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Sample records for 3h thymidine incorporation

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

    SciTech Connect

    Winding, A. )

    1992-08-01

    The incorporation of [[sup 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 [[sup 3]H] thymidine during growth. It is concluded that the [[sup 3]H] thymidine incorporation method underestimates bacterial growth in anaerobic environments.

  2. Calculation of cell production from ( sup 3 H)Thymidine incorporation with freshwater bacteria

    SciTech Connect

    Smits, J.D. ); Riemann, B. )

    1988-09-01

    The conversion factor for the calculation of bacterial production from rates of ({sup 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 {times} 10{sup 18} cells mol{sup {minus}1} of thymidine incorporated into the trichloroacetic acid (TCA) precipitate, when the generation time exceeded 20 h. At generation times of <20 h, the average conversion factor was 11.8 {times} 10{sup 18} cells mol{sup {minus}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 ({sup 3}H)thymidine into DNA is probably limited by uptake during period with generation times of <20 h and that freshwater bacterioplankton cell production sometimes is underestimated when a conversion factor of 2.15 {times} 10{sup 18} cells mol{sup {minus}1} of thymidine incorporated is used.

  3. Validation of ( sup 3 H)thymidine incorporation and its application to detecting natural transformation in the marine environment

    SciTech Connect

    Jeffrey, W.H.

    1989-01-01

    The ({sup 3}H)thymidine incorporation method to estimate bacterial DNA synthesis and heterotrophic production was examined by investigating the four major factors and assumptions associated with the technique. When compared to fluorometrically determined rates of DNA synthesis, ({sup 3}H)thymidine incorporation consistently underestimated DNA synthesis by 6 to 8-fold, indicating the inability of ({sup 3}H)thymidine incorporation and isotope dilution assays to accurately determine the amount of thymine bases incorporated into DNA. Non-specific labeling of macromolecules other than DNA was ubiquitous and the percentage of radioactivity incorporated into DNA was inversely related to total rates thymidine incorporation but independent of any other parameter examined. The use of specific inhibitors and a comparison of (methyl-{sup 3}H)thymidine with (6-{sup 3}H)thymidine indicated that non-specific labelling was not the result of a demethylation reaction but the result of ({sup 3}H)thymine catabolism. Four of the 41 marine bacterial isolates examined were incapable of incorporating thymidine into DNA and lacked thymidine transport and thymidine kinase activity. Transformation of an E. coli tdk into one of these organisms resulted in high levels of thymidine kinase activity but no capacity to incorporate or transport thymidine by this organism.

  4. A study on the lack of (Methyl- sup 3 H) thymidine uptake and incorporation by chemolithotrophic bacteria

    SciTech Connect

    Johnstone, B.H.; Jones, R.D. )

    1989-01-01

    Five chemolithotrophic bacteria were tested for their ability to incorporate (methyl-{sup 3}H) thymidine. None of the bacteria incorporated the label, even after incubation for 24 hours. The inability of these bacteria to incorporate thymidine appears to be due to an absence of transport mechanisms for exogenous nucleosides. As a result of these findings, it is concluded that activities deduced from labeled thymidine incorporation measurements probably do not include the activity of chemolithotrophic bacteria.

  5. Determining (/sup 3/H)thymidine incorporation into bacterioplankton DNA: improvement of the method by DNase treatment

    SciTech Connect

    Servais, P.; Martinez, J.; Vives-Rego, J.

    1987-08-01

    Determination of (/sup 3/H)thymidine incorporation into bacterial DNA versus other macromolecules is usually achieved by NaOH and hot trichloroacetic acid hydrolysis. This procedure was found not to be specific enough. An alternative method founded on DNase treatment is proposed. Under the new method, the fraction of thymidine incorporated into DNA ranged from 10 to 83%.

  6. Angiotensin II stimulates /sup 3/H-leucine and /sup 3/H-thymidine incorporation in cultured vascular smooth muscle cells

    SciTech Connect

    Dwyer, S.D.; Smith, J.B.

    1987-05-01

    Angiotensin II (ANG) stimulates the hydrolysis of phosphatidylinositol bisphosphate with the consequent formation of inositol trisphosphate and diacylglycerol in cultured smooth muscle cells derived from rat aorta. They have observed the effects of ANG on protein and DNA synthesis by measuring the incorporation of /sup 3/H-leucine and /sup 3/H-thymidine, respectively, into acid-precipitable material. Aortic muscle cells were grown to confluence in medium containing 10% fetal bovine serum (FBS) and incubated for 24 hours in serum-free medium to arrest growth. Then fresh serum-free medium was added with the following additions: ANG (100 nM), insulin (2 ..mu..g/ml), or 10% FBS. After an additional 24 hours the cells were pulse labeled for 30 min with either /sup 3/H-leucine or /sup 3/H-thymidine. FBS increased /sup 3/H-leucine incorporation by -2.5 fold and /sup 3/H-thymidine incorporation by 7-10 fold. ANG or insulin increased /sup 3/H-leucine incorporation by 40-50%, and the combination of ANG and insulin was nearly as effective as 10% FBS. ANG stimulated /sup 3/H-thymidine incorporation by -2.5 fold. Insulin, which was less effective than ANG, increased /sup 3/H-thymidine incorporation by about 50%. ANG and insulin added together synergistically increased /sup 3/H-thymidine incorporation by 5-6 fold. An ANG antagonist, Sarl,leu8-ANG, at 2 ..mu..M markedly decreased /sup 3/H-thymidine incorporation in the presence of ANG and insulin.

  7. Effects of toxic substances on natural bacterial assemblages determined by means of ( sup 3 H)thymidine incorporation

    SciTech Connect

    Riemann, B., Lindgaard-Jorgensen, P. )

    1990-01-01

    The effects of 3,5-dichlorophenol, 2,4-dinitrophenol, and potassium dichromate on natural bacterial assemblages were examined by means of ({sup 3}H)thymidine incorporation into trichloroacetic acid-insoluble material. Results from a large number of coastal marine and freshwater samples suggest the following. (i) The effects of the three toxicants included reductions in the bacterial cell number as well as changes in rates of ({sup 3}H)thymidine incorporation and in ({sup 3}H)thymidine incorporation per cell. The concentrations that inhibited ({sup 3}H)thymidine incorporation by 50% ranged from 3 to 11 mg liter{sup {minus}1} for 3,5-dichlorophenol, 5 to 10 mg liter{sup {minus}1} for 2,4-dinitrophenol, and 21 to 123 mg liter{sup {minus}1} for potassium dichromate, with a tendency to higher values in bacterial assemblages from more eutrophic environments. (ii) The effects of 3,5-dichlorophenol and potassium dichromate determined by ({sup 3}H) leucine incorporation into bacterial protein were similar or larger than those obtained from ({sup 3}H) thymidine incorporation. (III) Two to four hours of exposure to the toxicants was necessary before stable maximum effects were found in ({sup 3}H)thymidine incorporation. (IV) Storage of natural environmental samples should be avoided, since tests with water stored for 1 to 3 days sometimes produced results different from results obtained from in situ tests. (V) The effects of 3,5-dichlorophenol, 2,4-dinitrophenol, and potassium dichromate on natural bacterial assemblages were relatively constant during periods with different growth rates in the assemblages, during various periods of the year, and between samples from freshwater and marine localities.

  8. Fluoride stimulates ( sup 3 H)thymidine incorporation and alkaline phosphatase production by human osteoblasts

    SciTech Connect

    Khokher, M.A.; Dandona, P. )

    1990-11-01

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

  9. Autoradiographic studies of (methyl-/sup 3/H)thymidine incorporation in a cyanobacterium (Microcystis wesenbergii)-bacterium association and in selected algae and bacteria

    SciTech Connect

    Bern, L.

    1985-01-01

    The present investigation showed by means of autoradiography that the cyanobacterium Microcystis wesenbergii did not incorporate (/sup 3/H)thymidine at nanomolar concentrations, whereas its associated heterotrophic bacteria appearing in the gelatinous cover of the cyanobacterium became labeled. Several other tested cyanobacteria and algae did not incorporate (/sup 3/H)thymidine.

  10. Inhibition of macrophage DNA synthesis by immunomodulators. II. Characterization of the suppression by muramyl dipeptide or lipopolysaccharide (/sup 3/H)thymidine incorporation into macrophages

    SciTech Connect

    Nagao, S.; Ikegami, S.; Tanaka, A.

    1984-12-01

    Guinea pig peritoneal exudate macrophages actively incorporated (/sup 3/H)thymidine into trichloroacetic acid-insoluble fraction in vitro. The incorporation of (/sup 3/H)thymidine was almost completely inhibited by aphidicolin, an inhibitor of DNA polymerase alpha and an autoradiograph showed heavy labeling in nuclei of 15% of macrophage populations. These results indicate that the observed thymidine incorporation was due to a nuclear DNA synthesis. The (/sup 3/H)thymidine incorporation was markedly suppressed when macrophages were activated by immunoadjuvants such as muramyl dipeptide (MDP) or bacterial lipopolysaccharide (LPS). The suppression of (/sup 3/H)thymidine incorporation by MDP was neither due to the decrease in thymidine transport through the cell membrane, nor due to dilution by newly synthesized cold thymidine. An autoradiograph revealed that MDP markedly decreased the number of macrophages the nuclei of which were labeled by (/sup 3/H)thymidine. These results suggest that the suppression of (/sup 3/H)thymidine incorporation by the immunoadjuvants reflects a true inhibition of DNA synthesis. The inhibition of DNA synthesis by MDP was also observed in vivo. Further, it was strongly suggested that the inhibition was not caused by some mediators, such as prostaglandin E2, released from macrophages stimulated by the immunoadjuvants but caused by a direct triggering of the adjuvants at least at the early stage of activation. Cyclic AMP appears to be involved in the inhibitory reaction.

  11. Different effects of BrdU and 3H-Thymidine incorporation into DNA on cell proliferation, position and fate

    PubMed Central

    Duque, Alvaro; Rakic, Pasko

    2011-01-01

    As markers of DNA synthesis, 3H-Thymidine (3H-dT), and the later developed analog, Bromodeoxyuridine (BrdU) have revolutionized our ability to identify dividing cells and follow their fate in various tissues, including the nervous system. However, the effect of the incorporation of these molecules into DNA on cell proliferation, migration, differentiation and function is not fully understood. Here, we compare the number and distribution of labeled cells in the cerebral cortex of postnatal macaque monkeys exposed to either 3H-dT or BrdU as embryos. The large size and prolonged brain development in this species allows higher resolution of cellular events and more accurate discrimination between the two methods. Our analysis revealed substantial differences in the number and distribution of labeled cells. The data indicate that random incorporation of the thymidine analogue BrdU into the genes of dividing cells makes the fate of postmitotic neurons more prone to unpredictable errors than the incorporation of the more natural DNA constituent nucleotide 3H-dT. These findings have implications for the interpretation of results obtained by BrdU as an index of the number of neurons produced, their migration, placement, subsequent connectivity, function and survival. PMID:22016554

  12. Quantifying 3H-thymidine incorporation rates by a phylogenetically defined group of marine planktonic bacteria (Bacteriodetes phylum).

    PubMed

    van Mooy, Benjamin A S; Devol, Allan H; Keil, Richard G

    2004-10-01

    The rate of [(3)H-methyl] thymidine ((3)H-TdR) incorporation into DNA has been applied extensively to measure cell production by bacterial communities in aquatic environments. Here we describe a method to quantify (3)H-TdR incorporation by specific, phylogenetically defined members of the bacterial community. The method involves selectively capturing DNA from targeted groups of bacteria and then quantifying its (3)H radioactivity. The method was applied to measure (3)H-TdR incorporation by the members of the phylum Bacteriodetes whose members, which include the Cytophaga-Flavobacter cluster, are ubiquitous in coastal waters. (3)H-labelled DNA from Bacteriodetes was selectively biotinylated in PCR-like reactions that contained a Bacteriodetes-specific 16S rRNA gene primer, thermostable DNA polymerase and biotinylated dUTP. The biotinylated DNA was then captured on streptavidin-coated beads and its (3)H radioactivity determined by scintillation counting. We have termed this method 'selective nucleic acid polymerase-biotinylation and capture' or 'SNAP-BAC'. Internal (33)P-labelled DNA standards were used to quantify the recovery of (3)H-labelled DNA from the SNAP-BAC reactions. The method was verified by successfully targeting Bacteriodetes in simple laboratory mixtures of (3)H-labelled DNA extracted from pure cultures of Bacteriodetes and gamma-proteobacteria. Field application of this method in Puget Sound and off the Washington coast determined that Bacteriodetes were responsible for 56 +/- 17% and 32 +/- 5% of community (3)H-TdR incorporation (1.3 +/- 0.3 and 9.9 +/- 1.7 pmol l(-1) h(-1)) at these two locations. PMID:15344931

  13. Inhibitory effect of somatostatin on the basal and TSH-stimulated /sup 3/H-thymidine incorporation into rat thyroid lobes incubated in vitro

    SciTech Connect

    Zerek-Melen, G.; Lewinski, A.

    1988-07-15

    The effects of somatostatin on the spontaneous and TSH--stimulated incorporation of tritiated thymidine into the rat thyroid lobes incubated in vitro were investigated. The rate of /sup 3/H-thymidine incorporation was used as an index of thyroid follicular cells (TFC) proliferation. It was shown that: 1) somatostatin, at a concentration of 10(-7)M, decreased /sup 3/H-thymidine incorporation into DNA of TFC, 2) the highest somatostatin concentration, as tested in this study (10(-6)M), produced a similar decreasing effect; the decrease, in this case, did not attain significance vs. controls, 3) somatostatin, when employed together with TSH, suppressed the stimulatory effect of the latter hormone on /sup 3/H-thymidine incorporation into DNA of thyroid lobes.

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

    SciTech Connect

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

    1982-06-20

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

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

    SciTech Connect

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

    1990-05-01

    One assumption made in bacterial production estimates from ({sup 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.

  16. Identification of a non-specific inhibitor of (methyl/sup 3/H)thymidine incorporation into DNA of normal and pre-neoplastic cells

    SciTech Connect

    Overbye, L.J.

    1989-01-01

    Growth in mammalian cells is controlled by a variety of factors, both stimulatory and inhibitory. Bullough et al. (2) first proposed the idea that endogenous mitotic inhibitors may play a role in controlling cellular proliferation. Inhibitors may be tissue-specific (9) or non-specific (for review see Lozzio et al. (5)). Although many inhibitors are proteins of high molecular weights, several low molecular weight inhibitors of (/sup 3/H) thymidine incorporation (and therefore DNA synthesis) have been identified (1,3,6). Unlabelled thymidine has also been noted to be inhibitory (4). This paper describes a non-specific, unstable inhibitor that is found in media conditioned by neoplastic epithelial cells and some normal fibroblasts and inhibits (/sup 3/H) thymidine incorporation into normal and pre-neoplastic cells. The inhibitory activity has not been isolated and could consists of more than one compound. 9 refs., 1 tab.

  17. Peculiarities of the incorporation of (/sup 3/H)thymidine into AT-rich regions of DNA during replicative synthesis

    SciTech Connect

    Khudolii, G.A.; Khakimov, K.A.; Gorelova, T.V.; Akif'ev, A.P.

    1985-11-01

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

  18. Effect of photoperiod on the rate of 3H-thymidine incorporation of epididymal principal cells in adult Syrian hamsters

    SciTech Connect

    Johnson, L.; Bartke, A. )

    1991-04-01

    Photoperiod-induced cycles of gonadal regression and recrudescence in the Syrian hamster were used to determine if epididymal growth in adults involves mitotic activity of principal cells. In Experiment 1, the following groups of adult hamsters were examined: induced recrudescing (5L:19D (5 hr light and 19 hr dark) for 13 wk followed by 14L:10D for at least 3 wk), spontaneous recrudescing (5L:19D for 25 wk), and active gonadal state (14:10D). In Experiment 2, adult hamsters were divided into the following groups: induced recrudescing, active, and regressed (5L:19D for 16 wk). Hamsters received subcutaneous injections of 0.5 microCi 3H-thymidine/g body weight three times/wk for 3 wk. The epididymis was fixed in a glutaraldehyde followed by osmium, embedded in Epon 812, and sectioned at 1 micron. Slides were dipped in Kodak NTB-3 emulsion, exposed for 2 or 3 months, developed, and evaluated for isotopic labeling of principal and basal cell nuclei by scoring 500 to 1,000 nuclei. In Experiment 1, the percentages of labeled principal cell nuclei for the induced recrudescing, spontaneous recrudescing, and active groups were 26 {plus minus} 2%, 23 {plus minus} 5%, and 9 {plus minus} 1%, respectively. Considering the intermittent availability of 3H-thymidine during 21 days, this represents daily recruitment of 6.3%, 5.6%, and 2.2%, respectively. In Experiment 2, the percentages of labeled principal cell nuclei for induced recrudescing, active, and regressed groups were 12 {plus minus} 4%, 3 {plus minus} 1%, and 4 {plus minus} 1%, respectively. There was no effect of photoperiod on labeling pattern of basal cells (1.5 {plus minus} 0.6%, 1.2 {plus minus} 0.1%, 0.4 {plus minus} 0.1% for the three photoperiod groups, respectively).

  19. Asbestos and benzo(a)pyrene act synergistically to induce squamous metaplasia and incorporation of (/sup 3/H)thymidine in hamster tracheal epithelium

    SciTech Connect

    Mossman, B.T.; Eastman, A.; Bresnick, E.

    1984-11-01

    When exposed to either crocidolite asbestos (single 1-h exposure to 0.4 mg/ml medium) or the polycyclic aromatic hydrocarbon, benzo(a)pyrene (BaP) (less than or equal to 2.5 micrograms/ml medium, 1x weekly for 4 weeks), the epithelium of hamster tracheal explants exhibits insignificant amounts of squamous metaplasia, an atypical lesion, in comparison to amounts observed in untreated tissues. Incorporation of (/sup 3/H)thymidine, an indication of DNA synthesis by epithelial cells, likewise is unchanged. However, the extent of squamous metaplasia and numbers of labeled basal and suprabasal cells are increased substantially when BaP and asbestos are added in combination. These results suggest an important mechanism of co-carcinogenesis involving chemical and physical carcinogens and support epidemiologic observations documenting an increased risk of bronchogenic carcinoma in asbestos workers who smoke.

  20. Modifying effects of a single injection of phenobarbital on the inhibitory action of benzo(a)pyrene on 3H-thymidine incorporation into various organs of the mouse.

    PubMed

    Hellman, B

    1986-01-01

    The effects of the procarcinogen benzo(a)pyrene and the enzyme inducer phenobarbital on the DNA turnover in various organs of male C57BL mice were evaluated by measuring the incorporation of [6-3H]thymidine. When injected intraperitoneally 48 h before sacrifice, benzo(a)pyrene (28.8 mg/kg body weight) inhibited the incorporation of 3H-thymidine into the spleen, thymus, testis, and small intestine. A corresponding analysis with phenobarbital sodium (75 mg/kg b.w.) revealed reduced incorporation of 3H-thymidine into the pancreas (after 24 h) and bone marrow (after 72 h). The effects of combining benzo(a)pyrene and phenobarbital was examined by injecting the latter agent either 24 h before or after the polycyclic hydrocarbon. A previous injection of phenobarbital resulted in a potentiation of the inhibitory action of benzo(a)pyrene in the spleen. However, when phenobarbital was given after benzo(a)pyrene, there was a five-fold increase of the 3H-thymidine incorporation into the liver in comparison to the controls given vehicle. The demonstration that a single injection of phenobarbital has modifying effects on the 3H-thymidine incorporation both when administered alone and in combination with benzo(a)pyrene indicates that enzyme inducers may influence the outcome in genotoxicity tests. PMID:3767600

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

    SciTech Connect

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

    1991-06-01

    The action of somatostatin (SRIH) on {sup 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{sup {minus} 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.

  2. Degradation or consumption of exogenous thymidine in absence or presence of exogenous deoxycytidine: Effect on DNA chain elongation and (/sup 3/H)thymidine incorporation in control and uv-irradiated CHO-K1 cells

    SciTech Connect

    Newman, C.N.; Hagler, M.

    1986-04-01

    When CHO-K1 cells monolayers are grown in Ham's F-12 culture medium containing 10% fetal calf serum (medium A) exogenous thymidine (dThd) is degraded to thymine by a putative dThd phosphorylase. Thymine is then poorly incorporated into cellular DNA. When 2 mM deoxycytidine (dCyd) is added to medium A (medium B) no degradation of exogenous dThd occurs; rather dThd is consumed in the synthesis of DNA, presumably via a dThd kinase or other nucleoside salvage pathway. Differences in the kinetics of DNA synthesis, measured by (/sup 3/H)dThd pulse-incorporation or by alkaline sucrose velocity sedimentation, are observed in the two media. In comparison to cells in medium A, DNA chain elongation rates of cells in medium B are faster but total DNA synthesis in these cells, measured by incorporation of (/sup 3/H)dThd, appears to be 2- to 3-fold more sensitive to the inhibitory effects of 10 Jm/sup -2/ uv-radiation. 23 refs., 3 figs.

  3. Specificity and Efficiency of Thymidine Incorporation in Escherichia coli Lacking Thymidine Phosphorylase

    PubMed Central

    Fangman, Walton L.

    1969-01-01

    A mutant of Escherichia coli lacking the catabolic enzyme thymidine phosphorylase readily incorporates exogenous thymidine into deoxyribonucleic acid (DNA) even when provided at concentrations as low as 0.2 μg/ml. Incorporation by this prototrophic strain occurs specifically into DNA, since, with radioactively labeled thymidine, (i) more than 98% is incorporated into alkali-stable material, (ii) at least 90% is recovered as thymine after brief formic acid hydrolysis, and (iii) at least 90% is incorporated into material with the buoyant density of DNA. During growth in medium containing thymidine, the bacteria obtain approximately half of their DNA thymines from the exogenous thymidine and half from endogenous synthesis. The thymines and cytosines of DNA can be simultaneously and specifically labeled by thymidine-2-14C and uridine-5-3H, respectively. The mutant, which does not degrade thymidine, retains the ability to degrade the thymidine analogue 5-bromodeoxyuridine. PMID:4905532

  4. Effect of D-valine and cytosine arabinoside on (/sup 3/H)thymidine incorporation in rat and rabbit epididymal epithelial cell cultures

    SciTech Connect

    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 (/sup 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.

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

    PubMed

    Martirosyan, Varsik; Baghdasaryan, Naira; Ayrapetyan, Sinerik

    2013-03-01

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

  6. Sensitivity of early mouse embryos to (/sup 3/H)thymidine

    SciTech Connect

    Spindle, A.; Wu, K.; Pedersen, R.A.

    1982-12-01

    Effects of intranuclear radiation on the developmental capacity of early mouse embryos were studied by exposing embryos to (/sup 3/H)thymidine and counting the number of embryos forming blastocysts, trophoblast outgrowths, inner cell masses (ICMs), and two-layer ICMs (differentiated into primary endoderm and ectoderm). When embryos were cultured from the 2-cell stage for 8 days in the continuous presence of (/sup 3/H)thymidine, concentrations as low as 0.2 nCi/ml reduced the number of embryos forming two-layer ICMs. At 1 nCi/ml, the number of both ICMs and two-layer ICMs were reduced, and at 10 nCi/ml the number of embryos developing to all three post-blastocyst endpoints was reduced. Blastocyst formation was not affected even at the highst concentration (/sup 3/H)thymidine and then cultured further in unlabelled medium, the effects were similar to those of 8-day exposure. When embryos were exposed to (/sup 3/H)thymidine for 24 h at various developmental stages, effects were less severe than when they were exposed continuously for 3 or 8 days, and the sensitivity of embryos differed between stages. The 24-h exposure of immunosurgically isolated ICMS to (/sup 3/H)thymidine revealed that the high sensitivity of the ICM to (/sup 3/H)thymidine persists through the late blastocyst stage and declines progressively thereafter. Autoradiography indicated that the change in radiosensitivity of embryos or ICMs is generally related to their ability to incorporate (/sup 3/H)thymidine into the DNA.

