30 min. TPP 66 was identified by 2-D PAGE, autoradiography, and HV electrophoresis of the hydrolyzed protein. Concentrations of DMSO from 1% to 50% induced the tyrosine phosphorylation of TPP 66 with maximal stimulation seen at 20%. DMSO alone did not activate the T cells (measured by [ 3 H] thymidine incorporation) when tested at high concentrations for 30 sec to 10 min. (longer incubations were markedly toxic) or low concentrations for 12 to 48 hrs. Low concentrations of DMSO 0.1%-0.5% did however, markedly augment [ 3 H] thymidine incorporation induced by PHA or Con A. These data suggest that tyrosine phosphorylation of TPP 66 alone may not constitute sufficient signal for the activation sequence to begin but the phosphorylation of this soluble substrate may be a critical factor in the propagation of the activation sequence

  18. Investigation into the incorporation of H3-labelled thymidine into the DNA of a C3H-tumor after irradiation with 35 MeV electrons

    International Nuclear Information System (INIS)

    Pfersdorff, J.

    1978-01-01

    The radiation effect on the DNA synthesis of an injected tumor in C 3 H-mice is studied. The tumor is a mammary adenocarcinoma injected into the neck of the animals. A betatron of Brown, Boverie and Cie. of the type Asklepitton 35 is used as radiation source for the generation of fast electrons with an energy of 35 MeV applied to the tumor in the animals through an annular tube of 4 cm in diameter, in the given doses. The tumor-doubling rate during the exponential growth phase is computed to be td = 3.65 days. During irradiation of the tumor, the DNA synthesis is determined by measuring the incorporation of H 3 TdR into the DNA. The results show that the radiation effect on the DNA synthesis is enhanced by every exposure, whereas the additional damage decreases and a recovery can be detected after the first two exposures according to the damage. This study confirms the results of former investigations on tissue cultures. The decisive effect of the radiation is the disturbance of DNA synthesis. Analogous effects can be expected in the irradiation of human tumor cells, though no conclusions can be drawn with regard to fractionation, due to the fast tumor doubling rate of 3.7 days. (orig./MG) [de

  19. Thalidomide increases human keratinocyte migration and proliferation.

    Science.gov (United States)

    Nasca, M R; O'Toole, E A; Palicharla, P; West, D P; Woodley, D T

    1999-11-01

    Thalidomide is reported to have therapeutic utility in the treatment of pyoderma gangrenosum, Behçet's disease, aphthous ulcers, and skin wounds. We investigated the effect of thalidomide on human keratinocyte proliferation and migration, two early and critical events in the re-epithelialization of skin wounds. Thalidomide at concentrations less than 1 microM did not affect keratinocyte viability. Using a thymidine incorporation assay, we found that thalidomide, at therapeutic concentrations, induced more than a 2. 5-fold increase in the proliferative potential of the cells. Keratinocyte migration was assessed by two independent motility assays: a colloidal gold assay and an in vitro scratch assay. At optimal concentrations, thalidomide increased keratinocyte migration on a collagen matrix more than 2-fold in the colloidal gold assay and more than 3-fold in the scratch assay over control. Although pro-migratory, thalidomide did not alter the level of metalloproteinase-9 secreted into culture medium. Thalidomide did, however, induce a 2-4-fold increase in keratinocyte-derived interleukin-8, a pro-migratory cellular autocrine factor. Human keratinocyte migration and proliferation are essential for re-epithelialization of skin wounds. Interleukin-8 increases human keratinocyte migration and proliferation and is chemotactic for keratinocytes. Therefore, thalidomide may modulate keratinocyte proliferation and motility by a chemokine-dependent pathway.

  20. Growth kinetics of four human breast carcinomas grown in nude mice

    DEFF Research Database (Denmark)

    Spang-Thomsen, M; Rygaard, K; Hansen, L

    1989-01-01

    with cell generation times of 42 to 60 hours. The three receptor-positive tumors had slower growth rate, larger tumor volume doubling time, and smaller growth fraction and labelling index than the receptor-negative tumor. However, no single proliferation parameter was sufficient to characterize the growth......The immune-deficient nude mouse with human tumor xenografts is an appropriate model system for performing detailed growth kinetic examinations. In the present study one estrogen and progesterone receptor-negative (T60) and three receptor-positive (Br-10, MCF-7, T61) human breast cancer xenografts...... in nude mice were investigated. The proliferative tumor characteristics were examined by growth curves, thymidine labelling technique, and flow cytometric DNA analysis performed on fine-needle aspirations. The results showed that the tumors had growth kinetics comparable to other human tumor types...

  1. Explant culture of human peripheral lung. I. Metabolism of benzo[alpha]pyrene

    DEFF Research Database (Denmark)

    Stoner, G.D.; Harris, C.C.; Autrup, Herman

    1978-01-01

    the predominant alveolar epithelial cell type. Lamellar inclusion bodies were released from the type 2 cells and accumulated in the alveolar spaces. The metabolism of benzo[alpha]pyrene (BP) in human lung explants cultured for up to 7 days was investigated. Human lung explants had measurable aryl hydrocarbon......Human lung explants have been maintained in vitro for a period of 25 days. Autoradiographic studies indicated that the broncholar epithelial cells, type 2 alveolar epithelial cells, and stromal fibroblasts incorporated 3H-thymidine during the culture. After 7 to 10 days, type 2 cells were...... hydroxylase activity and could metabolize BP into forms that were bound to cellular DNA and protein. Peripheral lung had significantly lower aryl hydrocarbon hydroxylase activity than cultured bronchus but both tissues had similar binding levels of BP to DNA. Radioautographic studies indicated that all cell...

  2. Potential use of carbon-11 labeled thymidine (TdR) for studying the effect of therapy on prostatic adenocarcinoma in vivo

    International Nuclear Information System (INIS)

    Conti, P.S.; Kleinert, E.L.; Schma, B.; Herr, H.W.; Whitmore, W.F. Jr.

    1984-01-01

    Alterations in tumor growth, such as those which occur during therapeutic manipulation, may be followed by measuring variations in radiolabeled TdR uptake. In order to study such parameters in vivo using external imaging techniques, the authors have synthesized TdR labeled with cyclotron produced carbon-11, a short-lived (T1/2=20.4 min) positron-emitting radionuclide. The Copenhagen rat bearing the transplantable Dunning R3327G prostatic adenocarcinoma can be used as a model for poorly differentiated carcinoma of the prostate in humans. The tissue distribution of C-14 TdR was studied in untreated tumor rats and in tumor rats receiving a combination of difluoromethyl ornithine and methylglyoxal-bis-guanylhydrazone, effective inhibitors of polyamine biosynthesis. The tissue distribution at 45 min post-injection (5 rats/group) was determined by calculating the relative concentration (RC) of radioactivity in blood and tissue samples (RC=dpm found per gm tissue/dpm injected per gm animal mass). The mean RC in untreated tumor was 2.55 +- 0.46, compared to 0.85 +- 0.12 in treated tumor. Tumor/blood, tumor/muscle and tumor/prostate ratios were 3.07, 7.08, and 6.89 in untreated tumor, and 1.23, 3.04, and 2,93 in treated tumor. The differences in RC for the untreated and treated tumors suggest that external imaging with C-11 TdR may be useful for monitoring the effects of therapy on tumors in vivo

  3. 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.

  4. 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.

  5. Tritiated thymidine incorporation and the development of an interstitial lesion in the bronchiolar-alveolar regions of the lungs of normal and complement deficient mice after inhalation of chrysotile asbestos

    International Nuclear Information System (INIS)

    McGavran, P.D.; Butterick, C.J.; Brody, A.R.