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

    SciTech Connect

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

    1983-03-01

    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 (/sup 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. Labeling cells in microtiter plates for determination of [3H]thymidine uptake.

    PubMed

    Shevach, E M

    2001-05-01

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

  9. Catabolism of tritiated thymidine by aquatic microbial communities and incorporation of tritium into RNA and protein

    SciTech Connect

    Brittain, A.M.; Karl, D.M. )

    1990-05-01

    The incorporation of tritiated thymidine by five microbial ecosystems and the distribution of tritium into DNA, RNA, and protein were determined. Nonspecific labeling was greatest in sediment samples, for which {>=}95% of the tritium was recovered with the RNA and protein fractions. The percentage of tritium recovered in the DNA fraction ranged from 15 to 38% of the total labeled macromolecules recovered. Nonspecific labeling was independent of both incubation time and thymidine concentration over very wide ranges. We also evaluated the specificity of (2-{sup 3}H) adenine incorporation into adenylate residues in both RNA and DNA in parallel with the ({sup 3}H) thymidine experiments and compared the degree of nonspecific labeling by ({sup 3}H) adenine with that derived from ({sup 3}H)thymidine. Rapid catabolism of tritiated thymidine was evaluated by determining the disappearance of tritiated thymidine from the incubation medium and the appearance of degradation products. Degradation product formation, including that of both volatile and nonvolatile compounds, was much greater than the rate of incorporation of tritium into stable macromolecules. The standard degradation pathway for thymidine coupled with utilization of Krebs cycle intermediates for the biosynthesis of amino acids, purines, and pyrimidines readily accounts for the observed nonspecific labeling in environmental samples.

  10. Perturbation of DNA replication and cell cycle progression by commonly used ( sup 3 H)thymidine labeling protocols

    SciTech Connect

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

    1990-04-01

    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 ({sup 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 ({sup 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 ({sup 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.

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

    PubMed Central

    Chrzanowski, Thomas H.

    1988-01-01

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

  12. 2-Arylquinazolin-4(3H)-ones: A novel class of thymidine phosphorylase inhibitors.

    PubMed

    Javaid, Sumaira; Saad, Syed Muhammad; Perveen, Shahnaz; Khan, Khalid Mohammed; Choudhary, M Iqbal

    2015-12-01

    Thymidine phosphorylase (TP) over expression plays an important role in several pathological conditions, such as rheumatoid arthritis, chronic inflammatory diseases, psoriasis, and tumor angiogenesis. In this regard, a series of twenty-five 2-arylquinazolin-4(3H)-one derivatives 1-25 were evaluated for thymidine phosphorylase inhibitory activity. Six compounds 5, 6, 20, 2, 23, and 3 were found to be active against thymidine phosphorylase enzyme with IC50 values in the range of 42.9-294.6μM. 7-Deazaxanthine (IC50=41.0±1.63μM) was used as a standard inhibitor. Compound 5 showed a significant activity (IC50=42.9±1.0μM), comparable to the standard. The enzyme kinetic studies on the most active compounds 5, 6, and 20 were performed for the determination of their modes of inhibition, and dissociation constants Ki. All active compounds were found to be largely non-cytotoxic against the mouse fibroblast 3T3 cell line. This study identifies a novel class of thymidine phosphorylase inhibitors which may be further investigated as leads to develop therapeutic agents. PMID:26547232

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

    SciTech Connect

    Fallon, R.D.; Newell, S.Y.

    1986-11-01

    Thymidine incorporation by the microbial community on standing dead leaves of Spartina alterniflora did not obey many of the assumptions inherent in the use of the technique in planktonic systems. Incorporation rates of (methyl-/sup 3/H)thymidine were nonsaturable over a wide concentration range (10/sup 1/ to 10/sup 5/ nM). Owing to metabolism by both fungi and bacteria, a major fraction of the radiolabel (mean, 48%) appeared in protein. Extraction of the radiolabeled macromolecules were inefficient, averaging 8.8%. Based on an empirically derived conversion factor, 4 x 10/sup 18/ cells mol of thymidine/sup -1/, doubling times ranged from 4 to 69 h for the epiphytic bacterial assemblage.

  14. Rapid Initiation of Thymidine Incorporation into Deoxyribonucleic Acid in Vegetative Tobacco Stem Segments Treated with Indole-3-acetic Acid 1

    PubMed Central

    Wardell, William L.

    1975-01-01

    The short term effect of 11.4 μm indoleacetic acid on the incorporation of (methyl-3H)thymidine into DNA in vegetative tobacco (Nicotiana tabacum cv. Wis. 38) stem segments has been investigated. In segments that are defoliated, inverted, and kept in the dark for 7 hours, indoleacetic acid very rapidly (about 60 minutes) and strikingly initiates thymidine incorporation into DNA. The time required before enough indoleacetic acid (2.8 μm) to enhance thymidine incorporation moves into a segment has been found to be about 35 minutes. The initiation response time for segment tissue that already contains 2.8 μm indoleacetic acid should be no more than about 25 minutes. The rate of labeled thymidine incorporation into DNA is affected by physiological treatments of segments. Moving segments from the light into the dark or defoliating segments or inverting defoliated segments decreases the rate of thymidine incorporation. For segments given all three treatments, indoleacetic acid restores the rate of thymidine incorporation as compared to controls. Darkness, or defoliation or inversion of segments, therefore, may decrease thymidine incorporation into DNA by effecting reduced auxin levels in stem segments. PMID:16659268

  15. Neurogenesis in the vomeronasal epithelium of adult garter snakes: 3. Use of /sup 3/H-thymidine autoradiography to trace the genesis and migration of bipolar neurons

    SciTech Connect

    Wang, R.T.; Halpern, M.

    1988-10-01

    Use of 3H-thymidine autoradiography and unilateral vomeronasal (VN) axotomy has permitted us to demonstrate directly the existence of VN stem cells in the adult garter snake and to trace continuous bipolar neuron development and migration in the normal VN and deafferentated VN epithelium in the same animal. The vomeronasal epithelium and olfactory epithelium of adult garter snakes are both capable of incorporating 3H-thymidine. In the sensory epithelium of the vomeronasal organ, 3H-thymidine-labeled cells were initially restricted to the base of the undifferentiated cell layer in animals surviving 1 day following 3H-thymidine injection. With increasing survival time, labeled cells progressively migrated vertically within the receptor cell column toward the apex of the bipolar neuron layer. In both the normal and denervated VN epithelium, labeled cells were observed through the 56 days of postoperative survival. In the normal epithelium, labeled cells were always located within the matrix of the intact receptor cell columns. However, labeled cells of the denervated epithelium were always located at the apical front of the newly formed cell mass following depletion of the original neuronal cell population. In addition, at postoperative days 28 and 56, labeled cells of the denervated VN epithelium achieved neuronal differentiation and maturation by migrating much farther away from the base of the receptor cell column than the labeled cells on the normal, unoperated contralateral side. This study directly demonstrates that basal cells initially incorporating 3H-thymidine are indeed stem cells of the VN epithelium in adult garter snakes.

  16. Glucose-dependent insulinotropic peptide stimulates thymidine incorporation in endothelial cells: role of endothelin-1

    NASA Technical Reports Server (NTRS)

    Ding, Ke-Hong; Zhong, Qing; Isales, Carlos M.; Iscules, C. M. (Principal Investigator)

    2003-01-01

    We have previously characterized the receptor for glucose-dependent insulinotropic polypeptide (GIPR) in vascular endothelial cells (EC). Different EC types were found to contain distinct GIPR splice variants. To determine whether activation of the GIPR splice variants resulted in different cellular responses, we examined GIP effects on human umbilical vein endothelial cells (HUVEC), which contain two GIPR splice variants, and compared them with a spontaneously transformed human umbilical vein EC line, ECV 304, which contains four GIPR splice variants. GIP dose-dependently stimulated HUVEC and ECV 304 proliferation as measured by [3H]thymidine incorporation. GIP increased endothelin-1 (ET-1) secretion from HUVEC but not from ECV 304. Use of the endothelin B receptor blocker BQ-788 resulted in an inhibition of [3H]thymidine incorporation in HUVEC but not in ECV 304. These findings suggest that, although GIP increases [3H]thymidine incorporation in both HUVEC and ECV 304, this proliferative response is mediated by ET-1 only in HUVEC. These differences in cellular response to GIP may be related to differences in activation of GIPR splice variants.

  17. Further verification of the isotope dilution approach for estimating the degree of participation of (/sup 3/H)thymidine in DNA synthesis in studies of aquatic bacterial production

    SciTech Connect

    Bell, R.T.

    1986-11-01

    The optimal concentration of (/sup 3/H)thymidine (i.e., the maximal degree of participation in DNA synthesis) as determined by adding increasing amounts of labeled thymidine at the same specific activity was similar to the concentration of thymidine inhibiting the de novo pathway as determined by isotope dilution plots. These experiments provide further verification of the isotope dilution approach for determining the degree of participation of (/sup 3/H)thymidine in DNA synthesis.

  18. Catabolism of Tritiated Thymidine by Aquatic Microbial Communities and Incorporation of Tritium into RNA and Protein †

    PubMed Central

    Brittain, Andrew M.; Karl, David M.

    1990-01-01

    The incorporation of tritiated thymidine by five microbial ecosystems and the distribution of tritium into DNA, RNA, and protein were determined. All microbial assemblages tested exhibited significant labeling of RNA and protein (i.e., nonspecific labeling), as determined by differential acid-base hydrolysis. Nonspecific labeling was greatest in sediment samples, for which ≥95% of the tritium was recovered with the RNA and protein fractions. The percentage of tritium recovered in the DNA fraction ranged from 15 to 38% of the total labeled macromolecules recovered. Nonspecific labeling was independent of both incubation time and thymidine concentration over very wide ranges. Four different RNA hydrolysis reagents (KOH, NaOH, piperidine, and enzymes) solubilized tritium from cold trichloroacetic acid precipitates. High-pressure liquid chromatography separation of piperidine hydrolysates followed by measurement of isolated monophosphates confirmed the labeling of RNA and indicated that tritium was recovered primarily in CMP and AMP residues. We also evaluated the specificity of [2-3H]adenine incorporation into adenylate residues in both RNA and DNA in parallel with the [3H]thymidine experiments and compared the degree of nonspecific labeling by [3H]adenine with that derived from [3H]thymidine. Rapid catabolism of tritiated thymidine was evaluated by determining the disappearance of tritiated thymidine from the incubation medium and the appearance of degradation products by high-pressure liquid chromatography separation of the cell-free medium. Degradation product formation, including that of both volatile and nonvolatile compounds, was much greater than the rate of incorporation of tritium into stable macromolecules. The standard degradation pathway for thymidine coupled with utilization of Krebs cycle intermediates for the biosynthesis of amino acids, purines, and pyrimidines readily accounts for the observed nonspecific labeling in environmental samples. PMID

  19. Toxicity of zinc on photosynthesis and thymidine incorporation -- A comparison between periphyton and epipsammon communities

    SciTech Connect

    Paulsson, M.; Blanck, H.; Nystroem, B.; Admiraal, W.; Ivorra, N.; Lehmann, V.; Guasch, H.; Sabatar, S.

    1995-12-31

    The toxicity of zinc on epipsammon and periphyton communities was investigated in the river Dommel, Belgium. One, of the sites was located upstream and another downstream a tributary, heavily contaminated with heavy metals. The third site was located within the tributary. The periphyton communities were established during two weeks on artificial substrata while epipsammon was collected directly from the three sites. Two different endpoints were chosen: {sup 14}C-carbon dioxide incorporation and {sup 3}H-thymidine incorporation into macromolecules, resistant to alkaline hydrolysis, reflecting photosynthesis and bacterial growth respectively. Water from a less polluted site in the river Dommel was used both for the experiments and for the preparation of zinc stock solutions. The toxicity of the water from the examined sites was also investigated. Photosynthesis was generally not inhibited by zinc up to a concentration of 1 mM, while thymidine incorporation was inhibited. The sensitivity to zinc of bacteria was highest upstream and lowest in the tributary. ``Site water`` from the tributary as well as downstream was found to inhibit photosynthesis, while thymidine incorporation was only inhibited by ``site water`` for epipsammon within the tributary. At each site there was generally no difference in the response to zinc between the periphyton and epipsammon communities.

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

    SciTech Connect

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

    1987-01-01

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

  1. Evaluation of (/sup 3/H)thymidine uptake method for studying growth of spiroplasmas under various conditions

    SciTech Connect

    Bastian, F.O.; Baliga, B.S.; Pollock, H.M.

    1988-10-01

    (/sup 3/H)thymidine uptake and colony counts are quantitative and inexpensive methods for studying Spiroplasma growth. Using these techniques, we demonstrated subtle effects on the growth of suckling mouse cataract agent of medium alterations, inoculum size, and freezing of cultures. In addition, suckling mouse cataract agent multiplied more actively under aerobic than under anaerobic conditions. These techniques have wide application for the study of Spiroplasma growth and will be useful for the development of a defined medium.

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

    SciTech Connect

    Pawlikowski, M.; Lewinski, A.; Sewerynek, E.; Szkudlinski, M.; Kunert-Radek, J.; Wajs, E. )

    1990-02-14

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

  3. Induction of sister chromatid exchange in preimplantation mouse embryos in vitro by /sup 3/H-thymidine or ultraviolet light in combination with caffeine

    SciTech Connect

    Mueller, W.U.S.; Spindle, A.

    1986-01-01

    Preimplantation mouse embryos were exposed in vitro to /sup 3/H-thymidine (25, 100, or 250 Bq/ml) or ultraviolet (UV) light (1.35 or 4.05 J/m2), either alone or in combination with caffeine (1 mM with /sup 3/H-thymidine and 0.5 mM with UV light). Exposure to /sup 3/H-thymidine lasted for 2 days, from the two-cell stage to the late morula/early blastocyst stage, and UV radiation was applied acutely at the late morula/early blastocyst stage. The effects were quantified by the sister chromatid exchange (SCE) assay. All three agents induced SCEs when used singly. /sup 3/H-thymidine was effective in inducing SCEs only at 250 Bq/ml, whereas UV light was effective at both fluences. Although caffeine did not induce SCEs when it was added before exposure to bromodeoxyuridine (BrdUrd), which is used to visualize SCEs, it did induce SCEs when present during the entire culture period (/sup 3/H-thymidine experiments) or during incubation in BrdUrd (UV experiments). Caffeine markedly enhanced the SCE-inducing effect of UV light but did not influence the effect of /sup 3/H-thymidine.

  4. Hemicastration causes and testosterone prevents enhanced uptake of (/sup 3/H)thymidine by Sertoli cells in testes of immature rats

    SciTech Connect

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

    1984-02-01

    Rat pups were hemicastrated and uptake of (/sup 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 (/sup 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.

  5. Double labeling autoradiography. Cell kinetic studies with /sup 3/H- and /sup 14/C-thymidine

    SciTech Connect

    Schultze, B.

    1981-01-01

    Examples of the multiple applicability of the double labeling method with /sup 3/H- and /sup 14/C-TdR are demonstrated. Double labeling with /sup 3/H- and /sup 14/C-TdR makes it possible to determine the cycle and its phases with high precision by modifying the usual percent labeled mitoses method with a single injection of /sup 3/H-TdR. In addition, data is provided on the variances of the transit times through the cycle phases. For example, in the case of the jejunal crypt cells of the mouse, the transit times through successive cycle phases are uncorrelated. In the case of glial cells the double labeling method provides cell kinetic parameters despite the paucity of proliferating glial cells. In the adult untreated animal, glial cell mitoses are so rare that the percent labeled mitoses method can not be utilized. However, the S-phase duration can be measured by double labeling and the cycle time can be determined by the so-called method of labeled S phases. With the latter method the passage through the S phase of the /sup 3/H-TdR-labeled S phase cells can be registered by injecting /sup 14/C-TdR at different time intervals following /sup 3/H-TdR application. In this way an S-phase duration of about 10 hr and a cycle time of about 20 hr was found for glial cells in the adult untreated mouse. An exchange of glial cells between the growth fraction and the nongrowth fraction has also been shown by double labeling. A quite different application of the double labeling method with 3H- and /sup 14/C-TdR is the in vivo study of the cell cycle phase-specific effect of drugs used in chemotherapy of tumors. The effect of vincristine on these cells has been studied. Vincristine affects cells in S and G2 in such a manner that they are arrested during the next metaphase and subsequently become necrotic. It has no effect on G1 cells.

  6. Temporal pattern of incorporation of /sup 3/H precursors into pituitary glycoproteins and their subsequent release

    SciTech Connect

    Grotjan, H.E. Jr.

    1982-04-01

    The temporal pattern of incorporation of various /sup 3/H precursors into glycoproteins by rat anterior pituitaries incubated in vitro and the release of /sup 3/H-glycoproteins was examined. (/sup 3/H)Leucine incorporation was linear with respect to time and (/sup 3/H)leucine-containing macromolecules appeared in the media in about 1 hr. The temporal pattern of (/sup 3/H)mannose incorporation and release was similar. (/sup 3/H)Galactose and (/sup 3/H)fucose were incorporated after apparent time of delays of approximately 15 min and soon thereafter (20-25 min) appeared in the medium in /sup 3/H-glycoproteins. Thus, these precursors appear to be added as terminal residues. (/sup 3/H)Glucosamine exhibited a pattern intermediate between (/sup 3/H)leucine and (/sup 3/H)fucose whereas (/sup 3/H)GlcNAc appeared to be incorporated as a terminal residue.

  7. Induction of mutations by tritiated water and 3H-thymidine in Drosophila melanogaster assayed by the somatic zeste-white eye mutation system.

    PubMed

    Rasmuson, A; Xamena, N; Creus, A; Marcos, R

    1988-01-01

    In order to study the mutagenic effect of exposure to tritium, Drosophila melanogaster larvae were treated with tritiated water (3H2O) or tritiated thymidine (3H-TdR) during development. Dose rates ranged from 0.0058 to 0.058 rad/h per nucleus for 3H-TdR and from 0.049 to 0.122 rad/h for 3H2O. Induction of mutations was measured by the appearance of somatic mutations in the eyes of an unstable strain of Drosophila melanogaster. Both substances caused a significant increase in mutation frequency. With the assumption that each mutation observed in this assay is caused by one DNA break, the effectiveness of tritium to create DNA breaks is estimated to be 0.20 breaks per decay for 3H-TdR and 0.27 breaks per decay for 3H2O. PMID:3128734

  8. DNA synthesis and tritiated thymidine incorporation by heterotrophic freshwater bacteria in continuous culture

    SciTech Connect

    Ellenbroek, F.M.; Cappenberg, T.E. )

    1991-06-01

    Continuous cultivation of heterotrophic freshwater bacteria was used to assess the relationship between DNA synthesis and tritiated thymidine incorporation. In six different continuous cultures, each inoculated with a grazer-free mixed bacterial sample from Lake Vechten (The Netherlands), tritiated thymidine incorporation into a cold trichloroacetic acid precipitate and bacterial cell production were measured simultaneously. Empirical conversion factors were determined by division of both parameters. They ranged from 0.25 {times} 10{sup 18} to 1.31 {times} 10{sup 18} cells mol of tritiated thymidine{sup {minus}1}. In addition, DNA concentrations were measured by fluorometry with Heochst 33258. The validity of this technique was confirmed. Down to a generation time of 0.67 day, bacterial DNA content showed little variation, with values of 3.8 to 4.9 fg of DNA cell{sup {minus}1}. Theoretical conversion factors, which can be derived from DNA content under several assumptions, were between 0.26 {times} 10{sup 18} and 0.34 {times} 10{sup 18} cells mol of thymidine{sup {minus}1}. Isotope dilution was considered the main factor in the observed discrepancy between the conversion factors. In all experiments, a tritiated thymidine concentration of 20 nM was used. It was concluded that the observed difference resulted from intracellular isotope dilution which cannot be detected by current techniques for isotope dilution analysis.

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

    PubMed

    Thorn, P M; Ventullo, R M

    1988-07-01

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

  10. Rapid aquatic toxicity assay utilizing labeled thymidine incorporation in sea urchin embryos

    SciTech Connect

    Jackim, E.; Nacci, D.

    1984-01-01

    Aquatic toxicity was evaluated in the sea urchin embryo (Arbacea punctulata) by the inhibition of tritiated thymidine incorporation after a brief exposure to toxic chemicals. Arbacia is a useful surrogate species for assay: well-studied, easily cultured and fertile virtually year round. The simplicity and speed of this test system lends itself to screening large numbers of compounds, mixtures or water samples.

  11. Chemically induced transplantable malignant fibrous histiocytoma of the rat. Analyses with immunohistochemistry, immunoelectron microscopy and (3H)thymidine autoradiography

    SciTech Connect

    Kato, T.; Takeya, M.; Takagi, K.; Takahashi, K. )

    1990-05-01

    Malignant fibrous histiocytoma was produced in rats by injection of 9,10-dimethyl-1,2-benzanthracene into their knee joints. The original tumors consisted mainly of fibroblast-like cells and histiocyte-like cells, often intermixed with bizarre giant cells, and they frequently showed the storiform-pleomorphic pattern. By immunohistochemistry, anti-rat macrophage monoclonal antibodies, TRPM-3, RM-1, and Ki-M2R, and anti-rat leukocyte common antigen reacted to the histiocyte-like cells but not to the fibroblast-like cells. By the single cell cloning method, we established six tumor cell lines, none of which reacted with the anti-rat macrophage monoclonal antibodies, possessed any Fc receptors, or conducted immune phagocytosis and Latex particle phagocytosis. The ultrastructure of the cloned tumor cells resembled that of long-term cultured dermal fibroblasts. Collagen production by the tumor cells was demonstrated immunohistochemically with a monoclonal antibody for type I collagen. Inoculation of the cloned tumor cells into rats produced tumors with the histology of malignant fibrous histiocytoma and induced prominent macrophage infiltration. In the rat tumors produced by the inoculation of (3H)thymidine labeled cells, no reactivity of tumor cells with the anti-rat macrophage monoclonal antibodies was observed. Transplantation of the cultured rat tumor cells into nude mice produced tumors similar in histology to the original rat malignant fibrous histiocytoma. Tumor cells in nude mice induced marked macrophage infiltration as detected by immunohistochemistry with the anti-mouse macrophage monoclonal antibody F4/80. No differentiation of tumor cells into macrophages was detected, since no cells were stained with biotinylated anti-rat macrophage monoclonal antibody TRPM-3.

  12. Assessment of [h]thymidine incorporation into DNA as a method to determine bacterial productivity in stream bed sediments.

    PubMed

    Kaplan, L A; Bott, T L; Bielicki, J K

    1992-11-01

    We performed several checks on the underlying assumptions and procedures of the thymidine technique applied to stream bed sediments. Bacterial production rates were not altered when sediments were mixed to form a slurry. Incubation temperature did affect production rates. Controls fixed and washed with formaldehyde had lower backgrounds than trichloroacetic acid controls. DNA extraction by base hydrolysis was incomplete and variable at 25 degrees C, but hydrolysis at 120 degrees C extracted 100% of the DNA, of which 84% was recovered upon precipitation. Production rates increased as thymidine concentrations were increased over 3 orders of magnitude (30 nM to 53 muM thymidine). However, over narrower concentration ranges, thymidine incorporation into DNA was independent of thymidine concentration. Elevated exogenous thymidine concentrations did not eliminate de novo synthesis. Transport of thymidine into bacterial cells occurred at least 5 to 20 times faster than incorporation of label into DNA. We found good agreement between production rates of bacterial cultures based upon increases in cell numbers and estimates based upon thymidine incorporation and amount of DNA per cell. Those comparisons emphasized the importance of isotopic dilution measurements and validated the use of the reciprocal plot technique for estimating isotopic dilution. Nevertheless, the thymidine technique cannot be considered a routine assay and the inability to measure the cellular DNA content in benthic communities restricts the accuracy of the method in those habitats. PMID:16348806

  13. Flow cytometric evaluation of lymphocyte transformation test based on 5-ethynyl-2'deoxyuridine incorporation as a clinical alternative to tritiated thymidine uptake measurement.

    PubMed

    Poujol, Fanny; Monneret, Guillaume; Friggeri, Arnaud; Rimmelé, Thomas; Malcus, Christophe; Poitevin-Later, Françoise; Pachot, Alexandre; Lepape, Alain; Textoris, Julien; Venet, Fabienne

    2014-12-15

    In clinical laboratories, the evaluation of lymphocyte proliferative response (lymphocyte transformation test-LTT) is routinely performed by the measurement of [(3)H]-thymidine uptake after stimulation. In this study we evaluated the performances of a recently developed non-radioactive test based on the detection by flow cytometry of 5-ethynyl-2'deoxyuridine (EdU) incorporation for the measurement of LTT in routine lab conditions. After definition of optimal protocol parameters, EdU incorporation test showed good repeatability and reproducibility. Moreover, this assay was flexible enough to fit important clinical laboratory constraints (delayed stimulation, low number of cells and delayed analysis after staining). Importantly, correlations between results obtained with EdU and [(3)H]-thymidine incorporation assays were excellent both in healthy volunteers and pediatric and septic patients. In particular, the two techniques identified patients presenting with altered LTT. Upon confirmation in a larger cohort of patients, EdU incorporation assay may be a relevant non-radioactive candidate for LLT in clinic. PMID:25450005

  14. Genetic incorporation of HSV-1 thymidine kinase into the adenovirus protein IX for functional display on the virion

    SciTech Connect

    Li Jing; Le, Long; Sibley, Don A.; Mathis, J. Michael; Curiel, David T. . E-mail: david.curiel@ccc.uab.edu

    2005-08-01

    Adenoviral vectors have been exploited for a wide range of gene therapy applications. Direct genetic modification of the adenovirus capsid proteins has been employed to achieve alteration of vector tropism. We have defined the carboxy-terminus of the minor capsid protein pIX as a locus capable of presenting incorporated ligands on the virus capsid surface. Thus, we sought to exploit the possibility of incorporating functional proteins at pIX. In our current study, we incorporated the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) within pIX to determine if a larger protein of this type could retain functionality in this context. Our study herein clearly demonstrates our ability to rescue viable adenoviral particles that display functional HSV-1 TK as a component of their capsid surface. DNA packaging and cytopathic effect were not affected by this genetic modification to the virus, while CAR-dependent binding was only marginally affected. Using an in vitro [{sup 3}H]-thymidine phosphorylation assay, we demonstrated that the kinase activity of the protein IX-TK fusion protein incorporated into adenoviral virions is functional. Analysis of cell killing after adenovirus infection showed that the protein IX-TK fusion protein could also serve as a therapeutic gene by rendering transduced cells sensitive to gancyclovir. Using 9-[4-[{sup 18}F]-fluoro-3-(hydroxymethyl)butyl]guanine ([{sup 18}F]-FHBG; a positron-emitting TK substrate), we demonstrated that we could detect specific cell binding and uptake of adenoviral virions containing the protein IX-TK fusion protein at 1 h post-infection. Our study herein clearly demonstrates our ability to rescue viable adenoviral particles that display functional HSV-1 TK as a component of their capsid surface. The alternative display of HSV-1 TK on the capsid may offer advantages with respect to direct functional applications of this gene product. In addition, the determination of an expanded upper limit of incorporable

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

    PubMed

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

    1995-01-01

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

  16. Stimulation of sugar uptake and thymidine incorporation in mouse 3T3 cells by calcium phosphate and other extracellular particles.

    PubMed Central

    Barnes, D W; Colowick, S P

    1977-01-01

    Evidence is presented that the marked stimulation of sugar uptake and thymidine incorporation by addition of extra Ca2+ to stationary phase mouse 3T3 cells in culture is phosphate dependent and due to the action of the calcium phosphate precipitate formed in the medium. The cells are similarly stimulated by a variety of particulate materials, including calcium pyrophosphate, barium sulfate, kaolin, and polystrene beads. The precipitate effects on sugar uptake are of the same magnitude as those seen with certain hormones (insulin, epidermal growth factor) or with fresh 10% calf serum. The effect of barium sulfate on thymidine incorporation is also of the same magnitude as seen with these hormones, but much less than half that found with fresh calf serum. The stimulation by barium sulfate or hormones of thymidine incorporation is not phosphate dependent. PMID:202958

  17. Stimulation of sugar uptake and thymidine incorporation in mouse 3T3 cells by calcium phosphate and other extracellular particles.

    PubMed

    Barnes, D W; Colowick, S P

    1977-12-01

    Evidence is presented that the marked stimulation of sugar uptake and thymidine incorporation by addition of extra Ca2+ to stationary phase mouse 3T3 cells in culture is phosphate dependent and due to the action of the calcium phosphate precipitate formed in the medium. The cells are similarly stimulated by a variety of particulate materials, including calcium pyrophosphate, barium sulfate, kaolin, and polystrene beads. The precipitate effects on sugar uptake are of the same magnitude as those seen with certain hormones (insulin, epidermal growth factor) or with fresh 10% calf serum. The effect of barium sulfate on thymidine incorporation is also of the same magnitude as seen with these hormones, but much less than half that found with fresh calf serum. The stimulation by barium sulfate or hormones of thymidine incorporation is not phosphate dependent. PMID:202958

  18. Uptake, incorporation and metabolism of ( sup 3 H)triolein in the isolated perfused rabbit heart

    SciTech Connect

    Weis, M.T.; Palazzo, A.J.; Williams, J.L. Jr.; Malik, K.U. )

    1990-08-01

    The purpose of these experiments was to study the uptake and metabolism of exogenous triglyceride in the isolated perfused rabbit heart. When infused into the rabbit heart, (9,10-3H(N))triolein was retained and incorporated into a lipid fraction that had the chromatographic mobility of authentic triolein. Incorporation of labeled triolein was not likely to be the result of a lipoprotein lipase-mediated lipolysis/resynthesis cycle, since: (i) The distribution of radioactivity following administration of (3H)oleic acid was markedly different from the distribution of radioactivity following the administration of (3H)triolein; (ii) heparin was administered to the rabbits at the time of sacrifice; and (iii) the hearts were perfused with a protein-free buffer for 20 min prior to the labelling period. When isoproterenol was administered to hearts labelled with (3H)triolein, there was an increased output of total radioactivity, composed of labelled free fatty acids, diacylglycerol and monoacylglycerol. In these same hearts, there was an increased output of glycerol in response to isoproterenol. However, following the administration of bradykinin or angiotensin II, neither the radioactivity nor the glycerol content of the perfusate was changed. These data suggest that (3H)triolein is selectively incorporated into the triglyceride pool of the isolated perfused rabbit heart. Furthermore, this (3H)triolein is available to hormonally-activated lipolytic enzymes.

  19. Plasmodium falciparum: assessment of in vitro growth by (/sup 3/H)hypoxanthine incorporation

    SciTech Connect

    Chulay, J.D.; Haynes, J.D.; Diggs, C.L.

    1983-02-01

    To evaluate rapidly Plasmodium falciparum growth in Vitro, (/sup 3/H)hypoxanthine was added to parasite microcultures and radioisotope incorporation was measured. When culture parameters were carefully controlled, (/sup 3/H)hypoxanthine incorporation was proportional to the number of parasitized erythrocytes present. Factors affecting (/sup 3/H)hypoxanthine incorporation included initial parasitemia, duration of culture, duration of radioisotope pulse, parasite stage, concentration of uninfected erythrocytes, the use of serum or plasma to supplement growth, and the concentration of a variety of purines in the culture medium. The method described can be used to measure inhibition of P. falciparum growth by immune serum and has previously been used to study antimalarial drug activity in vitro.

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

    SciTech Connect

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

    1990-07-01

    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.

  1. Premitotic DNA synthesis in the brain of the adult frog (Rana esculenta L. ): An autoradiographic sup 3 H-thymidine study

    SciTech Connect

    Bernocchi, G.; Scherini, E.; Giacometti, S.; Mares, V. )

    1990-12-01

    Replicative synthesis of DNA in the brain of the adult frog was studied by light microscope autoradiography. Animals collected during the active period (May-June) and in hibernation (January) were used. In active frogs, 3H-thymidine labelling occurred mainly in the ependymal cells which line the ventricles. The mean labelling index (LI%) was higher in the ependyma of the lateral and fourth ventricles than in the ependyma of the lateral diencephalon and tectal parts of the mesencephalon. In the recessus infundibularis and preopticus the number of labelled cells (LCs) was several times greater than in the lateral parts of the third ventricle. LCs were seen subependymally only occasionally. The incidence of LCs in the parenchyma of the brain was much lower in most regions than in the ventricular ependyma; LCs were mainly small and, from their nuclear morphology, they were glial cells. The LI% reached the highest value in the septum hippocampi and in the nucleus entopeduncularis. In these locations, LCs were larger and closer in size to the nerve cells of these regions. From comparison with data obtained earlier in the brain of mammals, it is evident that the distribution of proliferating cells in the olfactory and limbic system is phylogenetically conservative. The occurrence of pyknotic cells in the same areas which contain LCs, suggests that cell division reflects in part the process of cell renewal observed in mammals. However, proliferating cells could also be linked to the continuous growth observed in non-mammalian vertebrates. In hibernating frogs, LCs and pyknoses were not seen or were found occasionally, which further indicates the functional significance of both processes.

  2. Increased TH-thymidine incorporation into DNA of organ-cultured adrenal explants from rats injected with corticotropin and/or cysteamine

    SciTech Connect

    Sewerynek, E.; Szkudlinski, M.; Lewinski, A.; Kunert-Radek, J.

    1988-11-30

    The effect of a single injection of cysteamine /CySH/ - a sulfhydryl substance, known to deplete tissue content of somatostatin /SS/ - on TH-thymidine incorporation into DNA of rat adrenal explants incubated in vitro was investigated. It was shown that: 1/ Single in vivo injection of ACTH or of CySH increased TH-thymidine incorporation into DNA of the organ-cultured adrenals, 2/ Dexamethasone reduced the TH-thymidine uptake, but that decrease did not attain statistical significance versus controls.

  3. Microsystem to evaluate the incorporation of 3H-uridine in macrophage RNA

    SciTech Connect

    Varesio, L.; Naglich, J.; Brunda, M.J.; Taramelli, D.; Eva, A.

    1981-01-01

    A method is described for the evaluation of the total 3H-uridine incorporated by macrophages in vitro into trichloroacetic acid (TCA)-precipitable material. The technique is based upon solubilization of the macrophage monolayers by guanidine-HC1, followed by TCA precipitation. The recovery of RNA into the precipitate and the reproducibility of the results were strictly dependent on the use of filtered reagents and on incubation of the TCA precipitate for 2 or more hours at 4 degree C before harvesting. Treatment with quanidine-HC1 did not affect the recovery of labeled RNA. Moreover, we observed that radioactive precipitate had the characteristics of RNA, since its recovery was sensitive to the addition of unlabeled uridine in the culture medium and to the treatment of the macrophages with inhibitors of RNA synthesis, but not of protein synthesis. Tritiated uridine incorporation in microcultures of macrophages can be assessed with this technique, by processing the cells directly in the wells. The main advantages of this procedure are: 1) the radioactivity can be measured by semiautomatic cell harvesters, 2) a small number of macrophages are required, and 3) many samples can be processed simultaneously. Overall, the technique is simple, rapid, and could be successfully adapted to study other metabolic pathways.

  4. Active melanogenesis in non-S phase melanocytes in B16 melanomas in vivo investigated by double-tracer microautoradiography with 18F-fluorodopa and 3H-thymidine.

    PubMed Central

    Kubota, R.; Yamada, S.; Ishiwata, K.; Kubota, K.; Ido, T.

    1992-01-01

    3,4-Dihydroxy-2-[18F]fluoro-L-phenylalanine (2-[18F]FDOPA) and [6-3H]thymidine ([3H]Thd) were simultaneously injected into mice transplanted with B16 melanomas of FM3A mammary carcinoma. Melanogenesis was differentiated from DNA synthesis in the mitotic cell cycle by monitoring grain distribution with double-tracer microautoradiography. The percentages of pigmented cells were inversely proportional to those of [3H]Thd-labelled cells, indicating that the greater the number of melanocytes, the smaller was the number of proliferating cells. The number of grains produced by 2-[18F]FDOPA in the [3H]Thd-unlabelled melanocytes was significantly higher (P < 0.001) than the numbers in the [3H]Thd-labelled melanocytes and in nonmelanocytes. The [3H]Thd-unlabelled non-melanocytes and FM3A cells showed the lowest accumulation of 2-[18F]DOPA, which may have resulted from the basic amino acid demand by malignant neoplasms via amino acid transport. The [3H]Thd-labelled cells, regardless of whether they were pigmented or not, had slightly more grains with 2-[18F]FDOPA than the [3H]Thd-unlabelled non-melanocytes (P < 0.05), which may have resulted from the enhanced amino acid requirement for proliferation. Melanogenesis appeared to be activated only in the non-S phase of the mitotic cycle in melanocytes. Images Figure 2 PMID:1419597

  5. Synthesis of a 2'-Se-thymidine Phosphoramidite and Its Incorporation into Oligonucleotides for Crystal Structure Study

    SciTech Connect

    Sheng,J.; Jian, J.; Salon, J.; Huang, Z.

    2007-01-01

    To investigate nucleic acids with selenium derivatization for crystallography, we report the first synthesis of 2'-methylseleno-thymidine phosphoramidite and its incorporation into DNAs and RNAs by solid-phase synthesis with over 99% coupling yield. The d(GT{sub Se}GTACAC){sub 2} crystal structure was also determined at 1.40 {angstrom} resolution using Se phasing, revealing that this Se derivatization did not cause significant structure perturbation, consistent with our UV melting study. In addition, we observed that the Se modification largely facilitated the crystallization.

  6. A comparison between ((3)H)-thymidine incorporation and isothermal microcalorimetry for the assessment of antigen-induced lymphocyte proliferation.

    PubMed

    Murigande, C; Regenass, S; Wirz, D; Daniels, A U; Tyndall, A

    2009-01-01

    Lymphocyte transformation tests (LTT) are time-consuming radioactive assays used in the clinic for the determination of allergic drug reactions and extensively in basic immunological research. In the present study we propose an alternative method in the monitoring of T-cell responses by isothermal microcalorimetric (IMC) measurements of overall cellular heat production as a function of time. For mitogen-induced lymphocyte proliferation, we analyzed a concentration dependent effect of phytohemaglutinin (PHA) and both tests showed a good correlation. This was also the case for specific antigenic stimulation with Varidase(R) or tetanus toxoid. On the other hand, antigen-induced lymphocyte proliferation analyzed by pre and post influenza vaccine (Inflexal(R) V) samples, showed no such correlation. Our study suggests that IMC measurements, despite the advantages of simplicity, on-line recording of metabolic activity and no use of radioactivity, may be limited to monitoring mitogen-induced lymphocyte proliferation. PMID:19172486

  7. Evaluation of a range of anti-proliferative assays for the preclinical screening of anti-psoriatic drugs: a comparison of colorimetric and fluorimetric assays with the thymidine incorporation assay.

    PubMed

    George, Suja Elizabeth; Anderson, Rosaleen J; Cunningham, Anne; Donaldson, Michael; Groundwater, Paul W

    2010-06-01

    Established treatments for psoriasis are generally based on antiproliferative, anti-inflammatory, or differentiation-modifying activity, or a combination of these effects. New agents for the treatment of psoriasis could be identified by high-throughput screening (HTS) of large compound libraries using keratinocyte proliferation models. Although several new proliferation assays have been developed, the radioactive [(3)H]-thymidine incorporation assay is still considered to be the gold standard for the evaluation of keratinocyte proliferation in vitro. In this study, we compare a number of simple, and reliable, colorimetric (MTT, NRU, SRB, and CVS), and fluorimetric (CAM and AB) methods with the [(3)H]-thymidine incorporation assay for the measurement of keratinocyte proliferation in the exponential growth phase in 96-well formats. The concentrations that induced 50% growth inhibition (GI(50)) were determined by each assay for the established antipsoriatics, dithranol, and methotrexate. Strong correlations were observed between the percentage growth inhibitions determined by the radioactive and the colorimetric assays, with no significant differences (P > 0.05) between their GI(50) values. The colorimetric assays are thus suitable alternatives to the radioactive assay for quantifying keratinocyte growth inhibition. We have also validated the use of the HaCaT cell line as a representative of the hyperproliferative psoriatic epidermis, in the preclinical screening of experimental anti-psoriatic agents. PMID:20482335

  8. Eimeria tenella: parasite-specific incorporation of /sup 3/H-uracil as a quantitative measure of intracellular development

    SciTech Connect

    Schmatz, D.M.; Crane, M.S.; Murray, P.K.

    1986-02-01

    An assay has been developed using parasite-specific incorporation of /sup 3/H-uracil to assess the intracellular growth of Eimeria tenella in vitro. As shown by both scintillation counts and autoradiography, /sup 3/H-uracil was incorporated specifically into intracellular parasites from the onset of infection and continued throughout development of the first generation schizonts. Mature schizonts and first generation merozoites did not continue to incorporate additional /sup 3/H-uracil, indicating that RNA synthesis had halted in these stages. Based on these findings, a semi-automated microscale uracil incorporation assay was developed to determine parasite viability. This method should be useful for biochemical studies with intracellular parasites and for screening compounds for anticoccidial activity. The ease, rapidity, and quantitative nature of this assay contrasts favorably with standard morphometric approaches of determining parasite development. In addition, parallel studies using host cell incorporation of /sup 3/H-uridine have been introduced as a method of determining whether antiparasitic activity is direct or indirect in relation to effects on the host cell.

  9. RAPID AQUATIC TOXICITY ASSAY USING INCORPORATION OF TRITIATED-THYMIDINE INTO SEA URCHIN, 'ARBACIA PUNCTULATA', EMBRYO: EVALUATION OF TOXICANT EXPOSURE PROCEDURES

    EPA Science Inventory

    Toxicity of substances in seawater was measured using growth inhibition of embryonic sea urchins during a short period after fertilization. Growth of Arbacia punctulata embryos was monitored by incorporation of tritium-labeled thymidine. The paper presents a comparison of toxican...

  10. In vivo incorporation of tritium from 3H2O into pulmonary lipids of meal-fed and starved rats

    SciTech Connect

    Todhunter, D.A.; Scholz, R.W.

    1980-12-01

    In vivo fatty acid synthesis, as measured by tritium incorporation from 3H2O into fatty acids, was examined in the lungs of meal-fed and starved rats. In meal-fed animals, 74% of the radioactivity isolated from pulmonary lipids was found in the phospholipid fraction. Starving rats for 72 h markedly reduced in vivo 3H2O incorporation into pulmonary lipids. These studies demonstrated net in vivo synthesis of fatty acids in pulmonary tissue of rats using a method that is not complicated by potential differences in metabolic pool sizes or peculiarities of specific carbon substrate sources. Synthesis of fatty acids in vivo was affected by the nutritional state of the animal and citrate appears to be a significant source of cytoplasmic acetyl-CoA for de novo pulmonary lipogenesis in the fed rat.

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

    SciTech Connect

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

    1984-03-01

    Uptake of /sup 3/H after exposure of cells to (/sup 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.

  12. Temperature and solar radiation interactions on 3H-leucine incorporation by bacterioplankton in a subtropical estuary.

    PubMed

    Bullock, Avery K; Jeffrey, Wade H

    2010-01-01

    Although the effects of UV radiation are thought to be temperature independent, the photoinhibition of aquatic bacteria may be temperature dependent owing to enzymatic repair kinetics, an important consideration for climate change analyses. We examined the interactions between temperature and solar radiation in water samples collected from the Blackwater River, Pensacola Bay, and the coastal Gulf of Mexico (Florida) in July 2008. Subsamples were incubated in the dark for 20 h at either the in situ temperature, +5 degrees C from in situ or -5 degrees C from in situ after which they were amended with (3)H-leucine and irradiated in full sunlight at their respective temperatures and compared to samples incubated simultaneously in the dark. Temperature and light significantly affected (3)H-leucine incorporation at all locations and interactive effects between temperature and sunlight were found for Pensacola Bay and the Gulf. Generally, warmer waters reduced photoinhibition. The -5 degrees C treatment was always significantly more inhibited than the +5 degrees C treatment, but the in situ temperature and +5 degrees C and -5 degrees C treatments were not always significantly different. Photoinhibition reduction at warmer temperatures suggests specific effects on photobiology not observed in general cellular activity may be important in determining interactive ecosystem effects of climate change. PMID:20158671

  13. Axon reaction in hypoglossal and dorsal motor vagal neurons of adult rat: incorporation of (3H)leucine

    SciTech Connect

    Aldskogius, H.; Barron, K.D.; Regal, R.

    1984-07-01

    Pairs of adult rats received (/sup 3/H)leucine 0.25, 1, and 16 h before killing and zero to 164 days after unilateral cervical vagotomy and hypoglossal neurotomy. Grain counts and morphometric measurements were made on axotomized and uninjured neurons in histoautoradiographs of the medullary nuclei. Axotomized hypoglossal neurons, which largely survive the injury, both enlarged and incorporated increased amounts of tritiated leucine at each labeling interval, 3 through 28 days postoperatively. In the vagal dorsal motor nucleus (DMN), axotomized cells, which frequently die after neurotomy, enlarged slightly through 28 days postoperatively, then atrophied; DMN neurons increased amino acid uptake for a shorter period (days 7 through 14) than hypoglossal neurons. Axotomized DMN neurons did not sustain increased protein synthesis as long as their hypoglossal counterparts and seemed to fail to increase synthesis of structural proteins with long half-lives (16-h labeling interval). The frequently necrobiotic response of axotomized DMN neurons may relate to these phenomena. From these and earlier results, the authors conclude that axon reaction appears to differ fundamentally in peripheral and central neurons. This difference may have significance for research on regeneration in the central nervous system.

  14. Rapid aquatic toxicity assay using incorporation of tritiated-thymidine into sea urchin, Arbacia punctulata, embryo: evaluation of toxicant exposure procedures

    SciTech Connect

    Nacci, D.E.; Jackim, E.

    1985-01-01

    Toxicity of substances in seawater was measured using growth inhibition of embryonic sea urchins during a short period after fertilization. Growth of Arbacia punctulata embryos was monitored by incorporation of tritium-labeled thymidine. The paper presents a comparison of toxicant exposure procedures using the Arbacia embryo thymidine incorporation test. Toxicant exposure began before, at the time of, or after fertilization and continued for 4 h following fertilization. In addition to the eight organic chemicals tested for comparison to acute toxicity values for other species, several chemicals with embryotoxic potentials (tumor promoters and teratogens) were tested to determine differential sensitivities of exposed life-stages: unfertilized egg, fertilization, and early embryo. EC50 values for any one substance were not significantly changed by exposure modification. Toxicity values for exposures that included fertilization as well as early embryo growth were at least as sensitive as post-fertilization exposure values for all compounds tested except one. Because of technical ease and potential sensitivity, toxicant exposure that includes fertilization as well as early embryo growth (but not unfertilized egg exposure) is recommended for future testing.

  15. Crucial roles of thymidine kinase 1 and deoxyUTPase in incorporating the antineoplastic nucleosides trifluridine and 2'-deoxy-5-fluorouridine into DNA.