    1989-01-01

    Inhaled asbestos causes the proliferation of bronchiolar-alveolar epithelial and interstitial cells in rats and mice 19 to 72 hours after a single 5-hour exposure. This condition is associated with rapid macrophage accumulation and development of an interstitial fibrotic lesion at alveolar duct bifurcations. In an attempt to define the mechanisms mediating asbestos-induced cell proliferation and fibrogenesis, we studied mice exposed to chrysotile asbestos for five hours. The mice were normal and a congenic strain (B10.D2/oSn), deficient in the fifth component of complement (C5-). We knew that the latter exhibit a depressed asbestos-induced macrophage response and wanted to learn whether the depressed response correlated with measurements of cell proliferation and progression of an interstitial lesion. Sections of first alveolar duct bifurcations were prepared for light microscopic autoradiography and ultrastructural morphometry at varying times after animal exposure to asbestos. In sham-exposed C5+ and C5- animals, less than 1% of epithelial and interstitial cells of the terminal bronchioles and alveolar ducts incorporated tritiated thymidine (3H-TdR) at any time after exposure to asbestos. Between 19 and 72 hours after exposure, epithelial and interstitial cells in both strains of mice exhibited significantly increased levels of 3H-TdR incorporation. The response decreased by eight days postexposure, and 3H-TdR incorporation was normal one month after exposure. Similarly, morphometry showed that both the C5+ and C5- asbestos-exposed mice exhibited significant increases in the volume density of epithelial and interstitial cells 48 hours after exposure. However, one month after exposure, the normal C5+ asbestos-exposed mice developed a fibrotic lesion, whereas the C5- asbestos-exposed animals were no different from sham-exposed C5- controls

  6. Autoradiographic examinations on the synthesis of the virus herpes simplex in macrophages, rabbit kidney- and BHK-cells by labelling with 3H-thymidine, 3H-uridine and UDP-3H-galactose

    International Nuclear Information System (INIS)

    Lindermann, J.

    1979-01-01

    After injection, an autoradiographically measurable reduplication of virus-specific DNA takes place in NMRI-macrophages infected with HSV (herpes simplex virus). For this process, a sufficient duration of the 3 H-thymidine pulses is of essential importance. An infection with HSV of the Lennette strain induces in considerably more cells of a macrophage population the production of virus-specific DNA than the infection with HSV of the D-316 type. After injection a reduplication of virus-specific DNA occurs in more cells of a HSV-infected population of old macrophages than in that one consisting of young macrophages. In the 7th hour after injection a reduction of RNA synthesis was observed in the HSV-infected macrophage cultures. After HSV-infection, a synthesis of virus-specific DNA could not be detected autoradiographically in macrophages of C 57/bl mice. After HSV-infection the virus-specific DNA synthesis was also observed in activated macrophages of NMRI mice, which had been treated with thioglucollate 72 hours before cell extraction. In general, the previous thioglucollate treatment increased the susceptibility of the macrophages of infection with subsequent reduplication of herpes-specific DNA. Compared to other not infected but activated macrophages, the RNA synthesis after injection was found to be not (HSV Lennette) or only minutely (HSV D-316) reduced in HSV-infected cultures of activated macrophages. In the investigations about the utilisation of exogenic UDP- 3 H-galactose by BHK and rabbit cells it was observed that only few cells of the cultures provide an externally accessible galactosyl transferase activity. Due to the influence of cell-damaging mechanisms a cell proliferation is induced, which is accompanied by externally accessible galactosyl transferase activities in the cell cultures. (orig./MG) [de

  7. Bilateral lesions of suprachiasmatic nuclei affect circadian rhythms in [3H]-thymidine incorporation into deoxyribonucleic acid in mouse intestinal tract, mitotic index of corneal epithelium, and serum corticosterone

    International Nuclear Information System (INIS)

    Scheving, L.E.; Tsai, T.H.; Powell, E.W.; Pasley, J.N.; Halberg, F.; Dunn, J.

    1983-01-01

    Investigations into the role of the suprachiasmatic nuclei (SCN) in the coordination of circadian rhythms have presented differing results. Several reports have shown that ablation of the suprachiasmatic nuclei (SCNA) alters the phase and amplitude of rhythms but does not abolish them. The present study investigates the effect of SCNA on the rhythms in cell proliferation in various regions of the intestinal tract as measured by the incorporation of [ 3 H]-thymidine into deoxyribonucleic acid, in the mitotic activity of the corneal epithelium, and in serum corticosterone levels. The study involved mice with verified lesions of the SCN (six to 13 mice per time point) and control groups of both sham-operated and unoperated mice (seven of each per time point). The mice were killed in groups that represented seven time points over a single 24 hr span (3 hr intervals with the 0800 hr sampled both at start and end of the series). The tissues examined were the tongue, esophagus, gastric stomach, and colon for DNA synthesis, the corneal epithelium for mitotic index, and blood serum for corticosterone level. The most consistent result of SCNA was a phase advance in the rhythms in cell proliferation in the tongue, esophagus, gastric stomach, colon, and corneal epithelium. A reduction in rhythm amplitude occurred in the tongue, esophagus, and corneal epithelium; however, there was an amplitude increase for the stomach, colon, and serum corticosterone. The mesor (rhythm-adjusted mean) was increased by SCNA in all tissues except the corneal epithelium. These findings further support the role of the suprachiasmatic nuclear area in the control of rhythms in cell proliferation and corticosterone production, by acting as a ''phase-resetter'' and as a modulator of rhythm amplitude

  8. Sickle erythrocytes inhibit human endothelial cell DNA synthesis

    International Nuclear Information System (INIS)

    Weinstein, R.; Zhou, M.A.; Bartlett-Pandite, A.; Wenc, K.

    1990-01-01

    Patients with sickle cell anemia experience severe vascular occlusive phenomena including acute pain crisis and cerebral infarction. Obstruction occurs at both the microvascular and the arterial level, and the clinical presentation of vascular events is heterogeneous, suggesting a complex etiology. Interaction between sickle erythrocytes and the endothelium may contribute to vascular occlusion due to alteration of endothelial function. To investigate this hypothesis, human vascular endothelial cells were overlaid with sickle or normal erythrocytes and stimulated to synthesize DNA. The erythrocytes were sedimented onto replicate monolayers by centrifugation for 10 minutes at 17 g to insure contact with the endothelial cells. Incorporation of 3H-thymidine into endothelial cell DNA was markedly inhibited during contact with sickle erythrocytes. This inhibitory effect was enhanced more than twofold when autologous sickle plasma was present during endothelial cell labeling. Normal erythrocytes, with or without autologous plasma, had a modest effect on endothelial cell DNA synthesis. When sickle erythrocytes in autologous sickle plasma were applied to endothelial monolayers for 1 minute, 10 minutes, or 1 hour and then removed, subsequent DNA synthesis by the endothelial cells was inhibited by 30% to 40%. Although adherence of sickle erythrocytes to the endothelial monolayers was observed under these experimental conditions, the effect of sickle erythrocytes on endothelial DNA synthesis occurred in the absence of significant adherence. Hence, human endothelial cell DNA synthesis is partially inhibited by contact with sickle erythrocytes. The inhibitory effect of sickle erythrocytes occurs during a brief (1 minute) contact with the endothelial monolayers, and persists for at least 6 hours of 3H-thymidine labeling

  9. Tetrameric DABCO™-Bromine: an Efficient and Versatile Reagent ...

    African Journals Online (AJOL)

    NJD

    Reagent for Bromination of Various Organic Compounds. Majid M. Heravi,a* ... aDepartment of Chemistry, School of Sciences, Azzahra University, Vanak, Tehran, Iran. bChemistry ... Synthesis of "-bromo ketones and nitriles has also been ...

  10. Metallofullerenol Inhibits Cellular Iron Uptake by Inducing Transferrin Tetramerization.

    Science.gov (United States)

    Li, Jinxia; Xing, Xueqing; Sun, Baoyun; Zhao, Yuliang; Wu, Zhonghua

    2017-10-18

    Herein, A549 tumor cell proliferation was confirmed to be positively dependent on the concentration of Fe 3+ or transferrin (Tf). Gd@C 82 (OH) 22 or C 60 (OH) 22 effectively inhibited the iron uptake and the subsequent proliferation of A549 cells. The conformational changes of Tf mixed with FeCl 3 , GdCl 3 , C 60 (OH) 22 or Gd@C 82 (OH) 22 were obtained by SAXS. The results demonstrate that Tf homodimers can be decomposed into monomers in the presence of FeCl 3 , GdCl 3 or C 60 (OH) 22 , but associated into tetramers in the presence of Gd@C 82 (OH) 22 . The larger change of SAXS shapes between Tf+C 60 (OH) 22 and Tf+FeCl 3 implies that C 60 (OH) 22 is bound to Tf, blocking the iron-binding site. The larger deviation of the SAXS shape from a possible crystal structure of Tf tetramer implies that Gd@C 82 (OH) 22 is bound to the Tf tetramer, thus disturbing iron transport. This study well explains the inhibition mechanism of Gd@C 82 (OH) 22 and C 60 (OH) 22 on the iron uptake and the proliferation of A549 tumor cells and highlights the specific interactions of a nanomedicine with the target biomolecules in cancer therapy. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Single oligomer spectra probe chromophore nanoenvironments of tetrameric fluorescent proteins

    NARCIS (Netherlands)

    Blum, Christian; Meixner, Alfred J; Subramaniam, Vinod

    2006-01-01

    When analyzing the emission of a large number of individual chromophores embedded in a matrix, the spread of the observed parameters is a characteristic property for the particular chromophore-matrix system. To quantitatively assess the influence of the matrix on the single molecule emission

  12. Single Oligomer Spectra Probe Chromophore Nanoenvironments of Tetrameric Fluorescent Proteins

    NARCIS (Netherlands)

    Blum, Christian; Meixner, Alfred J.; Subramaniam, Vinod

    2006-01-01

    When analyzing the emission of a large number of individual chromophores embedded in a matrix, the spread of the observed parameters is a characteristic property for the particular chromophore−matrix system. To quantitatively assess the influence of the matrix on the single molecule emission

  13. Why are there two kinds of chain in tetrameric hemoglobins

    International Nuclear Information System (INIS)

    Ferreira, R.; Jacchieri, S.G.