    PubMed

    Sakamoto, Kazuki; Yokogawa, Tatsushi; Ueno, Hiroyuki; Oguchi, Kei; Kazuno, Hiromi; Ishida, Keiji; Tanaka, Nozomu; Osada, Akiko; Yamada, Yukari; Okabe, Hiroyuki; Matsuo, Kenichi

    2015-01-01

    Trifluridine (FTD) and 2'-deoxy-5-fluorouridine (FdUrd), a derivative of 5-fluorouracil (5-FU), are antitumor agents that inhibit thymidylate synthase activity and their nucleotides are incorporated into DNA. However, it is evident that several differences occur in the underlying antitumor mechanisms associated with these nucleoside analogues. Recently, TAS-102 (composed of FTD and tipiracil hydrochloride, TPI) was shown to prolong the survival of patients with colorectal cancer who received a median of 2 prior therapies, including 5-FU. TAS-102 was recently approved for clinical use in Japan. These data suggest that the antitumor activities of TAS-102 and 5-FU proceed via different mechanisms. Thus, we analyzed their properties in terms of thymidine salvage pathway utilization, involving membrane transporters, a nucleoside kinase, a nucleotide-dephosphorylating enzyme, and DNA polymerase α. FTD incorporated into DNA with higher efficiency than FdUrd did. Both FTD and FdUrd were transported into cells by ENT1 and ENT2 and were phosphorylated by thymidine kinase 1, which showed a higher catalytic activity for FTD than for FdUrd. deoxyUTPase (DUT) did not recognize dTTP and FTD-triphosphate (F3dTTP), whereas deoxyuridine-triphosphate (dUTP) and FdUrd-triphosphate (FdUTP) were efficiently degraded by DUT. DNA polymerase α incorporated both F3dTTP and FdUTP into DNA at sites aligned with adenine on the opposite strand. FTD-treated cells showed differing nuclear morphologies compared to FdUrd-treated cells. These findings indicate that FTD and FdUrd are incorporated into DNA with different efficiencies due to differences in the substrate specificities of TK1 and DUT, causing abundant FTD incorporation into DNA. PMID:25901475

  16. Crucial roles of thymidine kinase 1 and deoxyUTPase in incorporating the antineoplastic nucleosides trifluridine and 2′-deoxy-5-fluorouridine into DNA

    PubMed Central

    SAKAMOTO, KAZUKI; YOKOGAWA, TATSUSHI; UENO, HIROYUKI; OGUCHI, KEI; KAZUNO, HIROMI; ISHIDA, KEIJI; TANAKA, NOZOMU; OSADA, AKIKO; YAMADA, YUKARI; OKABE, HIROYUKI; MATSUO, KENICHI

    2015-01-01

    Trifluridine (FTD) and 2′-deoxy-5-fluorouridine (FdUrd), a derivative of 5-fluorouracil (5-FU), are antitumor agents that inhibit thymidylate synthase activity and their nucleotides are incorporated into DNA. However, it is evident that several differences occur in the underlying antitumor mechanisms associated with these nucleoside analogues. Recently, TAS-102 (composed of FTD and tipiracil hydrochloride, TPI) was shown to prolong the survival of patients with colorectal cancer who received a median of 2 prior therapies, including 5-FU. TAS-102 was recently approved for clinical use in Japan. These data suggest that the antitumor activities of TAS-102 and 5-FU proceed via different mechanisms. Thus, we analyzed their properties in terms of thymidine salvage pathway utilization, involving membrane transporters, a nucleoside kinase, a nucleotide-dephosphorylating enzyme, and DNA polymerase α. FTD incorporated into DNA with higher efficiency than FdUrd did. Both FTD and FdUrd were transported into cells by ENT1 and ENT2 and were phosphorylated by thymidine kinase 1, which showed a higher catalytic activity for FTD than for FdUrd. deoxyUTPase (DUT) did not recognize dTTP and FTD-triphosphate (F3dTTP), whereas deoxyuridine-triphosphate (dUTP) and FdUrd-triphosphate (FdUTP) were efficiently degraded by DUT. DNA polymerase α incorporated both F3dTTP and FdUTP into DNA at sites aligned with adenine on the opposite strand. FTD-treated cells showed differing nuclear morphologies compared to FdUrd-treated cells. These findings indicate that FTD and FdUrd are incorporated into DNA with different efficiencies due to differences in the substrate specificities of TK1 and DUT, causing abundant FTD incorporation into DNA. PMID:25901475

  17. Uptake of thymidine labeled on carbon 2: A potential index of liver regeneration by positron emission tomography

    SciTech Connect

    Vander Borght, T.M.; Lambotte, L.E.; Pauwels, S.A.; Dive, C.C. )

    1990-07-01

    Noninvasive measurement of liver regeneration with positron emission tomography has been attempted with 11C-thymidine; however, results were unsatisfactory using thymidine labeled on its methyl group. To evaluate whether the specificity of the method could be improved by modifying the labeling position of the tracer, thymidine labeled on its methyl group with 3H and thymidine labeled on its carbon 2 with 14C were injected in 22 hepatectomized rats either 1 hr (when DNA synthesis is not increased) or 24 hr after the surgical procedure (when the rate of DNA synthesis is maximal). Liver samples taken 10, 30 and 120 min after injection showed that, in contrast to 3H-radioactivity, 14C-radioactivity measured in whole tissue allowed a clear discrimination between regenerating and nonregenerating livers. In addition, 14C-radioactivity measured in whole tissue of regenerating livers correlated with the DNA radioactivity 10, 30 and 120 min after injection of the tracer. In contrast, no such correlation was found with the methyl-labeled thymidine. Analysis of the radioactive material present in the non-DNA fraction using ion exchange disks and high-performance liquid chromatography showed that 2-C-labeled thymidine was incorporated into DNA without accumulation of labeled metabolites whereas, for the methyl-labeled thymidine, almost all radioactivity was related to degradative products. Therefore the evaluation of the liver regeneration with the 2-C-labeled thymidine, which does not require cellular fractionation, should be suited for noninvasive measurement with positron emission tomography.

  18. Dose and time dependent effects of morphine on the incorporation of (3H)valine into soluble brain and liver proteins

    SciTech Connect

    Roennbaeck, L.; Hansson, E.; Cupello, A.

    1983-03-01

    Morphine (10(-6)-10(-5) M) causes an increase in incorporation of (/sup 3/H)valine into soluble proteins during 4 hr in rat brain cortical slices, liver slices and cultivated astroglial cells. The effects are dose-dependent. They are neither cell specific nor strictly related to classical opiate receptors. Pulse-labeling with (/sup 3/H)valine for 60 min after incubation in 10(-6)-10(-5) M morphine, resolves time-dependent changes in incorporation, with both increases and decreases in protein metabolism.

  19. Inhibition of ovarian cancer cell proliferation in vivo and incorporation of /sup 3/H-thymidine in vitro after follicle regulatory protein administration

    SciTech Connect

    Rodgers, K.E.; Montz, F.J.; Scott, L.; Condon, S.; Fujimori, K.; diZerega, G.S.

    1989-01-01

    Follicle regulatory protein immunoreactivity and biologic activity were measured in ascites from a patient with juvenile granulosa cell tumor. Microscopic examination of immunohistochemical staining of a juvenile granulosa cell tumor with anti-follicle regulatory protein antisera showed homogeneous cytosolic expression of follicle regulatory protein throughout the tumor. Tumor cells were injected subcutaneously into nude mice. Partially purified follicle regulatory protein (50 micrograms/day) was then injected daily for 10 days, or for 25 days once the tumor became palpable. Treatment with follicle regulatory protein significantly slowed the rate of tumor growth with both treatments. To test the tissue specificity of the effect, a metastatic, well-differentiated endometrial adenocarcinoma was also grown in nude mice. Follicle regulatory protein treatment did not alter the rate of tumor growth. An in vitro clonigenic assay confirmed these in vivo results. Partially purified follicle regulatory protein had a biphasic effect on the proliferation of juvenile granulosa tumor cell but did not affect the proliferation of endometrial adenocarcinoma cells. Clonigenic assays were performed on five ovarian adenocarcinomas passaged in vitro, and these tumor cells exhibited a biphasic response to follicle regulatory protein. Immunoneutralization studies showed that this biphasic response was due to impurities in the follicle regulatory protein preparations. The longer the exposure of the tumor cells to follicle regulatory protein, the greater the degree of inhibition of proliferation. In summary, administration of follicle regulatory protein slowed tumor growth through a direct effect on the tumor cell rather than an indirect effect on the hormonal or immune status of the host.

  20. Regulatory effects of eicosanoids on thymidine uptake by vascular smooth muscle cells of rats

    SciTech Connect

    Uehara, Y.; Ishimitsu, T.; Kimura, K.; Ishii, M.; Ikeda, T.; Sugimoto, T.

    1988-12-01

    To define the roles of eicosanoids in vascular smooth muscle cells (VSMC) growth, we examined the effects of exogenous eicosanoids on (/sup 3/H)thymidine uptake by cultured VSMC of Wistar rats. Stable prostacyclin (PGI2) analog, OP-41483, significantly decreased the incorporation of (/sup 3/H)thymidine into deoxyribonucleic acid (DNA) of VSMC in a dose dependent manner from 10(-8) to 10(-4) M. Prostaglandin E2 (PGE2) and PGD2 ranging from 10(-8) to 10(-4) M also dose-dependently decreased the (/sup 3/H)thymidine uptake by VSMC. In contrast, stable thromboxane A2 analog, STA2, significantly increased the incorporation of (/sup 3/H)thymidine into DNA in a dose dependent manner from 10(-8) to 10(-4) M. The dose response curve of STA2 was shifted toward a lowered response when 10(-5) M PGI2 analog, PGE2 or PGD2 was added in the culture medium. Thus, it is indicated that vasodepressor eicosanoids decrease the proliferation of VSMC, whereas vasoconstrictor TXA2 enhances the VSMC growth. Vascular smooth muscle cells possibly autoregulate the cell proliferation through the eicosanoids generation.

  1. Islet cell thymidine kinase activity as indicator of islet cell proliferation in rat pancreas

    SciTech Connect

    Swenne, I. )

    1990-01-01

    The activity of thymidine kinase in homogenates of isolated rat islets of Langerhans was measured and correlated with the DNA replicatory activity of the islet cells. Adult and fetal rat islets were cultured in medium with 2.7 or 16.7 mM glucose or 16.7 mM glucose and 1 microgram/ml human growth hormone. In both types of islets, 16.7 mM glucose doubled (3H)thymidine incorporation compared with 2.7 mM glucose, and the addition of growth hormone caused a further increase in DNA replication. TK activity in the islets showed similar changes in response to glucose and growth hormone. The correlation between (3H)thymidine incorporation and TK activity was thus highly significant. Cell-cycle analysis of cultured fetal rat islets showed that TK activity was preferentially expressed during the S phase of the cell cycle. TK activity of freshly isolated islets declined with the age of the animal. In pancreatic sections, the islet cell autoradiographic labeling index after (3H)thymidine administration in vivo likewise declined with age and was correlated with the TK activity in freshly isolated islets. It is suggested that measurements of islet TK activity can be used as index of islet cell proliferation; this method has the distinct advantage of avoiding the cumbersome procedure of preparing and scoring autoradiograms.

  2. Characterization of thymus- and bone marrow-derived lymphocytes in rats by means of 3H-uridine incorporation.

    PubMed

    Klobusická, M; Babusíková, O; Koníková, E; Novotná, L

    1975-01-01

    Lymphocytes from various lymphoid organs and of the thoracic duct of normal and thymectomized rats, irradiated and reconstituted with syngeneic bone marrow were tested in vitro in a minimal non-enriched cultivation medium with 3H-uridine, and the percentage of uridine-labeled lymphocytes was determined. The highest number of heavily labeled small lymphocytes was found in the thymus and the thoracic duct, less in peripheral blood, the lymph nodes and the spleen, and the smallest numbers in the bone marrow. A reduced ability of uridine uptake was noted in the thymectomized animals. The method of immune rosette formation was used to determine the presence of B lymphocytes in the lymphoid rat population. The highest quantity of B lymphocytes was noted in bone marrow and the least in the thymus and the thoracic duct. Thymectomized animals had a significantly higher percentage of EAC rosettes than normal and sham-operated animals. The methods employed and existing literary data enabled us to identify the heavily uridine-labeled lymphocytes as T cells, while unlabeled lymphocytes are considered to be B cells. The difference in uriding uptake by rat lymphocytes may serve as one of the T lymphocyte markers in a heterologous lymphoid population. PMID:1082556

  3. Pertussis toxin treatment attenuates some effects of insulin in BC3H-1 murine myocytes

    SciTech Connect

    Luttrell, L.M.; Hewlett, E.L.; Romero, G.; Rogol, A.D.

    1988-05-05

    The effects of pertussis toxin (PT) treatment on insulin-stimulated myristoyl-diacylglycerol (DAG) generation, hexose transport, and thymidine incorporation were studied in differentiated BC3H-1 mycocytes. Insulin treatment caused a biphasic increase in myristoyl-DAG production which was abolished in myocytes treated with PT. There was no effect of PT treatment on basal (nonstimulated) myristoyl-DAG production. Insulin-stimulated hydrolysis of a membrane phosphatidylinositol glycan was blocked by PT treatment. ADP-ribosylation of BC3H-1 plasma membranes with (/sup 32/P)NAD revealed a 40-kDa protein as the major PT substrate in vivo and in vitro. The time course and dose dependence of the effects of PT on diacylglycerol generation correlated with the in vivo ADP-ribosylation of the 40-kDa substrate. Pertussis toxin treatment resulted in a 71% attenuation of insulin-stimulated hexose uptake without effect on either basal or phorbol ester-stimulated uptake. The stimulatory effects of insulin and fetal calf serum on (/sup 3/H)thymidine incorporation into quiescent myocytes were attenuated by 61 and 59%, respectively, when PT was added coincidently with the growth factors. Nonstimulated and EGF-stimulated (/sup 3/H)thymidine incorporation was unaffected by PT treatment. These data suggest that a PT-sensitive G protein is involved in the cellular signaling mechanisms of insulin.

  4. Transition State Analysis of Thymidine Hydrolysis by Human Thymidine Phosphorylase*

    PubMed Central

    Schwartz, Phillip A.; Vetticatt, Mathew; Schramm, Vern L.

    2010-01-01

    Human thymidine phosphorylase (hTP) is responsible for thymidine (dT) homeostasis and its action promotes angiogenesis. In the absence of phosphate, hTP catalyzes a slow hydrolytic depyrimidination of dT yielding thymine and 2-deoxyribose (dRib). Its transition state was characterized using multiple kinetic isotope effect (KIE) measurements. Isotopically enriched thymidines were synthesized enzymatically from glucose or (deoxy)ribose and intrinsic KIEs were used to interpret the transition state structure. KIEs from [1′-14C]-, [1-15N]-, [1′-3H]-, [2′R-3H]-, [2′S-3H]-, [4′-3H]-, [5′-3H]dTs provided values of 1.033 ± 0.002, 1.004 ± 0.002, 1.325 ± 0.003, 1.101 ± 0.004, 1.087 ± 0.005, 1.040 ± 0.003, and 1.033 ± 0.003, respectively. Transition state analysis revealed a stepwise mechanism with a 2-deoxyribocation formed early and a higher energetic barrier for nucleophilic attack of a water molecule on the high energy intermediate. An equilibrium exists between the deoxyribocation and reactants prior to the irreversible nucleophilic attack by water. The results establish activation of the thymine leaving group without requirement for phosphate. A transition state constrained to match the intrinsic KIEs was found using density functional theory. An active site histidine (His116) is implicated as the catalytic base for activation of the water nucleophile at the rate-limiting transition state. The distance between the water nucleophile and the anomeric carbon (rC-O) is predicted to be 2.3 Å at the transition state. The transition state model predicts that deoxyribose adopts a mild 3′-endo confirmation during nucleophilic capture. These results differ from the concerted bimolecular mechanism reported for the arsenolytic reaction PMID:20804144

  5. Radiation dose to trabecular bone marrow stem cells from 3H, 14C and selected α-emitters incorporated in a bone remodeling compartment

    NASA Astrophysics Data System (ADS)

    Nie, Huiling; Richardson, Richard B.

    2009-02-01

    A Monte Carlo simulation of repeated cubic units representing trabecular bone cavities in adult bone was employed to determine absorbed dose fractions evaluated for 3H, 14C and a set of α-emitters incorporated within a bone remodeling compartment (BRC). The BRC consists of a well-oxygenated vascular microenvironment located within a canopy of bone-lining cells. The International Commission on Radiological Protection (ICRP) considers that an important target for radiation-induced bone cancer is the endosteum marrow layer adjacent to bone surface where quiescent bone stem cells reside. It is proposed that the active stem cells and progenitor cells located above the BRC canopy, the 'BRC stem cell niche', is a more important radiation-induced cancer target volume. Simulation results from a static model, where no remodeling occurs, indicate that the mean dose from bone and bone surface to the 50 µm quiescent bone stem cell niche, the current ICRP target, was substantially lower (two to three times lower) than that to the narrower and hypoxic 10 µm endosteum for 3H, 14C and α-particles with energy range 0.5-10 MeV. The results from a dynamic model indicate that the temporal α-radiation dose to active stem/progenitor cells located in the BRC stem cell niche from the material incorporated in and buried by forming bone was 9- to 111-fold greater than the dose to the quiescent bone stem cell niche. This work indicates that the remodeling portion of the bone surface, rather than the quiescent (endosteal) surface, has the greatest risk of radiation-induced bone cancer, particularly from short-range radiation, due to the elevated dose and the radiosensitizing oxygen effect.

  6. Radioautographic visualization of differences in the pattern of (/sup 3/H)uridine and (/sup 3/H)orotic acid incorporation into the RNA of migrating columnar cells in the rat small intestine

    SciTech Connect

    Uddin, M.; Altmann, G.G.; Leblond, C.P.

    1984-05-01

    The epithelium of rat small intestine was radioautographed to examine whether RNA is synthesized by the salvage pathway as shown after (/sup 3/H)uridine injection or by the de novo pathway as shown after (/sup 3/H)orotic acid injection. The two modes of RNA synthesis were thus investigated during the migration of columnar cells from crypt base to villus top, and the rate of synthesis was assessed by counting silver grains over the nucleolus and nucleoplasm at six levels along the duodenal epithelium - that is, in the base, mid, and top regions of the crypts and in the base, mid, and top regions of the villi. Concomitant biochemical analyses established that, after injection of either (5-/sup 3/H)uridine or (5-/sup 3/H)orotic acid: (a) buffered glutaraldehyde fixative was as effective as perchloric acid or trichloroacetic acid in insolubilizing the nucleic acids of rat small intestine; (b) a major fraction of the nucleic acid label was in RNA, that is, 91% after (/sup 3/H)uridine and 72% after (/sup 3/H)orotic acid, with the rest in DNA; and (c) a substantial fraction of the RNA label was in poly A/sup +/ RNA (presumed to be messenger RNA). In radioautographs of duodenum prepared after (/sup 3/H)uridine injection, the count of silver grains was high over nucleolus and nucleoplasm in crypt base cells and gradually decreased at the upper levels up to the villus base. In the rest of the villus, the grain count over the nucleolus was negligible, while over the nucleoplasm it was low but significant.

  7. Trichomonas vaginalis thymidine kinase: purification, characterization and search for inhibitors.

    PubMed Central

    Strosselli, S; Spadari, S; Walker, R T; Basnak, I; Focher, F

    1998-01-01

    We report that a thymidine kinase (TK) activity is present in Trichomonas vaginalis and can be separated from the deoxyribonucleoside phosphotransferase. T. vaginalis TK, purified 11200-fold to apparent homogeneity, has a molecular mass of 31500 Da. It phosphorylates not only thymidine (Km 0.18 microM) but also deoxycytidine (Km 0.88 microM) and deoxyuridine (Km 0.14 microM). In contrast with T. vaginalis deoxyribonucleoside phosphotransferase, the TK activity is strongly inhibited by novel deoxyuridine analogues such as 5-methyl-4'-thio-2'-deoxyuridine (MTdU) (Ki 20 nM) and 5-iodo-4'-thio-2'-deoxyuridine (ITdU) (Ki 24 nM). MTdU and ITdU are phosphorylated by T. vaginalis TK in vitro. In vivo they inhibit [3H]thymidine incorporation in T. vaginalis cultured cells and T. vaginalis growth (IC50 7.5 and 24 microM respectively; minimal lethal dose 100 microM). Thus the TK inhibitors described here demonstrate the key role of T. vaginalis TK for protozoal growth and viability and indicate TK as a new target for the design of antitrichomonal drugs. PMID:9693096

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

    PubMed

    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. PMID:19154348

  9. Effect of epidermal growth factor (EGF) on (/sup 3/H)TdR incorporation into DNA in ad lib fed and fasted CD2F1 mice

    SciTech Connect

    Scheving, L.A.; Tsai, T.H.; Scheving, L.E.; Hoke, W.S.

    1987-03-01

    The effect of EGF on the incorporation of (/sup 3/H)TdR into DNA (DNA synthesis) was determined in the esophagus, liver, pancreas, and kidney in mice standardized to 12 hours (hr) of light alternating with 12 hr of darkness. A question asked was whether intraperitoneally administered EGF could alter the circadian patterns of DNA synthesis in these organs. The most marked effects of EGF were: an increase in DNA synthesis but only after a specific duration of time after treatment, ranging from 8 to 23 hr, which differed for each tissue, a similarity in the response of the esophagus in both ad lib fed and fasted mice, but not in the response of the liver, where the stimulatory effect of EGF observed in fed mice was dramatically reduced in fasted ones, and an advance in the phasing of the circadian rhythm in DNA synthesis of the esophagus by about 12 hr. In addition, no sex differences in fasted animals were found under the conditions of this study.

  10. Effect of lipopolysaccharide on thymidine salvage as related to macrophage activation.

    PubMed Central

    Harada, Y; Nagao, S; Nakamura, M; Okada, F; Tanigawa, Y

    1995-01-01

    Lipopolysaccharide (LPS), known as one of the potent activators of macrophages, has inhibitory effects on the proliferation of normal macrophages and macrophage-like cell lines. We report here that LPS dose- and time-dependently suppressed the tritiated thymidine ([3H]TdR) incorporation into the acid-insoluble fraction with a significant inverse correlation to the tumour necrosis factor-alpha (TNF) production in the J774.1 macrophage cell line. Among the three tested enzymes involved in DNA synthesis, only thymidine kinase (TK) activity decreased progressively in parallel with the decline in [3H]TdR incorporation, reaching 97% inhibition within 12 hr of LPS treatment, while changes in the activities of other two enzymes, DNA polymerase alpha and thymidylate synthase (TS), were less significant. On the other hand, LPS inhibited the cell proliferation only incompletely, as judged by 62% inhibition of cell growth at 36 hr. Even in the experiments done in a TdR-free medium, cell growth was inhibited by LPS to the same extent, suggesting that TK was not directly involved in the proliferation of J774 cells. LPS also inhibited the conversion of TdR to thymidine monophosphate (TMP) in murine peritoneal exudate macrophages (PEM). Thus LPS-induced suppression of TdR salvage related to TNF production is common in both normal and neoplastic macrophages, and therefore may be of potential importance in the process of macrophage activation. PMID:7751001

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

    SciTech Connect

    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.

  12. Use of Radiolabelled Thymidine and Leucine To Estimate Bacterial Production in Soils from Continental Antarctica

    PubMed Central

    Tibbles, B. J.; Harris, J. M.

    1996-01-01

    Tritiated thymidine incorporation (TTI) into DNA was used to examine bacterial production in two soil types from the Robertskollen group of nunataks in northwestern Dronning Maud Land, providing the first estimates of bacterial production in soil habitats on the Antarctic continent. Although estimates of bacterial productivity in soils near to bird nests (344 (plusmn) 422 ng of C g [dry weight](sup-1) h(sup-1)) were higher than those for soils from beneath mosses (175 (plusmn) 90 ng of C g [dry weight](sup-1) h(sup-1); measured by TTI at 10(deg)C), these differences were not significant because of patchiness of bacterial activity (P > 0.05). TTI- and [(sup14)C]leucine ([(sup14)C]Leu)-derived estimates of bacterial production were similar when incubations of 3 h were used, although incubations as short as 1 h were sufficient for measurable uptake of radiolabel. Dual-label incorporation of [(sup3)H]thymidine ([(sup3)H]TdR) into DNA and [(sup14)C]Leu into protein indicated that TTI did not reflect bacterial production of in situ assemblages when incubations were longer than 3 h. Isotope dilution analysis indicated that dilution of the specific activity of exogenously supplied [(sup3)H]TdR by de novo synthesis of TdR precursor could be limited by additions of [(sup3)H]TdR at a concentration of 1 nmol per ca. 115 mg of soil. TTI exhibited a psychrotrophic response to variation in temperature, with a temperature optimum of ca. 15(deg)C and a Q(inf10) value for 0 to 10(deg)C of 2.41. PMID:16535246

  13. Effect of colchicine on rat small intestinal absorptive cells. II. Distribution of label after incorporation of (/sup 3/H)fucose into plasma membrane glycoproteins

    SciTech Connect

    Ellinger, A.; Pavelka, M.; Gangl, A.

    1983-12-01

    By means of radioautography the influence was tested of various periods (5, 15, 30, 40 min, 2 hr) of pretreatment with colchicine, administered intraperitoneally to rats at a dosage of 0.5 mg/100 g of body weight, on the intracellular pathway of (/sup 3/H)fucose in absorptive cells of the small intestine. Administration of colchicine for 30 min and longer time intervals causes delay in the insertion of (/sup 3/H)fucose into the oligosaccharide chains of glycoconjugates in the Golgi apparatus, and results in redistribution of the label apparent over the different portions of the plasma membrane. In controls, at 2 and 4 hr after administration of (/sup 3/H)fucose the apical plasma membrane is strongly labeled. Colchicine causes equalization of the reaction of apical and basolateral regions of the plasma membrane: the number of silver grains attributable to the apical plasma membrane is reduced; following treatment with colchicine, apical portions of the plasma membrane comprise 31.6 +/- 1.8% of the silver grains, 38.6 +/- 3.8% are attributable to basolateral membrane regions. The colchicine-induced equalization of the density of label of apical and basolateral regions of the plasma membrane, in addition to the occurrence of basolateral microvillus borders, suggests microtubules to be important in the maintenance of the polar organization of small intestinal absorptive cells.

  14. Tracer kinetic model for quantitative imaging of thymidine ultilization using [C-11] thymidine and PET

    SciTech Connect

    Mankoff, D.A.; Shields, A.F.; Lee, T.T.

    1994-05-01

    2-[C-11]thymidine, a marker of thymidine incorporation into DNA, is a PET tracer for assessing tumor proliferation. Quantitation of thymidine images is complicated by the presence of C-11 labeled metabolites, which include thymidine degradation products such as thymine, as well as labeled carbon dioxide (CO{sub 2}). We have therefore formulated and analyzed a compartmental model of tracer and metabolite distribution for the estimation of the thymidine incorporation rate (TIR), which is closely tied to the DNA synthetic rate. During [C-11]thymidine studies, the activities of intact thymidine (Tdr), labeled CO{sub 2} (CO{sub 2}), and labeled non-carbon dioxide metabolites (Met) are measured from blood samples. The model uses these blood time-activity curves as the inputs to three separate sets of compartments representing tissue Tdr, Met, and CO{sub 2}. There are 9 parameters to be estimated by optimization of the model, given the three input functions and a tissue time-activity curve obtained from PET images taken over the 60 minutes following injection. The TIR is estimated from the rate constants for transfer between the plasma and the Tdr tissue compartments. To simplify parameter estimation, we have determined through sensitivity analysis and simulations that 4 of the parameters can be fixed to physiological reasonable values without overly biasing the estimate of the TIR. The remaining 5 parameters, including those necessary to estimate the TIR, can be floated in the optimization and reliably determined. Simulations show that errors in the assumed values for the fixed parameters lead to worst-case errors in the TIR estimate on the order of 25-30%. We therefore conclude that quantitative imaging of tumor proliferation with [C-11]thymidine is feasible and may be advantageous in tumor imaging, particularly following the response of tumors to therapy.

  15. 3H-Penciclovir (3H-PCV) Uptake Assay

    PubMed Central

    Sekar, Thillai V; Paulmurugan, Ramasamy

    2016-01-01

    Thymidine Kinase from human Herpes simplex virus type 1 (HSV1-TK) in combination with specific substrate prodrug nucleotide analogue ganciclovir (GCV) has been widely used as suicidal therapeutic gene for cancer gene therapy. HSV1, and its mutant (HSV1-sr39TK) with improved substrate specificity, were used as reporter genes for PET-imaging of various biological functions in small animals, by combining with radiolabeled substrates such as 18F-FHBG and 124I-FIAU. 3H-Penciclovir (PCV) uptake assay is a method of choice used to determine the expression level of HSV1-TK in mammalian cells and tissues. HSV1-TK phosphorylate PCV and result in the formation of penciclovir monophosphate, and its subsequent phopsphorylation by cellular TK lead to the formation of penciclovir triphosphate, which is trapped selectively in cells expressing HSV-TK. 3H-Penciclovir enables the detection of penciclovir uptake of mammalian cells and tissues by radioactive procedures such as scintillation counting. Here we describe the protocol to carry out 3H-Penciclovir uptakes in mammalian cells.

  16. Organic Photosensitizers Incorporating Rigidified Dithieno[3,2-f:2',3'-h]quinoxaline Segment Tethered with Thiophene Substitutes for Dye-Sensitized Solar Cells.

    PubMed

    Ni, Jen-Shyang; Chiu, Tang-Yao; Kao, Wei-Siang; Chou, Hao-Ju; Su, Chao-Chin; Lin, Jiann T

    2016-09-01

    Metal-free D-π-RS-π-A type sensitizers, consisting of triphenylamine as the electron donor, 2,3-bis(3-(2-ethylhexyl)-5-methylthiophen-2-yl)dithieno[3,2-f:2',3'-h]quinoxaline (DTQT) as the rigidified conjugation spacer (RS), thiophene as the π-spacer, and 2-cyanoacrylic acid as the acceptor/anchor, have broad absorption spectra ranging from 350 to 550 nm and a high molar extinction coefficient up to >46 200 M(-1) cm(-1). Under simulated AM 1.5 G illumination, the dye-sensitized solar cells (DSSCs) fabricated from the dyes exhibited light-to-electricity conversions in the range of 6.78% to 8.27%. The best efficiency is slightly higher than that of N719-based standard DSSC (7.92%). The efficiency can be further boosted to 8.51% by optimizing the concentration of LiI electrolyte. PMID:27523392

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

    SciTech Connect

    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 (/sup 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.

  18. Further studies of the metabolic incorporation and covalent binding of inhaled (/sup 3/H)- and (/sup 14/C)formaldehyde in Fischer-344 rats: effects of glutathione depletion

    SciTech Connect

    Casanova, M.; Heck Hd'

    1987-06-15

    Glutathione (GSH) is required for the oxidation of formaldehyde (HCHO) to formate catalyzed by formaldehyde dehydrogenase (FDH). The effects of GSH depletion on the mechanisms of labeling of macromolecules in the rat nasal mucosa and bone marrow by /sup 3/HCHO and H/sup 14/CHO were investigated. Male rats were exposed for 3 hr to atmospheres containing /sup 3/HCHO and H/sup 14/CHO at concentrations of 0.9, 2, 4, 6, or 10 ppm, 1 day after a single 3-hr preexposure to the same concentration of unlabeled HCHO. Two hours prior to the second exposure, the animals were injected either with phorone (300 mg/kg, ip) or with corn oil. The concentration of nonprotein sulfhydryls in the nasal respiratory mucosa of phorone-injected rats was decreased to 10% of that of corn oil-injected rats. The metabolic incorporation of /sup 3/HCHO and H/sup 14/CHO into DNA, RNA, and proteins in the respiratory and olfactory mucosa and bone marrow (femur) was significantly decreased, and DNA-protein crosslinking was significantly increased in the respiratory mucosa of phorone-injected relative to corn oil-injected rats at all HCHO concentrations. DNA-protein crosslinks were not detected in the respiratory mucosa of corn oil-injected rats at 0.9 ppm. Evidence was obtained for the formation of adducts of HCHO with the RNA from the nasal respiratory mucosa of phorone-injected rats at concentrations above 0.9 ppm. Covalent binding of HCHO to macromolecules in the bone marrow was not detected. These results indicate that the GSH-dependent oxidation of HCHO catalyzed by FDH is an important defense mechanism against the covalent reactions of HCHO with nucleic acids in the respiratory mucosa.

  19. Flaviviruses Are Sensitive to Inhibition of Thymidine Synthesis Pathways

    PubMed Central

    Fischer, Matthew A.; Smith, Jessica L.; Shum, David; Stein, David A.; Parkins, Christopher; Bhinder, Bhavneet; Radu, Constantin; Hirsch, Alec J.; Djaballah, Hakim; Nelson, Jay A.

    2013-01-01

    Dengue virus has emerged as a global health threat to over one-third of humankind. As a positive-strand RNA virus, dengue virus relies on the host cell metabolism for its translation, replication, and egress. Therefore, a better understanding of the host cell metabolic pathways required for dengue virus infection offers the opportunity to develop new approaches for therapeutic intervention. In a recently described screen of known drugs and bioactive molecules, we observed that methotrexate and floxuridine inhibited dengue virus infections at low micromolar concentrations. Here, we demonstrate that all serotypes of dengue virus, as well as West Nile virus, are highly sensitive to both methotrexate and floxuridine, whereas other RNA viruses (Sindbis virus and vesicular stomatitis virus) are not. Interestingly, flavivirus replication was restored by folinic acid, a thymidine precursor, in the presence of methotrexate and by thymidine in the presence of floxuridine, suggesting an unexpected role for thymidine in flavivirus replication. Since thymidine is not incorporated into RNA genomes, it is likely that increased thymidine production is indirectly involved in flavivirus replication. A possible mechanism is suggested by the finding that p53 inhibition restored dengue virus replication in the presence of floxuridine, consistent with thymidine-less stress triggering p53-mediated antiflavivirus effects in infected cells. Our data reveal thymidine synthesis pathways as new and unexpected therapeutic targets for antiflaviviral drug development. PMID:23824813

  20. Elevated thymidine phosphorylase activity in psoriatic lesions accounts for the apparent presence of an epidermal growth inhibitor, but is not in itself growth inhibitory

    SciTech Connect

    Hammerberg, C.; Fisher, G.J.; Voorhees, J.J.; Cooper, K.D. )

    1991-08-01

    An apparent tissue-specific growth inhibitor, or chalone, obtained from psoriatic lesions was tentatively identified in the 100-kDa fraction based upon inhibition of DNA synthesis, as measured by (3H)-thymidine uptake by a squamous cell carcinoma cell line, SCC 38. This fraction, however, failed to inhibit SCC 38 cell growth when assessed directly in a neutral red uptake assay. Characterization of the inhibitor of (3H)-thymidine uptake revealed it to have biochemical properties identical to thymidine phosphorylase: (1) molecular weight close to 100 kDa, (2) isoelectric point of 4.2, and (3) thymidine phosphorylase enzyme activity. Thus, the authors conclude that its ability to inhibit (3H)-thymidine uptake was due to thymidine catabolism rather than inhibition of DNA synthesis or growth inhibition. Examination of thymidine phosphorylase activity in keratome biopsies from psoriatic and normal skin demonstrated a twentyfold increase in activity in psoriatic lesions relative to non-lesional or normal skin. This increase in metabolism of thymidine was due to thymidine phosphorylase rather than uridine phosphorylase activity. The correlation between increased thymidine phosphorylase activity and increased keratinocyte proliferation in vitro (cultured) and in vivo (psoriasis), suggests that this enzyme may play a critical role in providing the thymidine necessary for keratinocyte proliferation.

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

    SciTech Connect

    Waldman, A.S.; Milman, G.

    1984-08-01

    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 (/sup 3/H)thymidine into cell nuclei as measured by autoradiography.

  2. Thymidine secretion by hybridoma and myeloma cells

    SciTech Connect

    Spilsberg, Bjorn . E-mail: bjorn.spilsberg@biokjemi.uio.no; Rise, Frode; Petersen, Dirk; Nissen-Meyer, Jon

    2006-03-31

    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 {mu}M 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.

  3. Measurement of in vitro leucocyte mitogenesis in fish: ELISA based detection of the thymidine analogue 5'-bromo-2'-deoxyuridine

    USGS Publications Warehouse

    Gauthier, David T.; Cartwright, Deborah D.; Densmore, Christine L.; Blazer, Vicki; Ottinger, Christopher A.

    2003-01-01

    In this study we present a method for the measurement of in vitro mitogenesis in fish leucocytes that is based on the incorporation of the thymidine analogue 5′-bromo-2′-deoxyuridine (BrdU) into the DNA of replicating cells, followed by ELISA-based detection. This technique, adapted from methods developed for mammalian cells, operates on a similar biological principle to 3H-thymidine incorporation, but circumvents the logistical and safety issues inherent with the radioactive label. Because it directly measures DNA proliferation, the assay has advantages over other colorimetric methods that may be strongly influenced by leucocyte metabolic status. Using BrdU incorporation followed by ELISA, we evaluate the responsiveness of rainbow trout (Oncorhynchus mykiss [Walbaum]) leucocytes to the mammalian T-cell mitogen Concanavalin A (Con A) as well as the differential response of white perch (Morone americana [Gmelin]) leucocytes to Con A and pokeweed mitogen. Specific considerations intrinsic to the assay system are discussed, including the implications of utilising enzyme-based detection.

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

    SciTech Connect

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

    1984-11-01

    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 (/sup 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. Tritiated thymidine uptake in chondrocytes of chickens afflicted with tibial dyschondroplasia

    SciTech Connect

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

    1985-10-01

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

  6. Radioimmunoassays of plasma thymidine, uridine, deoxyuridine, and cytidine/deoxycytidine

    SciTech Connect

    Dudman, N.P.B.; Deveski, W.B.; Tattersall, M.H.N.

    1981-08-01

    Radioimmunoassay techniques have been developed for the assay of thymidine, uridine, deoxyuridine, and deoxycytidine. Plasma levels of the first three nucleosides have been measured, and an upper limit has been determined for the plasma concentration of deoxycytidine. The assays involve displacement of a (3H)pyrimidine nucleoside from the appropriate labeled rabbit immunoglobulin. By assaying a mixture of uridine and deoxyuridine in the presence and absence of borax, the concentrations of both nucleosides have been measured. In seven healthy adults, plasma levels of uridine were 21.1 +/- 8.4 ..mu..M (mean +/- SD) and of deoxyuridine were 0.62 +/- 0.39 ..mu..M. In cancer patients, thymidine levels were 7.5 +/- 2.7 x 10/sup -7/M. The upper limit for plasma deoxycytidine levels in six healthy adults was 0.71 +/- 0.1 ..mu..M.

  7. Thymidine Phosphorylase is Angiogenic and Promotes Tumor Growth

    NASA Astrophysics Data System (ADS)

    Moghaddam, Amir; Zhang, Hua-Tang; Fan, Tai-Ping D.; Hu, De-En; Lees, Vivien C.; Turley, Helen; Fox, Stephen B.; Gatter, Kevin C.; Harris, Adrian L.; Bicknell, Roy

    1995-02-01

    Platelet-derived endothelial cell growth factor was previously identified as the sole angiogenic activity present in platelets; it is now known to be thymidine phosphorylase (TP). The effect of TP on [methyl-^3H]thymidine uptake does not arise from de novo DNA synthesis and the molecule is not a growth factor. Despite this, TP is strongly angiogenic in a rat sponge and freeze-injured skin graft model. Neutralizing antibodies and site-directed mutagenesis confirmed that the enzyme activity of TP is a condition for its angiogenic activity. The level of TP was found to be elevated in human breast tumors compared to normal breast tissue (P < 0.001). Overexpression of TP in MCF-7 breast carcinoma cells had no effect on growth in vitro but markedly enhanced tumor growth in vivo. These data and the correlation of expression in tumors with malignancy identify TP as a target for antitumor strategies.

  8. Acute effect of prostaglandins and somatostatin on thymidine uptake of gastric mucosa cells

    SciTech Connect

    Alino, S.F.; Hilario, E. )

    1988-12-01

    This study compares the in vivo effect of exogenous administration of prostaglandin E2 (30 {mu}g/kg) and its precursor, arachidonic acid (30 {mu}g/kg), with the effect of indomethacin (5 mg/kg) on the 6-({sup 3}H)thymidine uptake of antral mucosa of mice by autoradiographical methods. Likewise, the effect of somatostatin (30 {mu}g/kg) on 6-({sup 3}H)thymidine uptake is studied. Evaluation of the number of labeled cells, in the histological sections of the gastric mucosa, showed that arachidonic acid, prostaglandin E2, and somatostatin induced an increase in the number of labeled cells (107, 44, and 45%, respectively), while indomethacin induced a decrease of 32% compared to the control group. These results suggest that prostaglandins may mediate stimulatory effects on thymidine uptake of gastric mucosa cells in the first step after drug administration.

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

    SciTech Connect

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

    1984-12-01

    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.

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

    PubMed

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

    1999-02-15

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

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

    SciTech Connect

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

    1989-06-01

    The thymidine kinase gene encoded by herpesvirus of turkeys has been identified and characterized. A viral mutant (ATR/sup 0/) resistant to 1-..beta..-D-arabinofuranosylthymine was isolated. This mutant was also resistant to 1-(2-fluoro-2-deoxy-..beta..-D-arabinofuronosyl)-5-methyluracil and was unable to incorporate (/sup 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.

  12. Synthesis and Crystallographic Analysis of 5-Se-Thymidine DNAs

    SciTech Connect

    Hassan, A.; Sheng, J; Jiang, J; Zhanbg, W; Huang, Z

    2009-01-01

    We investigated the possibility of the interaction of 5-CH3 of thymidine and its 5?-phosphate backbone (C-H O-PO3 interaction) in DNA via the insertion of the atomic probe (a selenium atom) into the exo-5-position of thymidine (5-Se-T). 5-Se-T was synthesized for the first time, via Mn(OAc)3 assisted electrophilic addition of CH3SeSeCH3 to 3?,5?-di-O-benzoyl-2?-deoxyuridine. The 5-Se-T phosphoramidite was subsequently synthesized and incorporated into DNA in over 99% coupling yield. Biophysical and structural investigations of the 5-Se-T DNAs revealed that the Se-modified and nonmodified DNAs are virtually identical. In addition, the crystallographic analysis of a 5-Se-T DNA strongly suggests a hydrogen-bond formation between the 5-CH3 and 5?-phosphate groups (CH3 PO4- interaction).

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

    PubMed Central

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

    2016-01-01

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

  14. Assessment of thymidine phosphorylase function: measurement of plasma thymidine (and deoxyuridine) and thymidine phosphorylase activity.

    PubMed

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

    2012-01-01

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

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

    SciTech Connect

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

    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.

  16. Cell killing and mutation to 6-thioguanine resistance after exposure to tritiated amino acids and tritiated thymidine in cultured mammalian cells (L5178Y)

    SciTech Connect

    Furuno-Fukushi, I.; Ueno, A.M.; Matsudaira, H.

    1987-06-01

    Cell killing and mutation to 6-thioguanine resistance were studied in growing mouse leukemia cells in culture after exposure to tritiated amino acids and tritiated thymidine. These effects varied widely among the tritiated compounds tested, being greatest for tritiated thymidine followed by tritiated arginine and tritiated lysine, in that order, for a given concentration of /sup 3/H expressed in kBq/ml of /sup 3/H in the medium. The differences between each tritiated amino acid disappeared almost totally when the effects were compared on the basis of the absorbed dose to the cells. The effects of tritiated thymidine, however, remained more than twofold greater compared to other tritiated compounds. These results indicate the importance of determining the absorbed dose for assessment of the radiotoxicity of tritiated organic compounds. For an exceptional case (tritiated thymidine), contribution of a mechanism(s) other than beta irradiation should also be taken into account.

  17. Effect of 5-Fluorouracil on Thymidine Phosphorylase Activity in Model Experiment.

    PubMed

    Stashkevich, M A; Khomutov, E V; Dumanskii, Yu V; Matvienko, A G; Zinkovich, I I

    2016-03-01

    Variations in thymidine phosphorylase activity in rat liver were studied in 1, 3, 6, 12, and 24 h after intraperitoneal bolus injection of 5-fluorouracil. Enzyme activity was measured by HPLC. A 2-fold decrease in enzyme activity was observed 3 h after 5-fluorouracil administration and persisted for 12 h. This additional effect of the cytostatic should be taken into account in choosing chemotherapy protocol. PMID:27021101

  18. Enhanced thymidine uptake causes the lowered thymidine requirement of D. discoideum auxotroph HPS 401

    SciTech Connect

    Hurley, D.L.; Deering, R.A. )

    1988-11-01

    Dictyostelium discoideum strain HPS 401 contains a spontaneous mutation that lowers the amount of thymidine required for cell growth relative to that of the auxotrophic parental strain HPS 400. Assays for enzymes related to thymidine metabolism reveal that none of the strains tested (HPS 401, HPS 400, and prototrophic HPS 83 cells) contain detectable thymidine phosphorylase activity and that the specific activity of thymidine kinase is the same in these three strains. Thin-layer chromatography of extracts from cells grown on radiolabeled thymidine shows that there is no detectable conversion of thymidine to thymine in any of these strains. These analyses show that HPS 401 has rapid intracellular accumulation of thymidine, while only slight uptake is observed with HPS 400 or wild-type strains. HPS 401 also shows greater uptake of uridine in comparison to HPS 400 and wild-type cells. Thymidylate uptake was the same for all three strains. Thus, the mutation giving rise to the HPS 401 phenotype selectively increases the uptake of thymidine into the cell, where it can be efficiently utilized for DNA synthesis by the salvage pathways of nucleotide metabolism.

  19. Effects of thymidine and uridine on the phosphorylation of 3'-azido-3'-deoxythymidine (zidovudine) in human mononuclear cells

    SciTech Connect

    Szebeni, J.; Patel, S.S.; Hung, K.; Wahl, L.M.; Weinstein, J.N. )

    1991-01-01

    The effects of thymidine and uridine on the phosphorylation of 3'-azido-3'-deoxythymidine (AZT) were studied in various human mononuclear cell preparations. Thymidine suppressed ({sup 3}H)AZT phosphorylation in the same concentration range (20 to 100 microM) in which it antagonizes the anti-human immunodeficiency virus activity of AZT. Uridine, in turn, had no influence on AZT phosphorylation, just as it has no effect on the anti-human immunodeficiency virus activity of AZT. These findings are consistent with a close relationship between the inhibition of AZT phosphorylation and the influence of physiological nucleosides on the antiviral activity of AZT.

  20. Artificial mutants generated by the insertion of random oligonucleotides into the putative nucleoside binding site of the HSV-1 thymidine kinase gene

    SciTech Connect

    Dube, D.K.; Parker, J.D.; French, D.C.; Cahill, D.S.; Dube, S.; Horwitz, M.S.Z.; Munir, K.M.; Loeb, L.A. )

    1991-12-24

    The authors have obtained 42 active artificial mutants of HSV-1 thymidine kinase by replacing codons 166 and 167 with random nucleotide sequences. Codons 166 and 167 are within the putative nucleoside binding site in the HSV-1 tk gene. The spectrum of active mutations indicates that neither Ile{sup 166} nor Ala{sup 167} is absolutely required for thymidine kinase activity. Each of these amino acids can be replaced by some but not all of the 19 other amino acids. The active mutants can be classified as high activity or low activity on two bases: (1) growth of Escherichia coli KY895 in the presence of thymidine and (2) uptake of thymidine by this strain, when harboring plasmids with the random insertions. E. coli KY895 harboring high-activity plasmids or wild-type plasmids can grow in the presence of low amounts of thymidine but are unable to grow in the presence of high amounts of thymidine. The high-activity plasmids also have an enhanced ({sup 3}H)dT uptake. The amounts of thymidine kinase activity in vitro in unfractionated extracts do not correlate with either growth at low thymidine concentration or the rate of thymidine uptake. Heat inactivation studies indicate that the mutant enzymes are without exception more temperature-sensitive than the wild-type enzyme. This thermolability could account for the less than expected thymidine kinase activity in the extracts and suggests that amino acid substitutions at Ile{sup 166} and Ala{sup 167} have produced major changes in protein stability.