    1981-01-01

    The homeotropic allosteric interactions responsible for the sigmoidal oxygen saturation curves of α 2 β 2 hemoglobins are shown to be larger than those of hypothetical hemoglobins obeying identical curves and built from equivalent chains, γ 4 . It is also shown that this ensures for the α 2 β 2 species a more dependable cooperativity, through a biologically significative temperature range. On the basis of these findings it is argued that the existence of two different globin chains is advantageous in an evolutionary sense. (Author) [pt

  14. Characterization of a TK6-Bcl-xL gly-159-ala Human Lymphoblast Clone

    Energy Technology Data Exchange (ETDEWEB)

    Chyall, L.: Gauny, S.; Kronenberg, A.

    2006-01-01

    TK6 cells are a well-characterized human B-lymphoblast cell line derived from WIL-2 cells. A derivative of the TK6 cell line that was stably transfected to express a mutated form of the anti-apoptotic protein Bcl-xL (TK6-Bcl-xL gly-159- ala clone #38) is compared with the parent cell line. Four parameters were evaluated for each cell line: growth under normal conditions, plating efficiency, and frequency of spontaneous mutation to 6‑thioguanine resistance (hypoxanthine phosphoribosyl transferase locus) or trifluorothymidine resistance (thymidine kinase locus). We conclude that the mutated Bcl-xL protein did not affect growth under normal conditions, plating efficiency or spontaneous mutation frequencies at the thymidine kinase (TK) locus. Results at the hypoxanthine phosphoribosyl transferase (HPRT) locus were inconclusive. A mutant fraction for TK6‑Bcl-xL gly-159-ala clone #38 cells exposed to 150cGy of 160kVp x-rays was also calculated. Exposure to x-irradiation increased the mutant fraction of TK6‑Bcl-xL gly-159-ala clone #38 cells.

  15. Effects of cyclophosphamide on in vitro human lymphocyte culture and mitogenic stimulation

    International Nuclear Information System (INIS)

    Sharma, B.S.

    1983-01-01

    Cyclophosphamide (CY) has been reported to be inactive in vitro under certain conditions. In the present study, CY was tested for its ability to inhibit human lymphocyte proliferation and to modulate lymphocyte response to mitogens in vitro. The inhibition of or the increase in 3 H-thymidine incorporation in mitogen-stimulated and unstimulated lymphocytes by CY was used as a measure of CY activity in vitro. The results demonstrate that lymphocytes from 10 different persons had a mean decrease of 74% in 3 H-thymidine incorporation in the presence of CY (P less than 0.005). The effect was maximal at a concentration of 160 micrograms/ml. A mean inhibition of 35 and 55% was caused by 10 and 40 micrograms/ml concentrations of CY, respectively. CY also was able to reduce the number of viable cells during 5 days in culture and had a profound effect on mitogen stimulation of lymphocytes. In all cases, CY modulated the stimulation of lymphocytes by phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM) either by augmenting or suppressing the responses. At low concentrations (10 micrograms/ml) it augmented mitogenic stimulation by 46 to 281%. At higher concentrations (20 to 160 micrograms/ml), CY had a suppressive effect with a maximum suppression of 99%. The CY-induced immunomodulation is perhaps caused by its action on the regulatory T cells. When tested in vitro, CY had inhibitory activity on T cells

  16. Immunoregulatory adherent cells in human tuberculosis: radiation-sensitive antigen-specific suppression by monocytes

    Energy Technology Data Exchange (ETDEWEB)

    Kleinhenz, M.E.; Ellner, J.J.

    1985-07-01

    In human tuberculosis, adherent mononuclear cells (AMC) selectively depress in vitro responses to the mycobacterial antigen tuberculin purified protein derivative (PPD). The phenotype of this antigen-specific adherent suppressor cell was characterized by examining the functional activity of adherent cells after selective depletion of sheep erythrocyte-rosetting T cells or OKM1-reactive monocytes. Adherent cell suppression was studied in the (/sup 3/H)thymidine-incorporation microculture assay by using T cells rigorously depleted of T cells with surface receptors for the Fc portion of IgG (T gamma cells) as antigen-responsive cells. PPD-induced (/sup 3/H)thymidine incorporation by these non gamma T cells was uniformly reduced (mean, 42% +/- 10% (SD)) when autologous AMC were added to non gamma T cells at a ratio of 1:2. Antigen-specific suppression by AMC was not altered by depletion of sheep erythrocyte-rosetting T cells or treatment with indomethacin. However, AMC treated with OKM1 and complement or gamma irradiation (1,500 rads) no longer suppressed tuberculin responses in vitro. These studies identify the antigen-specific adherent suppressor cell in tuberculosis as an OKM1-reactive, non-erythrocyte-rosetting monocyte. The radiosensitivity of this monocyte immunoregulatory function may facilitate its further definition.

  17. Immunoregulatory adherent cells in human tuberculosis: radiation-sensitive antigen-specific suppression by monocytes

    International Nuclear Information System (INIS)

    Kleinhenz, M.E.; Ellner, J.J.

    1985-01-01

    In human tuberculosis, adherent mononuclear cells (AMC) selectively depress in vitro responses to the mycobacterial antigen tuberculin purified protein derivative (PPD). The phenotype of this antigen-specific adherent suppressor cell was characterized by examining the functional activity of adherent cells after selective depletion of sheep erythrocyte-rosetting T cells or OKM1-reactive monocytes. Adherent cell suppression was studied in the [ 3 H]thymidine-incorporation microculture assay by using T cells rigorously depleted of T cells with surface receptors for the Fc portion of IgG (T gamma cells) as antigen-responsive cells. PPD-induced [ 3 H]thymidine incorporation by these non gamma T cells was uniformly reduced (mean, 42% +/- 10% [SD]) when autologous AMC were added to non gamma T cells at a ratio of 1:2. Antigen-specific suppression by AMC was not altered by depletion of sheep erythrocyte-rosetting T cells or treatment with indomethacin. However, AMC treated with OKM1 and complement or gamma irradiation (1,500 rads) no longer suppressed tuberculin responses in vitro. These studies identify the antigen-specific adherent suppressor cell in tuberculosis as an OKM1-reactive, non-erythrocyte-rosetting monocyte. The radiosensitivity of this monocyte immunoregulatory function may facilitate its further definition

  18. Changes of the proliferation kinetics of human bone marrow in vivo through hydroxyurea

    International Nuclear Information System (INIS)

    Ertl, E.

    1982-01-01

    A 10-hour oral continuous infusion with hydroxyurea (HU) at a non-toxic concentration was performed in 20 malignoma patients with undisturbed bone marrow. Bone marrow taken before, during and after HU-administration was examined for 3H-TdR incorporation by means of autoradiography and liquid scintimetry, for cell phase distribution by means of flow cytophotometry, morphologically and by means of CFUc. 3H-TdR incorporation into bone marrow cells dropped to 16% of the initial value under HU and rose to 156% 10 h after HU-effect terminated. Cytophotometry did not furnish any proof of a decrease of S-phase cells or increase of cells in G 1 -to-S-transition during HU. S-cells rise to 129% of the initial value 10 h after having fallen below minimum inhibition concentration. Under HU, there is an equal number of cells in S which incorporate much less 3H-thymidine; after HU more S-cells incorporate more 3H-thymidine than before HU. During HU action, DNA synthesis activity is reduced to 17% and reaches the initial value with 105% afterwards. In human bone marrow, hydroxyurea in non-toxic concentration causes a temporary DNA synthesis inhibition in terms of activity reduction and partial arrest in S. A stop-and-go of the cell cycle effected by HU does not occur; the effect is rather a slow-down of DNA synthesis. (orig./MG) [de

  19. Immune responses to implanted human collagen graft in rats

    International Nuclear Information System (INIS)

    Quteish, D.; Dolby, A.E.

    1991-01-01

    Immunity to collagen implants may be mediated by cellular and humoral immune responses. To examine the possibility of such immunological reactivity and crossreactivity to collagen, 39 Sprague-Dawley rats (female, 10 weeks old, approximately 250 g wt) were implanted subcutaneously at thigh sites with crosslinked, freeze-dried human placental type I collagen grafts (4x4x2 mm) which had been irradiated (520 Gray) or left untreated. Blood was obtained by intracardiac sampling prior to implantation or from normal rats, and at various times afterwards when the animals were sacrificed. The sera from these animals were examined for circulating antibodies to human, bovine and rat tail (type I) collagens by enzyme-linked immunosorbent assay (ELISA). Also, the lymphoblastogenic responses of spleen lymphocytes from the irradiated collagen-implanted animals were assessed in culture by measuring thymidine uptake with autologous and normal rat sera in the presence of human bovine type I collagens. Implantation of the irradiated and non-irradiated collagen graft in rats led to a significant increase in the level of circulating antibodies to human collagen. Also antibody to bovine and rat tail collagens was detectable in the animals implanted with irradiated collagen grafts but at a lower level than the human collagen. There was a raised lymphoblastogenic response to both human and bovine collagens. The antibody level and lymphoblastogenesis to the tested collagens gradually decreased towards the end of the post-implantation period. (author)

  20. Procalcitonin NH2-terminal cleavage peptide has no mitogenic effect on normal human osteoblast-like cells

    International Nuclear Information System (INIS)

    Hassager, C.; Bonde, S.K.; Anderson, M.A.; Rink, H.; Spelsberg, T.C.; Riggs, B.L.

    1991-01-01

    The NH2-terminal cleavage peptide of procalcitonin (N-proCT) recently was reported to be a bone cell mitogen. The authors have investigated the effect of N-proCT on the proliferation of normal human cells that have the phenotype of mature osteoblasts (hOB cells). N-proCT treatment for 24, 48, or 96 h in concentrations from 1 nM to 1 microM did not significantly increase [3H]thymidine uptake (means ranged from -19% to 38% of control, no significant differences) in hOB cells (6-10 cell strains per experiment) plated at four different densities. However, the hOB cells responded significantly to treatment with transforming growth factor β (3 ng/ml), bovine insulin (300 micrograms/ml), or 30% fetal calf serum, which were included in all experiments as positive controls. The [3H]thymidine uptake data were confirmed in a direct cell count experiment tested at 96 h. Thus they data do not support the hypothesis that N-proCT is a potent mitogen for normal human osteoblasts

  1. Alkylation of deoxyribonucleic acid by carcinogens dimethyl sulphate, ethyl methanesulphonate, N-ethyl-N-nitrosourea and N-methyl-N-nitrosourea. Relative reactivity of the phosphodiester site thymidylyl(3'-5')thymidine.

    Science.gov (United States)

    Swenson, D H; Lawley, P D

    1978-01-01

    1. The ethyl phosphotriester of thymidylyl(3'-5')thymidine, dTp(Et)dT, was identified as a product from reaction of DNA with N-ethyl-N-nitrosourea, by procedures parallel to those reported previously for the methyl homologue produced by N-methyl-N-nitrosourea. 2. Enzymic degradation to yield alkyl phosphotriesters from DNA alkylated by these carcinogens and by dimethyl sulphate and ethyl methanesulphonate was studied quantitatively, and the relative yields of the triesters dTp(Alk)dT were determined. The relative reactivity of the phosphodiester group dTpdT to each of the four carcinogens was thus obtained, and compared with that of DNA overall, or with that of the N-7 atom of guanine in DNA. Relative reactivity of the phosphodiester group was lowest towards dimethyl sulphate, the least electrophilic of the reagents used, and was highest towards N-ethyl-N-nitrosourea, the most electrophilic reagent. 3. The nature of the alkyl group transferred also influenced reactivity of the phosphodiester site, since this site was relatively more reactive towards ethylation than would be predicted simply from the known Swain-Scott s values of the alkylating agents. It was therefore suggested that the steric accessibility of the weakly nucleophilic phosphodiester group on the outside of the DNA macromolecule favours its reaction with ethylating, as opposed to methylating, reagents. 4. Taking a value of the Swain-Scott nucleophilicity (n) of 2.5 for an average DNA nucleotide unit [Walles & Ehrenberg (1969) Acta Chem. Scand. 23, 1080-1084], a value of n of about 1 for the phosphodiester group was deduced, and this value was found to be 2-3 units less than that for the N-7 atom of guanine in DNA. 5. The reactivity of DNA overall was markedly high towards the alkylnitrosoureas, despite their relatively low s values. This was ascribed to an electrostatic factor that favoured reaction of the negatively charged polymer with alkyldiazonium cation intermediates. PMID:208508

  2. Auger electron emitter against multiple myeloma - targeted endo-radio-therapy with {sup 125}I-labeled thymidine analogue 5-iodo-4'-thio-2'-deoxyuridine

    Energy Technology Data Exchange (ETDEWEB)

    Morgenroth, Agnieszka, E-mail: amorgenroth@ukaachen.de [Nuclear Medicine Clinic, University Ulm, Albert-Einstein-Allee 23, D-89081 Ulm (Germany); Nuclear Medicine Clinic, University Aachen, RWTH, Pauwelsstrasse 30, D-52074 Aachen (Germany); Dinger, Cornelia; Zlatopolskiy, Boris D.; Al-Momani, Ehab; Glatting, Gerhard [Nuclear Medicine Clinic, University Ulm, Albert-Einstein-Allee 23, D-89081 Ulm (Germany); Mottaghy, Felix M. [Nuclear Medicine Clinic, University Aachen, RWTH, Pauwelsstrasse 30, D-52074 Aachen (Germany); Reske, Sven N. [Nuclear Medicine Clinic, University Ulm, Albert-Einstein-Allee 23, D-89081 Ulm (Germany)

    2011-10-15

    Introduction: Multiple myeloma (MM) is a plasma cell malignancy characterized by accumulation of malignant, terminally differentiated B cells in the bone marrow. Despite advances in therapy, MM remains an incurable disease. Novel therapeutic approaches are, therefore, urgently needed. Auger electron-emitting radiopharmaceuticals are attractive for targeted nano-irradiation therapy, given that DNA of malignant cells is selectively addressed. Here we evaluated the antimyeloma potential of the Auger electron-emitting thymidine analogue {sup 125}I-labeled 5-iodo-4'-thio-2'-deoxyuridine ([{sup 125}I]ITdU). Methods: Cellular uptake and DNA incorporation of [{sup 125}I]ITdU were determined in fluorodeoxyuridine-pretreated KMS12BM, U266, dexamethasone-sensitive MM1.S and -resistant MM1.R cell lines. The effect of stimulation with interleukin 6 (IL6) or insulin-like growth factor 1 (IGF1) on the intracellular incorporation of [{sup 125}I]ITdU was investigated in cytokine-sensitive MM1.S and MM1.R cell lines. Apoptotic cells were identified using Annexin V. Cleavage of caspase 3 and PARP was visualized by Western blot. DNA fragmentation was investigated using laddering assay. Therapeutic efficiency of [{sup 125}I]ITdU was proven by clonogenic assay. Results: [{sup 125}I]ITdU was shown to be efficiently incorporated into DNA of malignant cells, providing a promising mechanism for delivering highly toxic Auger radiation emitters into tumor DNA. [{sup 125}I]ITdU had a potent antimyeloma effect in cell lines representing distinct disease stages and, importantly, in cell lines sensitive or resistant to the conventional therapeutic agent, but was not toxic for normal plasma and bone marrow stromal cells. Furthermore, [{sup 125}I]ITdU abrogated the protective actions of IL6 and IGF1 on MM cells. [{sup 125}I]ITdU induced massive damage in the DNA of malignant plasma cells, which resulted in efficient inhibition of clonogenic growth. Conclusion: These studies may provide a

  3. NMR studies of the exocyclic 1,N6-ethenodeoxyadenosine adduct (εdA) opposite thymidine in a DNA duplex. Nonplanar alignment of εdA(anti) and dT(anti) at the lesion site

    International Nuclear Information System (INIS)

    Kouchakdjian, M.; Patel, D.J.; Eisenberg, M.; Yarema, K.; Basu, A.; Essigmann, J.