  1. Microautoradiographic localisation of [3H]sucrose and [3H]mannitol in Robinia pseudoacacia pulvinar tissues during phytochrome-mediated nyctinastic closure.

    PubMed

    Moysset, L; Llambrich, E; López-Iglesias, C; Simón, E

    2006-11-01

    We have analysed the incorporation of [(3)H]sucrose and [(3)H]mannitol in pulvinar motor cells of Robinia pseudoacacia L. during phytochrome-mediated nyctinastic closure. Pairs of leaflets, excised 2 h after the beginning of the photoperiod, were fed with 50 mM [(3)H]sucrose or [(3)H]mannitol, irradiated with red (15 min) or far-red (5 min) light and placed in the dark for 2-3 h. Label uptake was measured in whole pulvini by liquid scintillation counting. The distribution of labelling in pulvinar sections was assessed by both light and electron microautoradiography. [(3)H]Sucrose uptake was twice that of [(3)H]mannitol incorporation in both red- and far-red-irradiated pulvini. In the autoradiographs, [(3)H]sucrose and [(3)H]mannitol labelling was localised in the area from the vascular bundle to the epidermis, mainly in vacuoles, cytoplasm, and cell walls. Extensor and flexor protoplasts displayed a different distribution of [(3)H]sucrose after red and far-red irradiation. Far-red light drastically reduced the [(3)H]sucrose incorporation in extensor protoplasts and caused a slight increase in internal flexor protoplasts. After red light treatment, no differences in [(3)H]sucrose labelling were found between extensor and flexor protoplasts. Our results indicate a phytochrome control of sucrose distribution in cortical motor cells and seem to rule out the possibility of sucrose acting as an osmoticum. PMID:17102931

  2. Thymidine labeling index: prognostic role in breast cancer.

    PubMed

    Bilir, Ayhan; Ozmen, V; Kecer, M; Eralp, Yesim; Cabioglu, Neslihan; Ahishali, Bulent; Agizhali, Bulent; Camlica, Hakan; Aydiner, Adnan

    2004-08-01

    The aim of this study is to evaluate the prognostic role of thymidine labeling index in patients with breast cancer. Cellular proliferation rates in 155 breast cancer specimens were investigated by 3H-thymidine labeling index (3H-TLI). Median age was 47 years (range: 23-76). At presentation, 11 patients (7.1%) had stage I disease, 76 (49%) had stage II, 64 (41.3%) had stage III disease, and 4 (2.6%) had metastatic involvement. Patients were placed in 2 groups based on their proliferative indices. The cut-off level was assigned as the median TLI value of the whole group. Correlations between proliferative activity of the tumors based on 3H-TLI levels and various previously established prognostic factors, as well as the influence of proliferative activity on survival as a clinical outcome, were analyzed. The mean and median TLI values for the whole group of patients were 4.36 +/- 4.96% and 2.76% (range: 0-23.6), respectively. There was a significant association of nuclear grade with TLI (P = 0.04). Patients who were alive with no sign of disease at the final follow-up examination had a significantly lower median TLI rate than those who were either alive with disease or those who had eventually died with disease progression (3.7% versus 1.9%, respectively; P = 0.04). Patients with locally advanced disease (N2 + N3 involvement) had a significantly higher median TLI rate than those with local nodal involvement (N1) (3.4% versus 1.7%, respectively, P = 0.026). Furthermore, TLI levels showed a significant association with overall survival in patients with node-negative disease (P = 0.02). Based on the results of this study, it can be concluded that TLI plays a significant prognostic role in a subset of patients with node-negative breast cancer. Furthermore, TLI appears to have a predictive value for the clinical outcome of patients with breast cancer. These findings may justify a more aggressive therapeutic approach in patients with high TLI levels. Further large

  3. Oxidative damage to DNA constituents by iron-mediated Fenton reactions--the thymidine family.

    PubMed

    Chattopadhyaya, Rajagopal

    2014-01-01

    The degradative products formed from the exposure of derivatives of thymidine to iron-mediated Fenton reactions were identified by chromatographic resolution and analysis by UV absorption spectroscopy, radio-labeling, and positive and negative mode fast atom bombardment mass spectrometry. Fe(2+)/H2O2 and Fe(3+)/H2O2 were utilized to generate oxy-radicals and the effects of ethanol and N2-flushing were studied. Substrates included thymidine, 3'- and 5'-dTMP, TpT, oligo(dT), and oligo(dT)·poly(dA) and the results are compared to those reported for ionizing radiation. It is evident from the comparisons of the products that the damaging radical species and the product distribution are perturbed by interaction of the iron atom with the various phosphomono- and diester species that are present. PMID:23252741

  4. Lack of stereospecificity of suid pseudorabies virus thymidine kinase.

    PubMed Central

    Maga, G; Verri, A; Bonizzi, L; Ponti, W; Poli, G; Garbesi, A; Niccolai, D; Spadari, S; Focher, F

    1993-01-01

    We have partially purified suid pseudorabies virus (PRV) thymidine kinase from infected thymidine kinase- mouse cells, and cytosolic swine thymidine kinase from lymphatic glands, and we have found that PRV thymidine kinase, unlike the host enzyme, shows no stereospecificity for D- and L-beta-nucleosides. In vitro, unnatural L-enantiomers, except L-deoxycytidine, function as specific inhibitors for the viral enzyme in the order: L-thymidine >> L-deoxyguanosine > L-deoxyuridine > L-deoxyadenosine. Contrary to human and swine thymidine kinases and like herpes simplex virus-1 and -2 thymidine kinases, PRV thymidine kinase phosphorylates both the natural (D-) and the unnatural (L-) thymidine enantiomers to their corresponding monophosphates with comparable efficiency. The kinetic parameters Vmax/Km for D- and L-thymidine are 3.7 and 2.3 respectively. Our results demonstrate that the lack of stereospecificity might be a common feature of the thymidine kinases that are encoded by human and animal herpes viruses. These observations could lead to the development of a novel class of antiviral drugs. PMID:8396911

  5. Lack of stereospecificity of suid pseudorabies virus thymidine kinase.

    PubMed

    Maga, G; Verri, A; Bonizzi, L; Ponti, W; Poli, G; Garbesi, A; Niccolai, D; Spadari, S; Focher, F

    1993-09-01

    We have partially purified suid pseudorabies virus (PRV) thymidine kinase from infected thymidine kinase- mouse cells, and cytosolic swine thymidine kinase from lymphatic glands, and we have found that PRV thymidine kinase, unlike the host enzyme, shows no stereospecificity for D- and L-beta-nucleosides. In vitro, unnatural L-enantiomers, except L-deoxycytidine, function as specific inhibitors for the viral enzyme in the order: L-thymidine > L-deoxyguanosine > L-deoxyuridine > L-deoxyadenosine. Contrary to human and swine thymidine kinases and like herpes simplex virus-1 and -2 thymidine kinases, PRV thymidine kinase phosphorylates both the natural (D-) and the unnatural (L-) thymidine enantiomers to their corresponding monophosphates with comparable efficiency. The kinetic parameters Vmax/Km for D- and L-thymidine are 3.7 and 2.3 respectively. Our results demonstrate that the lack of stereospecificity might be a common feature of the thymidine kinases that are encoded by human and animal herpes viruses. These observations could lead to the development of a novel class of antiviral drugs. PMID:8396911

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

    PubMed

    Sun, Ren; Wang, Liya

    2014-10-01

    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. PMID:25215937

  7. Comparative double-tracer whole-body autoradiography: uptake of 11C-, 18F- and 3H-labeled compounds in rat tumors.

    PubMed

    d'Argy, R; Paul, R; Frankenberg, L; Stålnacke, C G; Lundqvist, H; Kangas, L; Halldin, C; Någren, K; Roeda, D; Haaparanta, M

    1988-01-01

    The uptake of various labeled compounds by tumors was studied by double-tracer whole-body autoradiography (DTWBA) in rats. Each animal carried two types of tumors: mammary carcinomas and the Walker 256 carcinosarcomas. The markers used were [18F]- and [3H]fluorodeoxyglucose (glucose utilization), [3H]thymidine (cell proliferation), [11C]methionine (amino acid metabolism) and [11C]- and [3H]toremifene (estrogen-receptor-avid agents). In each experiment, the distribution of a substance labeled with short-lived radionuclide (11C or 18F) was compared with that of another substance labeled with a long-lived nuclide (3H). Quantification was done by combining computerized image analysis of the autoradiograms with liquid scintillation counting of punched tissue pieces obtained from the cryosections. The relationships between the uptakes of the various radiopharmaceuticals were recorded in tumors and normal tissues. The dynamics of [18F]fluorodeoxyglucose and [11C]methionine were determined in tumors and some selected tissues by positron emission tomography (PET). The uptake rate between fluorodeoxyglucose and thymidine in the mammary tumor was five times higher than the ratio in the Walker tumor. The corresponding figure for FDG/methionine was four times. Thymidine, compared with methionine, was twice as efficient. Thus, the mammary tumors were best imaged with FDG or thymidine. The non-steroid antiestrogen toremifene was taken up in very low amounts by these tumors. By DTWBA, experimental tumors may serve as their own control. PMID:2978293

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

    SciTech Connect

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

    1982-01-30

    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 /sup 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. Thymidine Phosphorylase in Cancer; Enemy or Friend?

    PubMed

    Elamin, Yasir Y; Rafee, Shereen; Osman, Nemer; O Byrne, Kenneth J; Gately, Kathy

    2016-04-01

    Thymidine phosphorylase (TP) is a nucleoside metabolism enzyme that plays an important role in the pyrimidine pathway.TP catalyzes the conversion of thymidine to thymine and 2-deoxy-α-D-ribose-1-phosphate (dRib-1-P). Although this reaction is reversible, the main metabolic function of TP is catabolic. TP is identical to the angiogenic factor platelet-derived endothelial-cell growth factor (PD-ECGF). TP is overexpressed in several human cancers in response to cellular stressful conditions like hypoxia, acidosis, chemotherapy and radiotherapy. TP has been shown to promote tumor angiogenesis, invasion, metastasis, evasion of the immune-response and resistance to apoptosis. Some of the biological effects of TP are dependent on its enzymatic activity, while others are mediated through cytokines like interleukin 10 (IL-10), basic fibroblast growth factor (bFGF) and tumour necrosis factor α (TNFα). Interestingly, TP also plays a role in cancer treatment through its role in the conversion of the oral fluoropyrimidine capecitabine into its active form 5-FU. TP is a predictive marker for fluoropyrimidine response. Given its various biological functions in cancer progression, TP is a promising target in cancer treatment. Further translational research is required in this area. PMID:26298314

  10. Photofragmentation of a DNA nucleoside thymidine; valence- vs. core ionization

    NASA Astrophysics Data System (ADS)

    Itälä, E.; Kooser, K.; Hägerth, T.; Rachlew, E.; Huels, M. A.; Kukk, E.

    2012-11-01

    The photofragmentation of free thymidine molecule has been studied using combined electron- and ion spectroscopy. The results show that valence- and core ionization processes produce very different fragmentation patterns and that regardless of the photon energy, the photoionization leads almost always to dissociation of the thymidine molecule.

  11. A fluorescent surrogate of thymidine in duplex DNA.

    PubMed

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

    2016-03-17

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

  12. Solvent type influence on thymidine UV-sensitivity

    NASA Astrophysics Data System (ADS)

    Nikolaev, A. I.; Paston, S. V.

    2015-12-01

    Thymine is the most sensitive DNA nucleobase to UV-irradiation. In the thymidine solution the photoreactions probability is in dependence on the solvent properties which determine stacking-mediated thymidine association and lifetime of excited states. In this work we investigated the degree of UV-irradiation induced thymidine damages in water, salt (NaCl) and ethanol solvents using UV absorption and circular dichroism (CD) spectrometry, and MS ESI method. In the explored systems thymidine association degree rose in the following solvent order: 1 M NaCl, water, ethanol. UV-absorbance and CD intensity fell in the greater extent in the ethanol and water-salt solutions than in water. So the experiment showed that the association degree of thymidine in the solution does not play a main role in its photosensitivity.

  13. Photoaffinity labeling of opiate receptors using intrinsically photoactive /sup 3/H-opiates

    SciTech Connect

    Kooper, G.N.; Levinson, N.R.; Copeland, C.F.; Bowen, W.D.

    1988-03-01

    Opiate receptors in rat and cow brain membranes have been labeled irreversibly using the intrinsic photolability of 3H-opiates. Membranes were incubated with 3H-ligand and then irradiated with UV light of 254 nm. Nonspecific binding was determined in the presence of 10 microM unlabeled levallorphan. Irreversible binding was defined as binding which survived heat or acid denaturation of membranes. Specific incorporation of label into denatured samples was observed only when unbound or loosely bound 3H-ligand was washed free from the membranes prior to irradiation. There was a general correlation between photosensitivity of the 3H-ligand and its ability to photolabel receptors. Hence, photolabeling presumably results by covalent attachment of highly reactive species generated during photochemical decomposition of ligand. With 3H-etorphine, optimal irradiation time was 5 min. In addition to 3H-etorphine, receptors could be labeled irreversibly with 3H-oxymorphone, 3H-dihydromorphine, and 3H-ethylketocyclazocine. Of the specific binding present in irradiated, nondenatured samples, 45-60% remained attached to receptors upon denaturation. 3H-Ethylketocyclazocine exhibited an 86% yield of incorporation. Signal-to-noise levels of 50-80% could be achieved in denatured samples. Therefore, this method provides a means of covalently labeling opiate receptors in high yield and with high signal-to-noise ratios. The opioid peptides, 3H-D-Ala2,D-Leu5-enkephalin, 3H-D-Ser2,Leu5,Thr6-enkephalin, 3H-D-Ala2,Met5-enkephalin amide, and 3H-D-Ala2,N-MePhe4,Gly-ol5-enkephalin, as well as the benzomorphan, 3H-bremazocine, apparently lack the structural characteristics which allow photolabeling.

  14. Structural investigation of the thymidine phosphorylase from Salmonella typhimurium in the unliganded state and its complexes with thymidine and uridine.

    PubMed

    Balaev, Vladislav V; Lashkov, Alexander A; Gabdulkhakov, Azat G; Dontsova, Maria V; Seregina, Tatiana A; Mironov, Alexander S; Betzel, Christian; Mikhailov, Al'bert M

    2016-03-01

    Highly specific thymidine phosphorylases catalyze the phosphorolytic cleavage of thymidine, with the help of a phosphate ion, resulting in thymine and 2-deoxy-α-D-ribose 1-phosphate. Thymidine phosphorylases do not catalyze the phosphorolysis of uridine, in contrast to nonspecific pyrimidine nucleoside phosphorylases and uridine phosphorylases. Understanding the mechanism of substrate specificity on the basis of the nucleoside is essential to support rational drug-discovery investigations of new antitumour and anti-infective drugs which are metabolized by thymidine phosphorylases. For this reason, X-ray structures of the thymidine phosphorylase from Salmonella typhimurium were solved and refined: the unliganded structure at 2.05 Å resolution (PDB entry 4xr5), the structure of the complex with thymidine at 2.55 Å resolution (PDB entry 4yek) and that of the complex with uridine at 2.43 Å resolution (PDB entry 4yyy). The various structural features of the enzyme which might be responsible for the specificity for thymidine and not for uridine were identified. The presence of the 2'-hydroxyl group in uridine results in a different position of the uridine furanose moiety compared with that of thymidine. This feature may be the key element of the substrate specificity. The specificity might also be associated with the opening/closure mechanism of the two-domain subunit structure of the enzyme. PMID:26919527

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

    PubMed

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

    2009-01-01

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

  16. Diamine incorporated compounds derived from polymeric nickel(II) fumarates and oxalates: Crystal structure, spectral and thermal properties of [Ni(en) 3](O 2C sbnd CH dbnd CH sbnd CO 2)·3H 2O and [Ni(en) 3](O 2C sbnd CO 2)

    NASA Astrophysics Data System (ADS)

    Padmanabhan, M.; Joseph, James C.; Huang, Xiaoying; Li, Jing

    2008-08-01

    Lewis-base mediated fragmentation of polymeric nickel(II) fumarate and oxalate are attempted using chelating σ-donor diamines like ethylenediamine (en) and 1,3-diaminopropane (dap) in various conditions which yielded [Ni(en) 3](fum)·3H 2O ( 1), [Ni(en) 3](ox) ( 2), [Ni(dap) 2(fum)] ( 3) and [Ni(dap)(ox)]·2H 2O ( 4). While 1 and 2 are molecular products each containing octahedral [Ni(en) 3] 2+ moieties and the anionic dicarboxylate species, 3 and 4 are dap-incorporated polymeric products. The fumarate derivative 1 containing [Ni(en) 3] 2+ moieties crystallizes in the monoclinic space group C2/ c with a = 17.899(4) Å, b = 11.747(2) Å, c = 10.748(2) Å, β = 125.59(3)°, V = 1837.7(6) Å 3, Z = 4, while the oxalate analogue 2 is seen to be in the trigonal space group P-31 c with a = 8.8770(13) Å, b = 8.8770(13) Å, c = 10.482(2) Å, γ = 120°, V = 715.3(2) Å 3, Z = 2. The octahedral [Ni(en) 3] units in both 1 and 2 are seen to be strongly H-bonded to the dicarboxylate moieties through the coordinated en units leading to a three-dimensional network. However, in 1 the water molecules also take part in the H-bonding and contribute to the overall 3D structure. In both 1 and 2 the crystal packing is done with the [Ni(en) 3] 2+ units with absolute configuration Λ( δδδ) and its mirror conformer with Δ configuration in exactly equal numbers. Spectral (IR and UV-Visible) and magnetic measurements were carried out and some of the ligand-field parameters like Dq, B and β were evaluated for all the four compounds. These values suggest the presence of octahedrally coordinated nickel(II) in all the four complexes. Spectral data suggest that 3 has the two chelating dap moieties and the fumarate coordinated in η 1 form through both its carboxylate moieties while 4 has one chelating dap and the oxalate moiety coordinated in η 4-bis-chelating form. Though both 1 and 2 are made of the same type of [Ni(en) 3] 2+ units their thermograms give entirely different thermal

  17. Studies on molecular recognition of thymidines with molecularly imprinted polymers

    NASA Astrophysics Data System (ADS)

    Chen, Zhen-He; Luo, Ai-Qin; Sun, Li-Quan

    2009-07-01

    Molecularly imprinted polymers (MIPs) with excellent molecular recognition ability have been used in chemical sensors, chromatographic separation and biochemical analyses. Thymidine is an important part of DNA for biomolecular recognition and the intermediate of many medicines. The polymers imprinted with the template of thymidine and 5'-Otosylthymidine have been prepared, using a non-proton solvent, acetonitrile as the porogen. Direct imprinting with thymidine could not form strong molecular interaction sites in this system. Relative MIPs were obtained by bulk polymerization and their adsorption capacities were investigated. The adsorption capacities of MIP (P2) and nonimprinted polymer (P20) for thymidine are 0.120 mg•g-1and 0.103 mg•g-1, respectively. The imprinting factor is 1.17. As 5'-O-tosylthymidine is more soluble than thymidine moiety in acetonitrile and give rise to more sites of molecular recognition. The results demonstrated that the imprinted polymers were able to bind and recognize thymidine moderately in acetonitrile. MIPs imprinted with 5'-O-tosylthymidine like nature enzymes displayed some recognition ability to its analogues. The insoluble derivatives in the non-proton solvent can be an effective template to prepare efficient imprinting recognition sites.

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

    SciTech Connect

    Buschhausen, G.; Wittig, B.; Graessmann, M.; Graessmann, A.

    1987-03-01

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

  19. Formation of cyclic 1,N2-propanodeoxyguanosine and thymidine adducts in the reaction of the mutagen 2-bromoacrolein with calf thymus DNA

    SciTech Connect

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

    1989-11-15

    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.

  20. Insertional activation of a promoterless thymidine kinase gene

    SciTech Connect

    Hiller, S.; Hengstler, M.; Kunze, M.; Knippers, R.

    1988-08-01

    A plasmid carrying a promoterless herpes complex virus thymidine kinase gene was transfected via calcium phosphate precipitation into LM (tk/sup -/) mouse fibroblast cells. The transfected gene was efficiently expressed, as the transfected cells grew perfectly well in selective hypoxanthine-aminopterin-thymidine medium, suggesting that the thymidine kinase-coding region became linked to a promoterlike element on integration into the recipient genome. To investigate the structure of the surrogate promoter, the authors first isolated the integrated gene from a genomic library. The nucleotide sequence of the DNA adjacent to the thymidine kinase-coding sequence was then determined. They found, first, that the integration of the transfected DNA apparently occurred by a blunt end ligation mechanism involving no obvious sequence similarities between integrated and recipient DNA and, second, that the 5'-flanking region included a TATA box, to CCAAT boxes, and a GC box element. However, the TATA box motif and the most proximal CCAAT box appeared to be sufficient of full promoter activity, as determined by the transfection efficiencies of appropriate plasmid constructs. Except for these canonical promoter elements, the surrogate promoter had no obvious similarities to known thymidine kinase gene promoters.

  1. Thymidine phosphorylase deficiency causes MNGIE: an autosomal recessive mitochondrial disorder.

    PubMed

    Hirano, M; Martí, R; Spinazzola, A; Nishino, I; Nishigaki, Y

    2004-10-01

    Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by mutations in the gene encoding thymidine phosphorylase (TP). The disease is characterized clinically by impaired eye movements, gastrointestinal dysmotility, cachexia, peripheral neuropathy, myopathy, and leukoencephalopathy. Molecular genetic studies of MNGIE patients' tissues have revealed multiple deletions, depletion, and site-specific point mutations of mitochondrial DNA. TP is a cytosolic enzyme required for nucleoside homeostasis. In MNGIE, TP activity is severely reduced and consequently levels of thymidine and deoxyuridine in plasma are dramatically elevated. We have hypothesized that the increased levels of intracellular thymidine and deoxyuridine cause imbalances of mitochondrial nucleotide pools that, in turn, lead to the mtDNA abnormalities. MNGIE was the first molecularly characterized genetic disorder caused by abnormal mitochondrial nucleoside/nucleotide metabolism. Future studies are likely to reveal further insight into this expanding group of diseases. PMID:15571233

  2. Thymidine phosphorylase mutations cause instability of mitochondrial DNA.

    PubMed

    Hirano, Michio; Lagier-Tourenne, Clotilde; Valentino, Maria L; Martí, Ramon; Nishigaki, Yutaka

    2005-07-18

    Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder characterized by ptosis and progressive external ophthalmoplegia, peripheral neuropathy, severe gastrointestinal dysmotility, cachexia and leukoencephalopathy. Muscle biopsies of MNGIE patients have revealed morphologically abnormal mitochondria and defects of respiratory chain enzymes. In addition, patients harbor depletion, multiple deletions, and point mutations of mitochondrial DNA (mtDNA). This disorder is caused by loss-of-function mutations in the gene encoding thymidine phosphorylase (TP) a cytosolic enzyme. In MNGIE patients, TP activity is very low or absent resulting in dramatically elevated levels of plasma thymidine and deoxyuridine. We have hypothesized that the increased levels of thymidine and deoxyuridine cause mitochondrial nucleotide pool imbalances that, in turn, generate mtDNA alterations. PMID:15975738

  3. Three-dimensional structure of thymidine phosphorylase from E. coli in complex with 3'-azido-2'-fluoro-2',3'-dideoxyuridine

    NASA Astrophysics Data System (ADS)

    Timofeev, V. I.; Abramchik, Yu. A.; Fateev, I. V.; Zhukhlistova, N. E.; Murav'eva, T. I.; Kuranova, I. P.; Esipov, R. S.