    1991-01-01

    Two-dimensional proton NMR studies are reported on the complementary d(C-A-T-G-T-G-T-A-C)·d(G-T-A-C-εA-C-A-T-G) nonanucleotide duplex (designated εdA·dT 9-mer duplex) containing 1,N 6 -ethenodeoxyadenosine (εdA), a carcinogen-DNA adduct, positioned opposite thymidine in the center of the helix. The authors NMR studies have focused on the conformation of the εdA·dT 9-mer duplex at neutral pH with emphasis on defining the alignment at the dT5·εdA14 lesion site. The through-space NOE distance connectivities establish that both dT5 and εdA14 adopt anti glycosidic torsion angles, are directed into the interior of the helix, and stack with flanking Watson-Crick dG4·dC15 and dG6·dC13 pairs. Furthermore, the d(G4-T5-G6)·d(C13-εA14-C15) trinucleotide segment centered about the dT5·εdA14 lesion site adopts a right-handed helical conformation in solution. Energy minimization computations were undertaken starting from six different alignments of dT5(anti) and εdA14(anti) at the lesion site and were guided by distance constraints defined by lower and upper bounds estimated from NOESY data sets on the εdA·dT 9-mer duplex. The NMR data are consistent with a nonplanar alignment of εdA14(anti) and dT5(anti) with dT5 displaced toward the flanking dG4·dC15 base pair within the d(G4-T5-G6)·d(C13-εA14-C15) segment of the εdA·dT 9-mer duplex

  4. Thymidylate synthase, dihydropyrimidine dehydrogenase, ERCC1, and thymidine phosphorylase gene expression in primary and metastatic gastrointestinal adenocarcinoma tissue in patients treated on a phase I trial of oxaliplatin and capecitabine

    International Nuclear Information System (INIS)

    Uchida, Kazumi; Danenberg, Peter V; Danenberg, Kathleen D; Grem, Jean L

    2008-01-01

    Over-expression of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) in tumor tissue is associated with insensitivity to 5-fluorouracil (5-FU). Over-expression of ERCC1 correlates with insensitivity to oxaliplatin (OX) therapy, while high thymidine phosphorylase (TP) levels predict for increased sensitivity to capecitabine (Xel). Biopsies of metastatic tumor were taken before OX (130 mg/m 2 day 1) given with Xel (1200–3000 mg/m 2 in two divided doses days 1–5 and 8–12) every 3-weeks. Micro-dissected metastatic and primary tumors were analyzed for relative gene expression by real-time quantitative polymerase chain reaction. The clinical protocol prospectively identified the molecular targets of interest that would be tested. Endpoints for the molecular analyses were correlation of median, first and third quartiles for relative gene expression of each target with response, time to treatment failure (TTF), and survival. Among 91 patients participating in this trial; 97% had colorectal cancer. The median number of prior chemotherapy regimens was 2, and most had prior 5-FU and irinotecan. In paired samples, median mRNA levels were significantly higher in metastatic versus primary tumor (-fold): TS (1.9), DPD (3.8), ERCC1 (2.1) and TP (1.6). A strong positive correlation was noted between DPD and TP mRNA levels in both primary (r = 0.693, p < 0.0005) and metastatic tissue (r = 0.697, p < 0.00001). There was an association between TS gene expression and responsive and stable disease: patients whose intratumoral TS mRNA levels were above the median value had significantly greater risk of early disease progression (43% vs 17%), but this did not translate into a significant difference in TTF. ERCC1 gene expression above the third quartile was associated with a shorter TTF (median 85 vs 162 days, p = 0.046). Patients whose TS mRNA levels in metastatic tumor tissue were below the median had a longer overall survival (median 417 vs 294 days, p = 0

  5. Some biological properties of human chorionic follicle stimulating hormone

    International Nuclear Information System (INIS)

    Tojo, Shimpei; Ashitaka, Yoshihiko; Maruo, Takeshi; Nishimoto, Hiroyuki

    1975-01-01

    The biological properties of human chorionic FSH (hCFSH) for rat ovaries were investigated. Highly purified hCFSH had similar response to the ovarian augmentation test as bovine FSH and significantly enhanced 3 H-thymidine uptake by granulosa cells and theca cells in the ovary of hypophysectomized rat. In contrast, highly purified hCG little responded to the ovarian augmentation test and had no effect on 3 H-thymidine uptake by the ovary. These results indicate that hCFSH may promote the follicular growth of ovary resulting from granulosa cell proliferation and its enlargement. In addition, freshly harvested porcine granulosa cells were employed in an in vitro system to investigate specific binding of hCFSH to ovarian receptor. Radioiodinated hCFSH ( 125 I-hCFSH) and hCG ( 125 I-hCG) were respectively incubated with cell suspensions. Binding of these hormone preparations was proportional to the cell number and increased with the time of incubation through 120 minutes. The binding ability of 125 I-hCFSH to the cells was greater than that of 125 I-hCG. Increasing concentrations of unlabeled hCFSH in the incubation mixture progressively inhibited the uptake of 125 I-hCFSH by granulosa cells. Unlabeled hCG was not able to compete with 125 I-hCFSH binding. The similar phenomenon to inhibit the binding of 125 I-hCG to the cells was also recognized in the presence of unlabeled hCG. These findings suggest that granulosa cell has at least two different types of receptor sites: one for hCFSH and the other for hCG. (auth.)

  6. Cytogenetic effects of tritium incorporated into DNA of human lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Beno, M [Inst. of Preventive and Clinical Medicine, 83301 Bratislava (Slovakia)

    1996-12-31

    In the reported in vitro experiments the numbers of chromosomal aberrations (CA) in correlation to the physical dose as assessed by determining the specific radioactivity of DNA have been followed in vitro human lymphocytes from adult donors. Lymphocytes from healthy adult donors of age from 20 to 59 of both sexes (24 males and 20 females) were isolated from blood by centrifugation. After washing the cells were irradiated from tritium incorporated during in vitro incubation in phytohemagglutinin containing medium with tritium labelled thymidine. Slides for standard CA counting have been done from every sample 48 hours after the begin cultivation. The CA were counted in at least 200 metaphases on each slide. Parallel samples of lymphocytes served for preparation smears for autoradiography to determine the labeling index. Other parallel samples were used for the determination of tritium concentration in DNA by the diphenylamine method, as well as determination of the specific radioactivity in lymphocyte DNA by scintillation counting. The dose absorbed in DNA was estimated using the conversion factor implicating that 37 kBq of tritium uniformly distributed per gram of tissue of unit density delivers a dose rate of 121.4 {sup m}i{sup G}y/hour. The contamination of cells by precursors of nucleic acids - like tritiated thymidine - causes an uneven distribution of doses in the cell population. A proportion of the population of cells remains unlabelled. The dose-response curve is flat showing signs of loss of heavily damaged cells and signs of repair of damage. Both these signs are based on the nature of biological processes which lead to internal contamination of cells and to expression of effects in terms of numbers of CA. (J.K.) 5 figs., 4 refs.

  7. Growth of human T lymphocyte colonies from whole blood: culture requirements and applications

    International Nuclear Information System (INIS)

    Knox, S.J.; Wilson, F.D.; Greenberg, B.R.; Shifrine, M.

    1982-01-01

    Growth of human lymphocyte colonies from whole blood following stimulation with PHA, Con A, or PPD is described. Individual colony cells were identified as T lymphocytes on the basis of surface marker and enzyme cytochemical characterizations. Colony formation increased as a power function over a wide range of cell concentrations above a critical minimal concentration. The whole blood culture system eliminates possible selective effects of lymphocyte colony techniques utilizing gradient-enriched lymphocyte fractions and more closely approximates the in vivo milieu. The whole blood colony method is more sensitive for the detection of low-level radiation effects on lymphocytes than widely used tests that measure 3 H-thymidine incorporation. In preliminary studies, researchers used the whole blood method to determine the relative radiosensitivity of lymphocytes from humans with various hematopoietic disorders, and observed abnormalities in mitogen responsiveness and colony formation in some of the patient groups. This method has wide application for studies in cellular and clinical immunology

  8. ALDH1A2 (RALDH2 genetic variation in human congenital heart disease

    Directory of Open Access Journals (Sweden)