    2013-11-01

    The three-dimensional structures of thymidine phosphorylase from E. coli containing the bound sulfate ion in the phosphate-binding site and of the complex of thymidine phosphorylase with sulfate in the phosphate-binding site and the inhibitor 3'-azido-2'-fluoro-2',3'-dideoxyuridine (N3F-ddU) in the nucleoside-binding site were determined at 1.55 and 1.50 Å resolution, respectively. The amino-acid residues involved in the ligand binding and the hydrogen-bond network in the active site occupied by a large number of bound water molecules are described. A comparison of the structure of thymidine phosphorylase in complex with N3F-ddU with the structure of pyrimidine nucleoside phosphorylase from St. Aureus in complex with the natural substrate thymidine (PDB_ID: 3H5Q) shows that the substrate and the inhibitor in the nucleoside-binding pocket have different orientations. It is suggested that the position of N3F-ddU can be influenced by the presence of the azido group, which prefers a hydrophobic environment. In both structures, the active sites of the subunits are in the open conformation.

  4. A novel thymidine phosphorylase mutation in a Spanish MNGIE patient.

    PubMed

    Gamez, Josep; Lara, Maria Carmen; Mearin, Fermin; Oliveras-Ley, Carlos; Raguer, Nuria; Olive, Montse; Leist, Andres T; Perello, Antonia; Perona, Monica; Cervera, Carlos; Andreu, Antonio Luis; Martí, Ramon; Hirano, Michio

    2005-01-15

    A 29-year-old Spanish man presented with chronic intestinal pseudo-obstruction, progressive external ophthalmoplegia, peripheral neuropathy, and diffuse leukoencephalopathy. This combination of clinical features is characteristic of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). Genetic analysis revealed a novel 18-base pair (bp) duplication (5044-5061 dup) in exon 8 of the thymidine phosphorylase (TP) gene. The mutation is predicted to produce a 6 amino acid insertion in the alpha-beta-domain of the protein. This 18-bp insertion in the thymidine phosphorylase gene is the first duplication mutation identified in MNGIE. PMID:15607208

  5. Vertical detachment energies of anionic thymidine: Microhydration effects

    NASA Astrophysics Data System (ADS)

    Kim, Sunghwan; Schaefer, Henry F.

    2010-10-01

    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 N1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)].

  6. RETROGRADE AXONAL TRANSPORT OF PHOSPHOINOSITIDES AFTER INTRANEURAL INJECTION OF [3H]MYO-INOSITOL INTO THE RAT SCIATIC NERVE

    EPA Science Inventory

    Although autoradiography has demonstrated local incorporation of [3H]inositol into axonal phospholipids after intraneural injection (Gould, 1976; Gould et at., 1987b), retrograde axonal transport of phosphatidylinositol has only been demonstrated after injection of lipid precurso...

  7. Diastereoselective synthesis of the major products resulting from the interaction between ionizing radiation and thymidine

    SciTech Connect

    Barvian, M.R.; Greenberg, M.M.

    1993-12-31

    5,6-Dihydro-5,6-dihydroxythymidine (thymidine glycol, 1) and 5,6-dihydro-5-hydroxy-thymidine (C5-thymidine hydrate, 2) are the major products attributed to the effects of {gamma}-radiolysis on thymidine under aerobic and hypoxic conditions, respectively. The authors have achieved the first chemical synthesis of 2 from the respective cis-glycols. Diastereoselective synthesis of 1 and 2 is achieved by carrying out a stereoselective dihydroxylation on the bis-t-butyl-dimethylsilyl ether of thymidine.

  8. HPLC-UV analysis of thymidine and deoxyuridine in plasma of patients with thymidine phosphorylase deficiency.

    PubMed

    Mohamed, Susan; Caporali, Leonardo; De Giorgio, Roberto; Carelli, Valerio; Contin, Manuela

    2014-02-15

    We present a simple, fast and validated method for the determination of the two nucleosides thymidine (dThd) and deoxyuridine (dUrd) in plasma of patients with symptoms suggestive of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), using high performance liquid chromatography coupled with ultraviolet spectrophotometric detection (HPLC-UV). Plasma sample (100μL) pretreatment was based on simple deproteinization by 1.2M perchloric acid, using theophylline as internal standard (I.S.). HPLC-UV analysis was carried out on a Synergi 4μm Hydro-RP, 150×4mm I.D. column, at room temperature. The mobile phase was a mixture of potassium dihydrogen phosphate buffer (20mM, pH 4.5) and acetonitrile (95:5, v/v), at an isocratic flow rate of 0.7mL/min. The UV detector was set at 267nm. The chromatographic run lasted 19min. Similar pyrimidine nucleotides and nucleosides do not interfere with the assay. Calibration curves were linear for both dThd and dUrd over a range of 0.5 to 5.0μg/mL. The limit of quantitation was 0.5μg/mL for both nucleosides and the absolute recovery was >90% for dThd, dUrd and the I.S. Both intra- and inter-assay precision and accuracy were lower than 10% at all tested concentrations. The proposed method was successfully applied to measure plasma concentrations of dThd and dUrd in two MNGIE patients. This assay simplifies both plasma pretreatment and chromatographic conditions of previously reported procedures and describes the first validated method for the determination of the two nucleotides in human plasma. PMID:24463401

  9. Differential binding of /sup 3/H-imipramine and /sup 3/H-mianserin in rat cerebral cortex

    SciTech Connect

    Dumbrille-Ross, A.; Tang, S.W.; Coscina, D.V.

    1981-11-16

    Drug competition profiles, effect of raphe lesion, and sodium dependency of the binding of two antidepressant drugs /sup 3/H-imipramine and /sup 3/H-mianserin to rat cerebral cortex homogenate were compared to examine whether the drugs bound to a common ''antidepressant receptor.'' Of the neurotransmitters tested, only serotonin displaced binding of both /sup 3/H-imipramine and /sup 3/H-mianserin. /sup 3/H-Mianserin binding was potently displaced by serotonin S/sub 2/ antagonists and exhibited a profile similar to that of /sup 3/H-spiperone binding. In the presence of the serotonin S/sub 2/ antagonist spiperone, antihistamines (H/sub 1/) potently displaced /sup 3/H-mianserin binding. /sup 3/H-Imipramine binding was displaced potently by serotonin uptake inhibitors. The order of potency of serotonergic drugs in displacing /sup 3/H-imipramine binding was not similar to their order in displacing /sup 3/H-spiperone or -3H-serotonin binding. Prior midbrain raphe lesions greatly decreased the binding of /sup 3/H-imipramine but did not alter binding of /sup 3/H-mianserin. Binding of /sup 3/H-imipramine but not /sup 3/H-mianserin was sodium dependent. These results show that /sup 3/H-imipramine and /sup 3/H-mianserin bind to different receptors. /sup 3/H-Imipramine binds to a presynaptic serotonin receptor which is probably related to a serotonin uptake recognition site, the binding of which is sodium dependent. /sup 3/H-Mianserin binds to postsynaptic receptors, possibly both serotonin S/sub 2/ and histamine H/sub 1/ receptors, the binding of which is sodium independent.

  10. Metabolism of myo-[2-3H]Inositol and scyllo-[R-3H]Inositol in Ripening Wheat Kernels 1

    PubMed Central

    Sasaki, Ken; Loewus, Frank A.

    1980-01-01

    Injection of myo-[2-3H]inositol or scyllo-[R-3H]inositol into the peduncular cavity of wheat stalks about 2 to 4 weeks postanthesis led to rapid translocation into the spike and accumulation of label in developing kernels, especially the bran fraction. With myo-[2-3H]inositol, about 50 to 60% of the label was incorporated into high molecular weight cell wall substance in the region of the injection. That portion translocated to the kernels was utilized primarily for cell wall polysaccharide formation and phytate biosynthesis. A small amount was recovered as free myo-inositol and galactinol. When scyllo-[R-3H]inositol was supplied, most of the label was translocated into the developing kernels where it accumulated as free scyllo-inositol and O-α-d-galactopyranosyl-scyllo-inositol in approximately equal amount. None of the label from scyllo-[R-3H]inositol was utilized for either phytate biosynthesis or cell wall polysaccharide formation. PMID:16661513

  11. Elevated plasma deoxyuridine in patients with thymidine phosphorylase deficiency.

    PubMed

    Martí, Ramon; Nishigaki, Yutaka; Hirano, Michio

    2003-03-28

    Mutations in the nuclear gene encoding thymidine phosphorylase (TP) cause mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), an autosomal recessive disease with mitochondrial dysfunction and mitochondrial DNA abnormalities. We have demonstrated alterations of thymidine (dThd) metabolism in MNGIE patients. Here, we report the accumulation of another substrate of TP, deoxyuridine (dUrd), whose circulating levels ranged from 5.5 to 24.4 microM (average 14.2) in MNGIE and were undetectable (<0.05 microM) in both TP mutation carriers and controls. The dramatic accumulation of dUrd may contribute to nucleotide pool imbalances and, together with the increased levels of dThd, is likely to contribute to the pathogenesis of MNGIE. PMID:12646159

  12. N3-substituted thymidine bioconjugates for cancer therapy and imaging

    PubMed Central

    Khalil, Ahmed; Ishita, Keisuke; Ali, Tehane; Tjarks, Werner

    2013-01-01

    The compound class of 3-carboranyl thymidine analogues (3CTAs) are boron delivery agents for boron neutron capture therapy (BNCT), a binary treatment modality for cancer. Presumably, these compounds accumulate selectively in tumor cells via intracellular trapping, which is mediated by hTK1. Favorable in vivo biodistribution profiles of 3CTAs led to promising results in preclinical BNCT of rats with intracerebral brain tumors. This review presents an overview on the design, synthesis, and biological evaluation of first- and second-generation 3CTAs. Boronated nucleosides developed prior to 3CTAs for BNCT and non-boronated N3-substituted thymidine conjugates for other areas of cancer therapy and imaging are also described. In addition, basic features of carborane clusters, which are used as boron moieties in the design and synthesis of 3CTAs, and the biological and structural features of TK1-like enzymes, which are the molecular targets of 3CTAs, are discussed. PMID:23617430

  13. Sequestration and microsomal C-25 hydroxylation of (/sup 3/H)-vitamin D3 by the rat liver

    SciTech Connect

    Gascon-Barre, M.; Elbaz, H.; Therrien-Ferland, D.

    1985-03-01

    A study of the vitamin D3 (D3) 25-hydroxylase was undertaken in an in vivo-in vitro model. (/sup 3/H)-D3 (0.7, 1.0, 10, or 100 nmol/100 g of body weight) was injected into the portal vein and the liver was excised 18 seconds later. The liver homogenate was then submitted to differential centrifugation and the amount of (/sup 3/H)-D3 incorporated in the subcellular fractions was evaluated. The microsomal fraction was also incubated in vitro and the appearance of (/sup 3/H)-25-hydroxyvitamin D3 (25(OH)D3) was determined by high performance liquid chromatography (HPLC). Results showed that the fractional liver (/sup 3/H)-D3 uptake varied between 37 percent and 48 percent of the dose injected. The intracellular distribution of (/sup 3/H)-D3 showed that most of the vitamin was incorporated into the microsomal fraction (45% to 50% of the intracellular (/sup 3/H)-D3) except at the highest dose of (/sup 3/H)-D3 where the cytosolic fraction contained the highest amount (56.4%) of the incorporated vitamin. Mathematical analysis of the intracellular (/sup 3/H)-D3 distribution showed that the microsomal fraction was the only subcellular fraction that was found to incorporate (/sup 3/H)-D3 in relation to the total liver uptake of the vitamin. The apparent Michaelis-Menten kinetics of the (/sup 3/H)-D3-25-hydroxylase showed that with substrate concentration of up to 88.5 nM, the apparent Km and Vmax were 28.2 nM and 25.8 fentomoles (fmol) X min-1 X mg microsomal pro-1, respectively, but the reaction lost considerable efficiency with higher substrate concentrations.

  14. Enhanced expression of thymidine kinase in human cells following ionizing radiation

    SciTech Connect

    Boothman, D.A.; Davis, T.W.; Sahijdak, W.M.

    1994-09-30

    We investigated the induction of thymidine kinase transcription and enzymatic activity, and the activation of transcription factors binding to the thymidine kinase promoter, in human normal compared to tumor cells in culture before and after ionizing radiation. Northern blot, dot-blot, and thymidine kinase enzyme assays were used to observe thymidine kinase transcript and enzymatic changes before and after radiation. Temporal expression of thymidine kinase transcripts following an optimal induction dose of radiation was also studied. Gel mobility shift assays were performed using a 95-base pair fragment of the thymidine kinase promoter (containing the CCAAT box) to analyze transcription factor binding. Thymidine kinase transcript and enzymatic levels were higher in human tumor compared to normal cells. In contrast, levels of x-ray-activated thymidine kinase transcription factors were not significantly different in human neoplastic compared to normal cells. Elevated x-ray-induced thymidine kinase transcripts, enzymatic levels, and transcription factors are consistent with the loss of stringent cell growth regulation associated with neoplastic cells. The induction of thymidine kinase following ionizing radiation may be exploited in chemotherapeutic strategies which use halogenated pyrimidines and/or in various gene therapy strategies. 26 refs., 4 figs., 1 tab.

  15. Computer-assisted combinatorial design of bicyclic thymidine analogs as inhibitors of Mycobacterium tuberculosis thymidine monophosphate kinase.

    PubMed

    Frecer, Vladimir; Seneci, Pierfausto; Miertus, Stanislav

    2011-01-01

    Thymidine monophosphate kinase (TMPK(mt)) is an essential enzyme for nucleotide metabolism in Mycobacterium tuberculosis, and thus an attractive target for novel antituberculosis agents. In this work, we have explored the chemical space around the 2',3'-bicyclic thymidine nucleus by designing and in silico screening of a virtual focused library selected via structure based methods to identify more potent analogs endowed with favorable ADME-related properties. In all the library members we have exchanged the ribose ring of the template with a cyclopentane moiety that is less prone to enzymatic degradation. In addition, we have replaced the six-membered 2',3'-ring by a number of five-membered and six-membered heterocyclic rings containing alternative proton donor and acceptor groups, to exploit the interaction with the carboxylate groups of Asp9 and Asp163 as well as with several cationic residues present in the vicinity of the TMPK(mt) binding site. The three-dimensional structure of the TMPK(mt) complexed with 5-hydroxymethyl-dUMP, an analog of dTMP, was employed to develop a QSAR model, to parameterize a scoring function specific for the TMPK(mt) target and to select analogues which display the highest predicted binding to the target. As a result, we identified a small highly focused combinatorial subset of bicyclic thymidine analogues as virtual hits that are predicted to inhibit the mycobacterial TMPK in the submicromolar concentration range and to display favorable ADME-related properties. PMID:21082329

  16. Retinoic acid treatment of fibroblasts causes a rapid decrease in ( sup 3 H)inositol uptake

    SciTech Connect

    Sinha, R.; Creek, K.E.; Silverman-Jones, C.; de Luca, L.M. )

    1989-04-01

    NIH 3T3 fibroblasts treated with all-trans-retinoic acid (RA) showed a dramatic decrease in the uptake of ({sup 3}H)inositol compared to solvent-treated controls. The onset of RA-induced inhibition of ({sup 3}H)inositol uptake was rapid with a 10-15% decrease occurring after 2-3 h of RA exposure and 60-70% reduction after 16 h of RA treatment. A progressive dose-dependent decrease in inositol uptake was found as the concentration of RA increased from 10{sup {minus}8} to 10{sup {minus}5} M and the effect was fully reversible within 48 h after RA removal. RA inhibition of inositol uptake was also observed in 3T3-Swiss and Balb/3T3 cells but not in two virally transformed 3T3 cell lines. Phlorizin, amiloride, and monensin inhibited inositol uptake by 66, 74, and 58%, respectively, and this inhibition was additive when the cells were treated with RA as well as these inhibitors. A decreased incorporation of ({sup 3}H)inositol into polyphosphoinositides was also observed in RA-treated cells but not to the same extent as for ({sup 3}H)inositol uptake. In conclusion, RA treatment of 3T3 fibroblasts decreases the uptake of ({sup 3}H)inositol by up to 70% within 8 to 10 h at near physiological concentrations in a reversible and specific manner.

  17. The whole of intramolecular H-bonding in the isolated DNA nucleoside thymidine. AIM electron density topological study

    NASA Astrophysics Data System (ADS)

    Yurenko, Yevgen P.; Zhurakivsky, Roman O.; Samijlenko, Svitlana P.; Ghomi, Mahmoud; Hovorun, Dmytro M.

    2007-10-01

    Based on comprehensive conformational analysis at the DFT B3LYP/6-31G(d,p) level of theory [Ye.P. Yurenko et al., J. Phys. Chem. B, 111 (2007) 9655] 13 types of intramolecular hydrogen bonds (171 in total number, from 1 to 3 H-bonds per conformer) were identified in the 92 allowed conformers of isolated thymidine, a DNA canonical nucleoside. Weak interactions CH⋯ O, OH⋯ HC and CH⋯ HC were analyzed by all the Koch and Popelier criteria of H-bonding. Quite satisfactory linear correlation was found between Grabowski complex measure of H-bond strength Δcom and Iogansen H-bond energy -Δ H for 17 OH⋯O hydrogen bonds.

  18. Short-term thymidine uptake in normal and neoplastic tissues: studies for PET

    SciTech Connect

    Shields, A.F.; Larson, S.M.; Grunbaum, Z.; Graham, M.M.

    1984-07-01

    Uptake of H-3 thymidine was studied in mice, both normal and with spontaneous lymphoma, and in the organs and tumors of dogs with spontaneous tumors. Uptake was compared with relative blood flow as measured by the distribution of C-14 iodoantipyrine. Initial distribution of thymidine in normal mice measured 20 sec after injection, correlated with the relative perfusion measurements; however, all measurements of thymidine uptake made between 1 and 60 min after injection showed no correlation with perfusion. A time-course study demonstrated that normal mouse organs with high rates of proliferation retained all the labeled thymidine initally taken up. Organs with low rates of proliferation retained all the labeled thymidine initially take up. Organs with low rates of proliferation lost their label in a nearly exponential wash-out. These studies provide further evidence of the feasibility of using C-11 thymidine for positron emission tomography (PET).

  19. A comparative study of dissociation of thymidine molecules following valence or core photoionization

    NASA Astrophysics Data System (ADS)

    Itälä, E.; Huels, M. A.; Rachlew, E.; Kooser, K.; Hägerth, T.; Kukk, E.

    2013-11-01

    We have studied the fragmentation of gas phase thymidine following valence and core ionization using synchrotron radiation, combined with electron energy and ion mass resolved detection in coincidence. To identify certain fragment masses 13C labelled thymidine was also used. We find that in large part, the photofragmentation of thymidine can be described as separated thymine and 2-deoxy-d-ribose cations. However, also the intact thymidine radical cation, formation of intact base and sugar cations from glycosidic bond cleavage and several new thymidine-specific fragments are observed after valence ionization. Conversely, at the photoionization of C 1s electrons neither parent thymidine cations nor any fragments above 55 amu were detected, and only ion pairs with small masses survive core ionization, Auger decay and the subsequent Coulomb separation of the DNA sub-unit. This demonstrates the genotoxic nature of soft x-rays which in cells induce complex clustered damage similar to those yielded by heavy particles.

  20. Mutant strains of Tetrahymena thermophila defective in thymidine kinase activity: Biochemical and genetic characterization

    SciTech Connect

    Cornish, K.V.; Pearlman, R.E.

    1982-08-01

    Three mutant strains, one conditional, of Tetrahymena thermophila were defective in thymidine phosphorylating activity in vivo and in thymidine kinase activity in vitro. Nucleoside phosphotransferase activity in mutant cell extracts approached wild-type levels, suggesting that thymidine kinase is responsible for most, if not all, thymidine phosphorylation in vivo. Thymidine kinase activity in extracts of the conditional mutant strain was deficient when the cells were grown or assayed or both at the permissive temperature, implying a structural enzyme defect. Analysis of the reaction products from in vitro assays with partially purified enzymes showed that phosphorylation by thymidine kinase and nucleoside phosphotransferase occurred at the 5' position. Genetic analyses showed that the mutant phenotype was recessive and that mutations in each of the three mutant strains did not complement, suggesting allelism.

  1. Comparison of (/sup 3/H)nicotine and (/sup 3/H)acetylcholine binding in mouse brain: regional distribution

    SciTech Connect

    Sershen, H.; Reith, M.E.; Hashim, A.; Lajtha, A.

    1985-06-01

    In a continuing study of nicotine binding sites, the authors determined the relative amount of nicotine binding and acetylcholine binding in various brain regions of C57/BL and of DBA mice. Although midbrain showed the highest and cerebellum the lowest binding for both (/sup 3/H)nicotine and (/sup 3/H)acetylcholine, the ratio of nicotine to acetylcholine binding showed a three-fold regional variation. Acetylcholine inhibition of (/sup 3/H)nicotine binding indicated that a portion of nicotine binding was not inhibited by acetylcholine. These results indicate important differences between the binding of (+/-)-(/sup 3/H)nicotine and that of (/sup 3/H)acetylcholine.

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

    SciTech Connect

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

    1982-06-01

    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.

  3. Characterization of a monoclonal antibody to thymidine glycol monophosphate

    SciTech Connect

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

    1990-11-01

    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.

  4. Synthesis and characterization of 3H-labelled tetrahydrobiopterin.

    PubMed Central

    Werner, E R; Schmid, M; Werner-Felmayer, G; Mayer, B; Wachter, H

    1994-01-01

    We synthesized [3'-3H]-5,6,7,8-tetrahydrobiopterin from [8,5'-3H]guanosine 5'-triphosphate ([8,5'-3H]GTP) using GTP cyclohydrolase (EC 3.5.4.16), 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase (EC 1.1.1.153). After purification by cation-exchange h.p.l.c. a solution of radiochemically pure (> 95%) [3'-3H]-5,6,7,8-tetrahydrobiopterin with a specific activity of 9.2 Ci/mmol was obtained. The product proved well suited for studying the binding of tetrahydrobiopterin to nitric-oxide synthase. PMID:7528005

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

    SciTech Connect

    Zaghloul, M.S.; Dorie, M.J.; Kallman, R.F.

    1994-07-01

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

  6. Fragmentation of Electrospray-Produced Deprotonated Ions of Oligodeoxyribonucleotides Containing an Alkylated or Oxidized Thymidine

    NASA Astrophysics Data System (ADS)

    Wang, Pengcheng; Williams, Renee T.; Guerrero, Candace R.; Ji, Debin; Wang, Yinsheng

    2014-07-01

    Alkylation and oxidation constitute major routes of DNA damage induced by endogenous and exogenous genotoxic agents. Understanding the biological consequences of DNA lesions often necessitates the availability of oligodeoxyribonucleotide (ODN) substrates harboring these lesions, and sensitive and robust methods for validating the identities of these ODNs. Tandem mass spectrometry is well suited for meeting these latter analytical needs. In the present study, we evaluated how the incorporation of an ethyl group to different positions (i.e., O 2, N3, and O 4) of thymine and the oxidation of its 5-methyl carbon impact collisionally activated dissociation (CAD) pathways of electrospray-produced deprotonated ions of ODNs harboring these thymine modifications. Unlike an unmodified thymine, which often manifests poor cleavage of the C3'-O3' bond, the incorporation of an alkyl group to the O 2 position and, to a much lesser extent, the O 4 position, but not the N3 position of thymine, led to facile cleavage of the C3'-O3' bond on the 3' side of the modified thymine. Similar efficient chain cleavage was observed when thymine was oxidized to 5-formyluracil or 5-carboxyluracil, but not 5-hydroxymethyluracil. Additionally, with the support of computational modeling, we revealed that proton affinity and acidity of the modified nucleobases govern the fragmentation of ODNs containing the alkylated and oxidized thymidine derivatives, respectively. These results provided important insights into the effects of thymine modifications on ODN fragmentation.