    Mesquita Sonia MF

    2009-11-01

    Full Text Available Abstract Background Signaling by the vitamin A-derived morphogen retinoic acid (RA is required at multiple steps of cardiac development. Since conversion of retinaldehyde to RA by retinaldehyde dehydrogenase type II (ALDH1A2, a.k.a RALDH2 is critical for cardiac development, we screened patients with congenital heart disease (CHDs for genetic variation at the ALDH1A2 locus. Methods One-hundred and thirty-three CHD patients were screened for genetic variation at the ALDH1A2 locus through bi-directional sequencing. In addition, six SNPs (rs2704188, rs1441815, rs3784259, rs1530293, rs1899430 at the same locus were studied using a TDT-based association approach in 101 CHD trios. Observed mutations were modeled through molecular mechanics (MM simulations using the AMBER 9 package, Sander and Pmemd programs. Sequence conservation of observed mutations was evaluated through phylogenetic tree construction from ungapped alignments containing ALDH8 s, ALDH1Ls, ALDH1 s and ALDH2 s. Trees were generated by the Neighbor Joining method. Variations potentially affecting splicing mechanisms were cloned and functional assays were designed to test splicing alterations using the pSPL3 splicing assay. Results We describe in Tetralogy of Fallot (TOF the mutations Ala151Ser and Ile157Thr that change non-polar to polar residues at exon 4. Exon 4 encodes part of the highly-conserved tetramerization domain, a structural motif required for ALDH oligomerization. Molecular mechanics simulation studies of the two mutations indicate that they hinder tetramerization. We determined that the SNP rs16939660, previously associated with spina bifida and observed in patients with TOF, does not affect splicing. Moreover, association studies performed with classical models and with the transmission disequilibrium test (TDT design using single marker genotype, or haplotype information do not show differences between cases and controls. Conclusion In summary, our screen indicates that

  9. Inhibition of human polymorphonuclear leukocyte function by components of human colostrum and mature milk.

    Science.gov (United States)

    Pickering, L K; Cleary, T G; Caprioli, R M

    1983-04-01

    To compare the effect of human colostrum (days 1 to 3 postpartum) and mature milk (days 170 +/- 24 postpartum) on the function of polymorphonuclear leukocytes (PMNL), Ficoll-Hypaque-separated PMNL from the blood of 60 healthy volunteers were incubated with whole colostrum, colostral lipid, and colostral aqueous phase from 30 mothers, or with mature whole milk and its separated components from 30 mothers, and tested for resting and zymosan-stimulated oxidative metabolism, functional activity, and the presence of Fc receptors. Stimulated oxygen consumption, quantitative nitroblue tetrazolium dye reduction, [1-(14)C]glucose utilization, and Fc receptors were significantly (P cells or cells exposed to the aqueous phase of colostrum. In contrast, PMNL exposed to whole mature milk or to its lipid or aqueous phase caused no significant decrease in any of these parameters when compared to nonexposed cells. In assays of phagocytosis, colostral PMNL or blood PMNL exposed to colostral lipid had a significant (P < 0.001) decrease in their ability to ingest [methyl-(3)H]thymidine-labeled Staphylococcus aureus when compared to non-lipid-exposed PMNL. Blood PMNL exposed to lipid from mature milk had no decrease in ability to ingest S. aureus. Analysis of total lipid and total and individual fatty acid content revealed a uniform increase in all components in mature milk when compared to colostrum. Lipid or lipid-soluble material present in human colostrum but not mature milk causes inhibition of phagocytosis and respiratory burst-related activities of PMNL.

  10. The gating mechanism of the human aquaporin 5 revealed by molecular dynamics simulations.

    Directory of Open Access Journals (Sweden)

    Lorant Janosi

    Full Text Available Aquaporins are protein channels located across the cell membrane with the role of conducting water or other small sugar alcohol molecules (aquaglyceroporins. The high-resolution X-ray structure of the human aquaporin 5 (HsAQP5 shows that HsAQP5, as all the other known aquaporins, exhibits tetrameric structure. By means of molecular dynamics simulations we analyzed the role of spontaneous fluctuations on the structural behavior of the human AQP5. We found that different conformations within the tetramer lead to a distribution of monomeric channel structures, which can be characterized as open or closed. The switch between the two states of a channel is a tap-like mechanism at the cytoplasmic end which regulates the water passage through the pore. The channel is closed by a translation of the His67 residue inside the pore. Moreover, water permeation rate calculations revealed that the selectivity filter, located at the other end of the channel, regulates the flow rate of water molecules when the channel is open, by locally modifying the orientation of His173. Furthermore, the calculated permeation rates of a fully open channel are in good agreement with the reported experimental value.

  11. Hydrogen-bond network and pH sensitivity in human transthyretin

    Energy Technology Data Exchange (ETDEWEB)

    Yokoyama, Takeshi, E-mail: tyokoya3@pha.u-toyama.ac.jp; Mizuguchi, Mineyuki; Nabeshima, Yuko [University of Toyama, 2630 Sugitani, Toyama 930-0914 (Japan); Kusaka, Katsuhiro; Yamada, Taro [Ibaraki University, 162-1 Shirakata, Tokai, Ibaraki 319-1106 (Japan); Hosoya, Takaaki [Ibaraki University, 162-1 Shirakata, Tokai, Ibaraki 319-1106 (Japan); Ibaraki University, 4-12-1 Naka-Narusawa, Hitachi, Ibaraki 316-8511 (Japan); Ohhara, Takashi [Comprehensive Research Organization for Science and Society, 162-1 Shirakata, Tokai, Ibaraki 319-1106 (Japan); Kurihara, Kazuo [Japan Atomic Energy Agency, 2-4 Shirakata, Tokai, Ibaraki 319-1195 (Japan); Tanaka, Ichiro [Ibaraki University, 162-1 Shirakata, Tokai, Ibaraki 319-1106 (Japan); Ibaraki University, 4-12-1 Naka-Narusawa, Hitachi, Ibaraki 316-8511 (Japan); Niimura, Nobuo [Ibaraki University, 162-1 Shirakata, Tokai, Ibaraki 319-1106 (Japan)

    2013-11-01

    The neutron crystal structure of human transthyretin is presented. Transthyretin (TTR) is a tetrameric protein. TTR misfolding and aggregation are associated with human amyloid diseases. Dissociation of the TTR tetramer is believed to be the rate-limiting step in the amyloid fibril formation cascade. Low pH is known to promote dissociation into monomer and the formation of amyloid fibrils. In order to reveal the molecular mechanisms underlying pH sensitivity and structural stabilities of TTR, neutron diffraction studies were conducted using the IBARAKI Biological Crystal Diffractometer with the time-of-flight method. Crystals for the neutron diffraction experiments were grown up to 2.5 mm{sup 3} for four months. The neutron crystal structure solved at 2.0 Å revealed the protonation states of His88 and the detailed hydrogen-bond network depending on the protonation states of His88. This hydrogen-bond network is involved in monomer–monomer and dimer–dimer interactions, suggesting that the double protonation of His88 by acidification breaks the hydrogen-bond network and causes the destabilization of the TTR tetramer. Structural comparison with the X-ray crystal structure at acidic pH identified the three amino acid residues responsible for the pH sensitivity of TTR. Our neutron model provides insights into the molecular stability related to amyloidosis.

  12. Interactions of human hemoglobin with charged ligand-functionalized iron oxide nanoparticles and effect of counterions

    Energy Technology Data Exchange (ETDEWEB)

    Ghosh, Goutam, E-mail: ghoshg@yahoo.com [UGC-DAE Consortium for Scientific Research, Mumbai Centre (India); Panicker, Lata [Bhabha Atomic Research Centre, Solid State Physics Division (India)

    2014-12-15

    Human hemoglobin is an important metalloprotein. It has tetrameric structure with each subunit containing a ‘heme’ group which carries oxygen and carbon dioxide in blood. In this work, we have investigated the interactions of human hemoglobin (Hb) with charged ligand-functionalized iron oxide nanoparticles and the effect of counterions, in aqueous medium. Several techniques like DLS and ζ-potential measurements, UV–vis, fluorescence, and CD spectroscopy have been used to characterize the interaction. The nanoparticle size was measured to be in the range of 20–30 nm. Our results indicated the binding of Hb with both positively as well as negatively charged ligand-functionalized iron oxide nanoparticles in neutral aqueous medium which was driven by the electrostatic and the hydrophobic interactions. The electrostatic binding interaction was not seen in phosphate buffer at pH 7.4. We have also observed that the ‘heme’ groups of Hb remained unaffected on binding with charged nanoparticles, suggesting the utility of the charged ligand-functionalized nanoparticles in biomedical applications.

  13. Human prostatic cancer cells, PC3, elaborate mitogenic activity which selectively stimulates human bone cells

    International Nuclear Information System (INIS)

    Perkel, V.S.; Mohan, S.; Herring, S.J.; Baylink, D.J.; Linkhart, T.A.

    1990-01-01

    Prostatic cancer typically produces osteoblastic metastases which are not attended by marrow fibrosis. In the present study we sought to test the hypothesis that prostatic cancer cells produce factor(s) which act selectively on human osteoblasts. Such a paracrine mechanism would explain the observed increase in osteoblasts, unaccompanied by an increase in marrow fibroblasts. To test this hypothesis we investigated the mitogenic activity released by the human prostatic tumor cell line, PC3. PC3 cells have been reported previously to produce mitogenic activity for cells that was relatively specific for rat osteoblasts compared to rat fibroblasts. However, the effects of this activity on human cells has not been examined previously. PC3-conditioned medium (CM) (5-50 micrograms CM protein/ml) stimulated human osteoblast proliferation by 200-950% yet did not stimulate human fibroblast proliferation ([3H]thymidine incorporation). PC3 CM also increased cell numbers in human osteoblast but not fibroblast cell cultures. To determine whether the osteoblast-specific mitogenic activity could be attributed to known bone growth factors, specific assays for these growth factors were performed. PC3 CM contained 10 pg insulin-like growth factor (IGF) I, less than 2 pg IGF II, 54 pg basic fibroblast growth factor, and 16 pg transforming growth factor beta/microgram CM protein. None of these growth factors alone or in combination could account for the observed osteoblast-specific PC3 cell-derived mitogenic activity. Furthermore, when 5 micrograms/ml PC3 CM was tested in combination with maximally effective concentrations of either basic fibroblast growth factor, IGF I, IGF II, or transforming growth factor beta, it produced an additive effect suggesting that PC3 CM stimulates osteoblast proliferation by a mechanism independent of these bone mitogens

  14. Assay for Epstein--Barr virus based on stimulation of DNA systhesis in mixed leukocytes from human umbilical cord blood

    International Nuclear Information System (INIS)

    Robinson, J.; Miller, G.

    1975-01-01

    Relationships between the rate of DNA synthesis in cultured human umbilical cord leukocytes and the multiplicity of added Epstein-Barr virus (EBV) were studied. At low multiplicities of approximately 0.1 transforming units/cell (approximately 10 physical particles/cell), inoculated cultures demonstrated increased rates of DNA synthesis, by comparison to uninoculated cultures, 3 days after inoculation. Stimulation of DNA synthesis was evident at progressively longer intervals after inoculations of 10-fold dilutions of virus. The rate of DNA synthesis, determined by short [ 3 H]thymidine pulses, reflected as small as twofold changes in multiplicity and thus can serve as a quantitative assay for the virus. Changes in the rate of DNA synthesis were evident before increases in cell number or alteration in morphology. Stimulation of DNA synthesis in umbilical cord leukocytes was inhibited by treatment of EBV with antibody and also in graded fashion, by progressive doses of uv irradiation to the virus. Induction of DNA synthesis by EBV was serum dependent. Estimates of the number of cells transformed were obtained by extrapolation from a standard curve relating known numbers of transformed cells to [ 3 H]thymidine incorporation and also by cloning cells after exposure to virus. At the low multiplicities of infection used in these experiments approximately 0.04 to 0.002 of the total cellular population was transformed. The high efficiency of cell transformation by EBV by comparison to other DNA tumor viruses is emphasized

  15. Effects of synthetic and naturally occurring flavonoids on mitogen-induced proliferation of human peripheral-blood lymphocytes

    International Nuclear Information System (INIS)

    Hirano, Toshihiko; Oka, Kitaro; Kawashima, Etsuko; Akiba, Mitsuo

    1989-01-01

    Examination was made of the effects of 17 synthetic and naturally occurring flavonoids on human lymphocyte proliferation in the presence of concanavalin A as a mitogen. Twelve of the flavonoids examined were mono-hydroxy of methoxy derivatives. The mitogen-induced response of lymphocytes was evaluated from the extent of the incorporation of [ 3 H]thymidine into cells in vitro. All the compounds showed inhibitory effects; 4.5-77.7% of [ 3 H] thymidine incorporation was blocked by an 1.0 μg/ml concentration. The viability of lymphocytes before and after treatment, as assessed by a dye exclusion test, indicated no change, and thus the flavonoids may inhibit DNA synthesis. The flavonoids possessing 5-hydroxyl, 5-methoxyl and 6-methoxyl groups, and those with cyclohexyl instead of phenyl substituent (i.e. 2-cyclohexyl-benzopyran-4-one), showed the greatest inhibition. The inhibitory effect of any one of them was less than one half that of prednisolone, but essentially the same or somewhat exceeding that of bredinine of azathioprine. It would thus appear that the well-known anti-inflammatory effects of flavonoids may possibly arise in part from the inhibition of the proliferative response of lymphocytes

  16. The effect of culture density and proliferation rate on the expression of ouabain-sensitive Na/K ATPase pumps in cultured human retinal pigment epithelium

    International Nuclear Information System (INIS)

    Burke, J.M.; Jaffe, G.J.; Brzeski, C.M.

    1991-01-01

    The number and activity of ouabain-sensitive Na/K ATPase pumps expressed by many cell types in vitro, including human retinal pigment epithelial cells (RPE), have been shown to decline with increasing culture density. Cell proliferation also declined as cultures became dense so it was unclear if pump number was modulated by cell proliferation or culture confluency. By exposing RPE cultures to various feeding regimens, using culture medium containing or lacking serum, it was possible to produce RPE cultures with a range of culture densities and growth rates. These were analyzed for proliferative activity by quantifying [ 3 H]thymidine incorporation and for Na/K ATPase pump number by measuring specific [ 3 H]ouabain binding. The results suggest that pump number is modulated by culture density and, further, that the density-dependent regulation of pump number requires serum. Although density-dependent modulation of culture growth is also serum requiring, cell proliferation and pump number did not appear to be related; cultures of similar density which differed significantly in growth rate had similar numbers of pumps. The view that elevated numbers of pumps were not necessarily found in proliferating cells was further supported by qualitative examination of radioautographs of cells dually labeled with [ 3 H]thymidine and [ 3 H]ouabain. Cycling cells which had [ 3 H]thymidine-labeled nuclei did not have notably higher labeling with [ 3 H]ouabain. However, [ 3 H]ouabain labeling, as an indicator of pump site number and distribution, did vary among cells in an RPE population and also within individual cells. This latter observation suggests that unpolarized RPE cells in sparse cultures may have regionally different requirements for ionic regulation

  17. Inhibition of the ERK phosphorylation plays a role in terbinafine-induced p21 up-regulation and DNA synthesis inhibition in human vascular endothelial cells

    International Nuclear Information System (INIS)

    Ho, P.-Y.; Hsu, S.-P.; Liang, Y.-C.; Kuo, M.-L.; Ho, Y.-S.; Lee, W.-S.

    2008-01-01

    Previously, we showed that terbinafine (TB) induces cell-cycle arrest in cultured human umbilical vein endothelial cells (HUVEC) through an up-regulation of the p21 protein. The aim of this study is to delineate the molecular mechanisms underlying TB-induced increase of p21 protein. RT-PCR analysis demonstrated that the mRNA levels of p21 and p53 were increased in the TB-treated HUVEC. The p21 promoter activity was also increased by TB treatment. Transfection of HUVEC with p53 dominant negative (DN) abolished the TB-induced increases of p21 promoter activity and protein level, suggesting that the TB-induced increase of p21 is p53-dependent. Western blot analysis demonstrated that TB decreased the levels of phosphorylated extracellular signal-regulated kinase (ERK). Over-expression of mitogen-activated protein kinase (MEK)-1, the immediate upstream activator kinase of ERK, abolished the TB-induced increases of p21 and p53 protein and decrease of thymidine incorporation. The ERK inhibitor (PD98059) enhanced the TB-induced inhibition of thymidine incorporation into HUVEC. Taken together, these data suggest that the decrease of ERK activity plays a role in the TB-induced up-regulation of p21 in HUVEC. On the other hand, pretreatment of the cells with geranylgeraniol (GGOH), farnesol (FOH), or Ras inhibitor peptide did not affect the TB-induced decrease of thymidine incorporation. Taken together, our results suggest that TB might cause a decrease of MEK, which in turn up-regulates p53 through the inhibition of ERK phosphorylation, and finally causes an increase of p21 expression and cell-cycle arrest

  18. The effect of culture density and proliferation rate on the expression of ouabain-sensitive Na/K ATPase pumps in cultured human retinal pigment epithelium

    Energy Technology Data Exchange (ETDEWEB)

    Burke, J.M.; Jaffe, G.J.; Brzeski, C.M. (Medical College of Wisconsin, Milwaukee (USA))

    1991-06-01

    The number and activity of ouabain-sensitive Na/K ATPase pumps expressed by many cell types in vitro, including human retinal pigment epithelial cells (RPE), have been shown to decline with increasing culture density. Cell proliferation also declined as cultures became dense so it was unclear if pump number was modulated by cell proliferation or culture confluency. By exposing RPE cultures to various feeding regimens, using culture medium containing or lacking serum, it was possible to produce RPE cultures with a range of culture densities and growth rates. These were analyzed for proliferative activity by quantifying ({sup 3}H)thymidine incorporation and for Na/K ATPase pump number by measuring specific ({sup 3}H)ouabain binding. The results suggest that pump number is modulated by culture density and, further, that the density-dependent regulation of pump number requires serum. Although density-dependent modulation of culture growth is also serum requiring, cell proliferation and pump number did not appear to be related; cultures of similar density which differed significantly in growth rate had similar numbers of pumps. The view that elevated numbers of pumps were not necessarily found in proliferating cells was further supported by qualitative examination of radioautographs of cells dually labeled with ({sup 3}H)thymidine and ({sup 3}H)ouabain. Cycling cells which had ({sup 3}H)thymidine-labeled nuclei did not have notably higher labeling with ({sup 3}H)ouabain. However, ({sup 3}H)ouabain labeling, as an indicator of pump site number and distribution, did vary among cells in an RPE population and also within individual cells. This latter observation suggests that unpolarized RPE cells in sparse cultures may have regionally different requirements for ionic regulation.

  19. Effect of irradiation on human T-cell proliferation: low dose irradiation stimulates mitogen-induced proliferation and function of the suppressor/cytotoxic T-cell subset

    International Nuclear Information System (INIS)

    Gualde, N.; Goodwin, J.S.

    1984-01-01

    Unfractionated human T cells exposed to 10-50 rad of X irradiation incorporated less [ 3 H]thymidine than nonirradiated T cells when subsequently cultured with PHA or Con A. The cytotoxic/suppressor T-cell subset, isolated as either OKT8(+) or OKT4(-) cells, demonstrated significantly enhanced [ 3 H]thymidine incorporation in PHA- or Con A-stimulated cultures after exposure to 10-50 rad, compared to unirradiated cells, while the proliferation of the OKT4(+) helper/inducer subset was inhibited by low dose irradiation. It has been previously reported that approximately 30% of the cytotoxic/suppressor subset also stains with OKM1. When the cytotoxic/suppressor subset was further subdivided into OKT4(-), OKM1(+), and OKT4(-), OKM1(-) cells, proliferation of the OKT4(-), OKM1(+) population was inhibited by exposure to 25 rad while proliferation of the OKT4(-), OKM1(-) population was stimulated. The increase in proliferation of the cytotoxic/suppressor T-cell subset after low dose irradiation is paralleled by an increase in suppressor activity of these cells. T cells exposed to 25 rad and then cultured with Con A for 48 hr caused greater inhibition of IgG production when added to fresh autologous lymphocytes stimulated by pokeweed mitogen than did unirradiated cells. Thus, low dose irradiation enhances both the proliferation and function of the human suppressor T-cell subset

  20. 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

  1. Stability of RNA and DNA in Bone Marrow Cells, Demonstrated with Tritiated Cytidine and Thymidine; Emploi de la Cytidine et de la Thymidine Tritiees pour Demontrer la Stabilite de l'ARN et l'ADN dans les Cellules de la Moelle Osseuse; 0421 0442 0430 0414 ; Estudio de la Estabilidad de los Acidos Ribonucleico y Desoxirribonucleico de las Celulas de la Medula Osea, Utilizando Citidina y Timidina Tritiadas

    Energy Technology Data Exchange (ETDEWEB)

    Bond, V. P.; Feinendegen, L. E.; Cronkite, E. P. [Medical Research Centre, Brookhaven National Laboratory, Upton, Long Island, NY (United States)

    1962-02-15

    DNA and RNA metabolism was studied using tritiated thymidine (H{sup 3}Th), a specific precursor for DNA, and tritiated cytidine (H{sup 3}C), a common precursor for both RNA and DNA. With H{sup 3}C, differential incorporation into RNA, DNA or the soluble pool was determined autoradiographically in the single cell, and/or chemically for cell populations by means of differential extraction using appropriate treatment with perchloric acid. Initial turnover studies in the Hela cell with H{sup 3}C indicated the precursor role of nuclear RNA for cytoplasmic RNA. Conservation and distribution of label in the RNA fraction was consistent with major macromolecular RNA stability, and continued incorporation of label into the DNA fraction was consistent with the presence of a late precursor for DNA. Similar findings were observed in the immature bone marrow cells of the rat studied over a period of several days after intravenous administration of H{sup 3}C. The amount of tritium activity in the acid-soluble' RNA and DNA fractions was followed chemically and/or autoradiographically. The three curves were found to be parallel from the first day after injection and parallel to curves for tritium label in DNA following H{sup 3}Th administration. The expected rate of fall off in label, calculated from kinetics of the rat bone marrow cell populations studied separately by H{sup 3}Th and autoradiography, assuming no turnover of RNA or DNA and loss of label only by loss of marrow cells by division and maturation, was in agreement with the slopes obtained. The results indicate that, once synthesized, soluble and macromolecular RNA is retained by the bone marrow cell in a manner similar to DNA. Newly formed RNA and DNA are diluted in the cells only through cell division. (author) [French] Les auteurs ont etudie le metabolisme de l'ADN et de l'ARN a l'aide de thymidine tritiee (Th-{sup 3}H), precurseur specifique de l'ADN, et de cytidine tritiee (Cy-{sup 3}H), precurseur a la fois de l

  2. Partial digestion with restriction enzymes of ultraviolet-irradiated human genomic DNA: a method for identifying restriction site polymorphisms

    International Nuclear Information System (INIS)

    Nobile, C.; Romeo, G.

    1988-01-01

    A method for partial digestion of total human DNA with restriction enzymes has been developed on the basis of a principle already utilized by P.A. Whittaker and E. Southern for the analysis of phage lambda recombinants. Total human DNA irradiated with uv light of 254 nm is partially digested by restriction enzymes that recognize sequences containing adjacent thymidines because of TT dimer formation. The products resulting from partial digestion of specific genomic regions are detected in Southern blots by genomic-unique DNA probes with high reproducibility. This procedure is rapid and simple to perform because the same conditions of uv irradiation are used for different enzymes and probes. It is shown that restriction site polymorphisms occurring in the genomic regions analyzed are recognized by the allelic partial digest patterns they determine

  3. A UV-sensitive human clonal cell line, RSa, which has low repair activity

    International Nuclear Information System (INIS)

    Suzuki, N.; Fuse, A.

    1981-01-01

    The repair activity of a human transformed cell line, RSa, which was found to be highly sensitive to the lethal effects of 254 mm far-ultraviolet radiation, was compared with that of HeLa cells by evaluating the range of UV-induced incorporation of [methyl- 3 H]thymidine ([ 3 H]dThd) or 5-[6- 3 H]bromodeoxyuridine ([ 3 H]BrdUrd) into deoxyribonucleic acid. Direct scintillation counting was used for measuring the extent of unscheduled DNA synthesis (UDS) in UV-irradiated cells, which were treated with hydroxyurea or with arginine deprivation. More quantitative measurements were made by using the density labeling and equilibrium centrifugation method for assaying repair replication. All the amounts of UDS and repair replication in RSa cells were markedly below those in HeLa cells. The possible relationships of the low repair activity to abnormally high UV sensitivity in RSa cells are discussed. (orig.)

  4. Cell-free assay measuring repair DNA synthesis in human fibroblasts

    International Nuclear Information System (INIS)

    Ciarrocchi, G.; Linn, S.

    1978-01-01

    Osmotic disruption of confluent cultured human fibroblasts that have been irradiated or exposed to chemical carcinogens allows the specific measurement of repair DNA synthesis using dTTP as a precursor. Fibroblasts similarly prepared from various xeroderma pigmentosum cell lines show the deficiencies of uv-induced DNA synthesis predicted from in vivo studies, while giving normal responses to methylmethanesulfonate. A pyrimidine-dimer-specific enzyme, T4 endonuclease V, stimulated the rate of uv-induced repair synthesis with normal and xeroderma pigmentosum cell lines. This system should prove useful for identifying agents that induce DNA repair, and cells that respond abnormally to such induction. It should also be applicable to an in vitro complementation assay with repair-defective cells and proteins obtained from repair-proficient cells. Finally, by using actively growing fibroblasts and thymidine in the system, DNA replication can be measured and studied in vitro

  5. High resolution autoradiographic studies of RNA, protein and DNA synthesis during human eosinophil granulocytopoiesis

    International Nuclear Information System (INIS)

    Wickramasinghe, S.N