  7. Partial chemical characterization of cyclopyrrolones ((/sup 3/H) suriclone) and benzodiazepines ((/sup 3/H)flunitrazepam) binding site: Differences

    SciTech Connect

    Zundel, J.L.; Blanchard, J.C.; Julou, L.

    1985-06-10

    Rat hippocampus membranes were treated with several protein modifying reagents (iodoacetamide, N-ethylmaleimide, tetranitromethane and N-acetylimidazole). The effects of these treatments on the binding sites of cyclopyrrolones ((/sup 3/H) suriclone), a new chemical family of minor tranquilizers, and benzodiazepines ((/sup 3/H) flunitrazepam) were investigated. Here the authors show that both ligands are similarly sensitive to cysteine alkylation: (/sup 3/H) suriclone and (/sup 3/H) flunitrazepam binding are reduced by iodoacetamide and slightly increased by N-ethylmaleimide. On the contrary they are clearly differentiated by tyrosine modification: (/sup 3/H) suriclone binding is not changed whereas (/sup 3/H) flunitrazepam binding is increased by tetranitromethane and decreased by N-acetylimidazole. The present findings and published evidence suggest cyclopyrrolones and benzodiazepines bind to distinct sites or to different allosteric forms of the benzodiazepine receptor. 28 references, 6 figures.

  8. Thymidine analogues to assess microperfusion in human tumors

    SciTech Connect

    Janssen, Hilde L.; Ljungkvist, Anna S.; Rijken, Paul F.; Sprong, Debbie; Bussink, Jan; Kogel, Albert J. van der; Haustermans, Karin M.; Begg, Adrian C. . E-mail: a.begg@nki.nl

    2005-07-15

    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.

  9. Glutaminase 1 inhibition reduces thymidine synthesis in NSCLC.

    PubMed

    Lee, Jae-Seon; Kang, Joon H; Lee, Seon-Hyeong; Lee, Chang-Hun; Son, Jaekyoung; Kim, Soo-Youl

    2016-08-26

    We found that non-small cell lung cancer (NSCLC) is remarkably sensitive to the regulation of glutamine supply by testing the metabolic dependency of 11 cancer cell lines against regulation of glycolysis, autophagy, fatty acid synthesis, and glutamine supply. Glutamine is known as a key supplement of cancer cell growth that is converted to α-ketoglutarate for anabolic biogenesis via glutamate by glutaminase 1 (GLS1). GLS1 inhibition using 10 μM of bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) showed about 50% cell growth arrest by SRB assay. By testing the synergistic effects of conventional therapeutics, BPTES combined with 5-fluorouracil (5-FU), an irreversible inhibitor of thymidylate synthase, significant effects were observed on cell growth arrest in NSCLC. We found that GLS1 inhibition using BPTES reduced metabolic intermediates including thymidine and carbamoyl phosphate. Reduction of thymidine and carbamoyl-phosphate synthesis by BPTES treatment exacerbated pyrimidine supply by combination with 5-FU, which induced cell death synergistically in NSCLC. PMID:27338638

  10. The nucleotide sequence of the chicken thymidine kinase gene and the relationship of its predicted polypeptide to that of the vaccinia virus thymidine kinase.

    PubMed Central

    Kwoh, T J; Engler, J A

    1984-01-01

    The entire DNA nucleotide sequence of a 3.0 kilobase pair Hind III fragment containing the chicken cytoplasmic thymidine kinase gene was determined. Oligonucleotide linker insertion mutations distributed throughout this gene and having known effects upon gene activity ( Kwoh , T.J., Zipser , D., and Wigler , M. 1983. J. Mol. Appl. Genet. 2, 191-200), were used to access regions of the Hind III fragment for sequencing reactions. The complete nucleotide sequence, together with the positions of the linker insertion mutations within the sequence, allows us to propose a structure for the chicken thymidine kinase gene. The protein coding sequence of the gene is divided into seven small segments (each less than 160 base pairs) by six small introns (each less than 230 base pairs). The proposed 244 amino acid polypeptide encoded by this gene bears strong homology to the vaccinia virus thymidine kinase. No homology with the thymidine kinases of the herpes simplex viruses was found. PMID:6328447

  11. Parkinson's disease: decreased density of /sup 3/H-imipramine and /sup 3/H-paroxetine binding sites in putamen

    SciTech Connect

    Raisman, R.; Cash, R.; Agid, Y.

    1986-04-01

    The density of high-affinity /sup 3/H-imipramine and /sup 3/H-paroxetine binding sites (two serotonin-uptake blockers) was decreased in the putamen of parkinsonian patients. The correlation between serotonin levels and the number of /sup 3/H-imipramine and /sup 3/H-paroxetine binding sites suggests that they are located on serotoninergic nerve terminals and could be used to study serotoninergic innervation in the human brain. Since imipramine and paroxetine are powerful antidepressants, these results furthermore suggest that decreased serotoninergic transmission may be implicated in the pathophysiology of depression in Parkinson's disease.

  12. Comparison of ( sup 3 H)Phencyclidine (( sup 3 H)PCP) and ( sup 3 H) N-(1-(2-thienyl) cyclohexyl)piperidine (( sup 3 H)TCP) binding properties to rat and human brain membranes

    SciTech Connect

    Vignon, J.; Chaudieu, I.; Allaoua, H.; Journod, L.; Javoy-Agid, F.; Agid, Y.; Chicheportiche, R.

    1989-01-01

    The investigation of ({sup 3}H)PCP and ({sup 3}H)TCP binding properties to rat cerebrum and cerebellum resulted in the demonstration of multiple binding sites for the two drugs. In the two tissue preparations PCP had a lower affinity than TCP. In membranes from the cerebrum an equal number of high affinity binding sites were present for ({sup 3}H)PCP and ({sup 3}H)TCP. However, low affinity binding sites were two times more numerous for ({sup 3}H)PCP than for ({sup 3}H)TCP. In the cerebellum, the number of high and low affinity sites labeled by the two radioligands was identical, but the number of high affinity sites was about 7 fold lower than in cerebrum. In human cerebral cortex samples ({sup 3}H)TCP also bound to two different sites. The number of high and low affinity sites were 12 and 3 times, respectively, less abundant than in the rat cerebrum. Low affinity sites were of higher affinity than corresponding sites in the rat brain. In the human cerebellum ({sup 3}H)TCP binding parameters were identical to those measured in the same region in the rat.

  13. IODINE MONOCHLORIDE FACILITATED DEGLYCOSYLATION, ANOMERIZATION, AND ISOMERIZATION OF 3-SUBSTITUTED THYMIDINE ANALOGUES

    PubMed Central

    Khalil, Ahmed; Ishita, Keisuke; Ali, Tehane; Tiwari, Rohit; Riachy, Ramy; Toppino, Antonio; Hasabelnaby, Sherifa; Sayfullin, Naum; Oliver, Allen G.; Gallucci, Judith; Huang, Zhenguo; Tjarks, Werner

    2014-01-01

    The reaction of thymidine, 3-mono-, and 3,3′,5′-trialkylsubstitued thymidine analogues with iodine monochloride (ICl) was investigated. Treatment with ICl resulted in rapid deglycosylation, anomerization, and isomerization of thymidine and 3-substituted thymidine analogues under various reaction conditions leading to the formation of the nucleobases as the major products accompanied by minor formation of α-furanosidic-, α-pyranosidic- and β-pyranosidic nucleosides. On the other hand, 3,3′,5′-trisubstitued thymidine analogues were only deglycosylated and anomerized. These results are similar to those observed for the acidic hydrolysis of the glycoside bond in nucleosides, but were presumably caused by the Lewis acid character of an iodine electrophile. PMID:25372994

  14. Fallout /sup 3/H ingestion in Akita, Japan

    SciTech Connect

    Hisamatsu, S.; Takizawa, Y.; Abe, T.; Katsumata, T.

    1987-09-01

    To study fallout /sup 3/H ingestion in Japan, 16 separate food group samples were collected from Akita during 1985. The /sup 3/H concentration in free water and that in a tissue-bound form were determined separately. The average /sup 3/H concentration in the tissue-bound form was 2.2 Bq L-1, 1.7 times higher than in the free water of the food. The ingestions of /sup 3/H in the tissue-bound form and as free water in the diet were 0.60 Bq d-1 and 1.0 Bq d-1, respectively. Cereals represented the food group that contributed the most to the ingestion of tissue-bound /sup 3/H. Total /sup 3/H ingestion was estimated to be 4.1 Bq d-1. The contribution of the tissue-bound form to the total ingestion was 15%, considerably lower than reported for Italian diets. The ratio of /sup 3/H ingestion in the tissue-bound form to the free water form in the diet was similar to the ratio reported for New York City.

  15. Transplantation of photoreceptors labeled with tritiated thymidine into RCS rats

    SciTech Connect

    Gouras, P.; Du, J.; Gelanze, M.; Kwun, R.; Kjeldbye, H.; Lopez, R. )

    1991-04-01

    Tritiated thymidine was administered to newborn rats to label photoreceptors, about 50% of which are still dividing. These photoreceptors were enzymatically dissociated and separated from the remainder of the retina after the infant rat matured. These labeled photoreceptors were then transplanted into a foreign host retina in the region of the outer nuclear layer. The hosts were ocular, albinotic, Royal College of Surgeons (RCS) rats, congenic to the normal donors and at least 4 months old, a time when virtually all the photoreceptors have degenerated from their retinas. The transplant site was examined at various times after transplantation by light microscope autoradiography. Labeled photoreceptor cell bodies were found in clusters in the outer nuclear layer region for as long as 3 months after transplantation surgery.

  16. Cell-Cycle Analyses Using Thymidine Analogues in Fission Yeast

    PubMed Central

    Anda, Silje; Boye, Erik; Grallert, Beata

    2014-01-01

    Thymidine analogues are powerful tools when studying DNA synthesis including DNA replication, repair and recombination. However, these analogues have been reported to have severe effects on cell-cycle progression and growth, the very processes being investigated in most of these studies. Here, we have analyzed the effects of 5-ethynyl-2′-deoxyuridine (EdU) and 5-Chloro-2′-deoxyuridine (CldU) using fission yeast cells and optimized the labelling procedure. We find that both analogues affect the cell cycle, but that the effects can be mitigated by using the appropriate analogue, short pulses of labelling and low concentrations. In addition, we report sequential labelling of two consecutive S phases using EdU and 5-bromo-2′-deoxyuridine (BrdU). Furthermore, we show that detection of replicative DNA synthesis is much more sensitive than DNA-measurements by flow cytometry. PMID:24551125

  17. Stereoselective synthesis of P-homochiral oligo(thymidine methanephosphonates).

    PubMed Central

    Lesnikowski, Z J; Jaworska, M; Stec, W J

    1988-01-01

    An approach to the stereoselective synthesis of P-homochiral oligo(thymidine methanephosphonates) is described. Fully protected (Rp)- and (Sp)-diastereomers of MMTrTPMeTAC (3) were prepared in the stereospecific reaction of P-chiral nucleotide component 5'-O-monomethoxytritylthymidine 3'-O-[O-(4-nitrophenyl)methanephosphonate] (1) and 3'-O-acetylthmydine (2) bearing activated 5'-hydroxyl function. Deprotection of the 5'-OH group in 3 and subsequent stepwise reactions of activated 5'-OH oligonucleotide components with (Rp)- or (Sp)- isomers of 1 gave the trinucleotide MMTrTPMeTPMeTAC (4) and, subsequently, the tetranucleotide MMTrTPMeTPMeTPMeTAC (5) possessing all (Rp)- or all (Sp)- configurations at their internucleotide methanephosphonate P-atoms. PMID:3211747

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

    PubMed

    Larsen, N B; Munch-Petersen, B; Piškur, J

    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 efficiently increase the sensitivity of human glioblastoma cells to AZT, and nude rats with intracranial glioblastoma grafts have shown significantly improved survival when treated with the toTK1/AZT system. We show in our paper that the strong suicidal effect of AZT together with toTK1 may be explained by reduced TTP-mediated feedback inhibition of the AZT phosphorylation. PMID:24940681

  19. Structural and Kinetic Characterization of Thymidine Kinase from Leishmania major

    PubMed Central

    Recio, Eliseo; Nettleship, Joanne E.; Rada, Heather; González-Pacanowska, Dolores; Wilson, Keith S.

    2015-01-01

    Leishmania spp. is a protozoan parasite and the causative agent of leishmaniasis. Thymidine kinase (TK) catalyses the transfer of the γ-phosphate of ATP to 2’-deoxythymidine (dThd) forming thymidine monophosphate (dTMP). L. major Type II TK (LmTK) has been previously shown to be important for infectivity of the parasite and therefore has potential as a drug target for anti-leishmanial therapy. In this study, we determined the enzymatic properties and the 3D structures of holo forms of the enzyme. LmTK efficiently phosphorylates dThd and dUrd and has high structural homology to TKs from other species. However, it significantly differs in its kinetic properties from Trypanosoma brucei TK since purines are not substrates of the enzyme and dNTPs such as dUTP inhibit LmTK. The enzyme had Km and kcat values for dThd of 1.1 μM and 2.62 s-1 and exhibits cooperative binding for ATP. Additionally, we show that the anti-retroviral prodrug zidovudine (3-azido-3-deoxythymidine, AZT) and 5’-modified dUrd can be readily phosphorylated by LmTK. The production of recombinant enzyme at a level suitable for structural studies was achieved by the construction of C-terminal truncated versions of the enzyme and the use of a baculoviral expression system. The structures of the catalytic core of LmTK in complex with dThd, the negative feedback regulator dTTP and the bi-substrate analogue AP5dT, were determined to 2.74, 3.00 and 2.40 Å, respectively, and provide the structural basis for exclusion of purines and dNTP inhibition. The results will aid the process of rational drug design with LmTK as a potential target for anti-leishmanial drugs. PMID:25978379

  20. Synthesis of N3-Substituted Carboranyl Thymidine Bioconjugates and their Evaluation as Substrates of Recombinant Human Thymidine Kinase 1

    PubMed Central

    Agarwal, Hitesh K.; McElroy, Craig A.; Sjuvarsson, Elena; Eriksson, Staffan; Darby, Michael V.; Tjarks, Werner

    2013-01-01

    Four different libraries of overall twenty three N3-substituted thymidine (dThd) analogues, including eleven 3-carboranyl thymidine analogues (3CTAs), were synthesized. The latter are potential agents for Boron Neutron Capture Therapy (BNCT) of cancer. Linker between the dThd scaffold and the m-carborane cluster at the N3-position of the 3CTAs contained amidinyl-(3e and 3f), guanidyl-(7e-7g), tetrazolylmethyl-(9b1/2-9d1/2), or tetrazolyl groups (11b1/2-11d1/2) to improve human thymidine kinase 1 (hTK1) substrate characteristics and water solubilities compared with 1st generation 3CTAs, such as N5 and N5-2OH. The amidinyl- and guanidyl-type N3-substitued dThd analogues (3a-3f and 7a-7g) had hTK1 phosphorylation rates of <30% relative to that of dThd, the endogenous hTK1 substrate, whereas the tetrazolyl-type N3-substitued dThd analogues 9a, 9b1/2-9d1/2 and 11a, 11b1/2-11d1/2) had relative phosphorylation rates (rPRs) of >40%. Compounds 9a, 9b1/2-9d1/2 and 11a, 11b1/2-11d1/2 were subjected to in-depth enzyme kinetics studies and the obtained rkcat/Km (kcat/Km relative to that of dThd) ranged from 2.5-26%. The tetrazolyl-type N3-substitued dThd analogues 9b1/2 and 11d1/2 were the best substrates of hTK1 with rPRs of 52.4% and 42.5% and rkcat/Km values of 14.9% and 19.7% respectively. In comparison, the rPR and rkcat/Km values of N5-2OH in this specific study were 41.5% and 10.8%, respectively. Compounds 3e and 3f were >1,900 and >1,500 times, respectively, better soluble in PBS (pH 7.4) than N5-2OH whereas solubilities for 9b1/2-9d1/2 and 11b1/2-11d1/2 were only 1.3 – 13 times better. PMID:23318906

  1. Fallout sup 3 H in human tissue at Akita, Japan

    SciTech Connect

    Hisamatsu, S.; Takizawa, Y.; Itoh, M.; Ueno, K.; Katsumata, T.; Sakanoue, M. )

    1989-10-01

    The {sup 3}H concentration in Japanese human tissue samples is reported in this paper. Four brain, 10 liver, and nine lung samples from 11 cases were collected from Akita Prefecture in northern Japan from January to July 1986. The median of free-water {sup 3}H concentration was similar in these tissues and agreed well with the concentrations in the diet, including tap water. The median specific activity ratio of tissue-bound {sup 3}H to free-water {sup 3}H was 1.1 and was slightly lower than that in the diet. The specific activity ratio was also lower than that reported in the United States and significantly lower than in Italy.

  2. Evidence for indirect involvement of thymidine kinase in excision repair processes in mouse cell lines

    SciTech Connect

    McKenna, P.G.; Yasseen, A.A.; McKelvey, V.J.

    1985-05-01

    Wild-type cells and thymidine kinase-deficient clones from two mouse lymphoma cell lines, P388 and L5178Y, were compared for sensitivity to killing by the mutagens, ultraviolet irradiation (UV), ethyl methane sulfonate (EMS), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Two out of three thymidine kinase-deficient P388 clones showed significantly enhanced sensitivity to killing by all three mutagens. This increased sensitivity to killing was also reflected in increased mutagenesis by the three mutagens. In the L5178Y cell line, wild-type cells showed little difference to two thymidine kinase-deficient clones in terms of mutagen sensitivity. This indicates that thymidine kinase may be significant for DNA repair processes in P388 but not in L5178Y cells. Unscheduled DNA synthesis (UDS) experiments were carried out on P388 and L5178Y wild-type cells and wild-type Friend leukemia cells (which are mutagen-sensitive when deficient in thymidine kinase). The UDS experiments showed the L5178Y cells were low in excision repair abilities relative to the P388 cells and the Friend cell clone. This indicates that the increased mutagen sensitivity in thymidine kinase-deficient P388 and clone 707 Friend cells may be due to thymidine kinase playing an indirect role in DNA excision repair, a process which is of little significance in the L5178Y cell line.

  3. N-H stretching excitations in adenosine-thymidine base pairs in solution: pair geometries, infrared line shapes, and ultrafast vibrational dynamics.

    PubMed

    Greve, Christian; Preketes, Nicholas K; Fidder, Henk; Costard, Rene; Koeppe, Benjamin; Heisler, Ismael A; Mukamel, Shaul; Temps, Friedrich; Nibbering, Erik T J; Elsaesser, Thomas

    2013-01-24

    We explore the N-H stretching vibrations of adenosine-thymidine base pairs in chloroform solution with linear and nonlinear infrared spectroscopy. Based on estimates from NMR measurements and ab initio calculations, we conclude that adenosine and thymidine form hydrogen bonded base pairs in Watson-Crick, reverse Watson-Crick, Hoogsteen, and reverse Hoogsteen configurations with similar probability. Steady-state concentration and temperature dependent linear FT-IR studies, including H/D exchange experiments, reveal that these hydrogen-bonded base pairs have complex N-H/N-D stretching spectra with a multitude of spectral components. Nonlinear 2D-IR spectroscopic results, together with IR-pump-IR-probe measurements, as also corroborated by ab initio calculations, reveal that the number of N-H stretching transitions is larger than the total number of N-H stretching modes. This is explained by couplings to other modes, such as an underdamped low-frequency hydrogen-bond mode, and a Fermi resonance with NH(2) bending overtone levels of the adenosine amino-group. Our results demonstrate that modeling based on local N-H stretching vibrations only is not sufficient and call for further refinement of the description of the N-H stretching manifolds of nucleic acid base pairs of adenosine and thymidine, incorporating a multitude of couplings with fingerprint and low-frequency modes. PMID:23234439

  4. The 3 H(d , γ) Reaction at Ec . m . <= 300 keV

    NASA Astrophysics Data System (ADS)

    Parker, C. E.; Brune, C. R.; Massey, T. N.; O'Donnell, J. E.; Richard, A. L.; Sayre, D. B.

    2015-04-01

    The 3 H(d , γ) 5He reaction has been measured using a 500-keV pulsed deuteron beam incident on a stopping titanium tritide target at the Edwards Accelerator Laboratory. The time-of-flight technique has been used to distinguish the γ-rays from neutrons in the bismuth germinate (BGO) γ-ray detector. A stilbene scintillator and an NE-213 scintillator have been used to detect the neutrons from the 3 H(d , n) α reaction using both the pulse-shape discrimination and time-of-flight techniques. A newly designed target holder with a silicon surface barrier detector to simultaneously measure α-particles to normalize the number of neutrons, along with a new titanium tritide target, was incorporated for subsequent measurements. The γ-rays have been measured at laboratory angles of 0 °, 45 °, 90 °, and 135 °. Information about the γ-ray energy distribution for the unbound ground state and first excited state of 5He can be obtained experimentally by comparing the BGO data to Monte Carlo simulations. The 3 H(d , γ) /3 H(d , n) branching ratio has also been measured. Data analysis is currently underway for the subsequent measurements. This work is supported in part by Lawrence Livermore National Laboratory and the U.S. D.O.E. (NNSA) through Grant No. DE-NA0001837.

  5. Stereospecific binding of 3H-phencyclidine in brain membranes.

    PubMed

    Hampton, R Y; Medzihradsky, F; Woods, J H; Dahlstrom, P J

    1982-06-21

    Phencyclidine (PCP) displaceable binding of 3H-PCP to glass-fiber filters was eliminated and total binding markedly reduced by initial treatment of the discs with 0.05% polyethyleneimine. Assessed with treated filters, unlabeled PCP displaced 3H-PCP in both rat and pigeon brain membranes with an EC50 of 1 microM. Of similar high inhibitory potency were dextrorphan, levorphanol, SKF 10047 and ketamine, while morphine, naloxone and etorphine had EC50 values higher then 1 mM. Using the dissociative anesthetic dexoxadrol and its inactive isomer levoxadrol as displacing agents, stereospecific binding of 3H-PCP was obtained in rat and pigeon brain membranes. The markedly higher potency of dexoxadrol, relative to levoxadrol, in displacing bound 3H-PCP is compatible with behavioral data for these enantiomers. However, they were equipotent in displacing 3H-PCP bound to glass-fiber filters in the absence of tissue. Heat denaturation, but not freezing, abolished stereospecific binding of 3H-PCP, which was also absent in rat liver membranes. The stereospecific binding component in brain displayed biphasic saturability at 60-70 nM and 300-400 nM, respectively. PMID:7109842

  6. (/sup 3/H)-beta-endorphin binding in rat brain

    SciTech Connect

    Houghten, R.A.; Johnson, N.; Pasternak, G.W.

    1984-10-01

    The binding of (/sup 3/H)-beta-endorphin to rat brain homogenates is complex. Although Scatchard analysis of saturation studies yields a straight line, detailed competition studies are multiphasic, suggesting that even at low concentrations of the compound, the /sup 3/H-ligand is binding to more than one class of site. A portion of (/sup 3/H)-beta-endorphin binding is sensitive to low concentrations of morphine or D-Ala2-Leu5-enkephalin (less than 5 nM). The inhibition observed with each compound alone (5 nM) is the same as that seen with both together (each at 5 nM). Thus, the binding remaining in the presence of both morphine and the enkephalin does not correspond to either mu or delta sites. The portion of (/sup 3/H)-beta-endorphin binding that is inhibited under these conditions appears to be equally sensitive to both morphine and the enkephalin and may correspond to mu1 sites. Treating membrane homogenates with naloxonazine, a mu1 selective antagonist, lowers (/sup 3/H)-beta-endorphin binding to the same degree as morphine and D-Ala2-Leu5-enkephalin alone or together. This possible binding of (/sup 3/H)-beta-endorphin to mu1 sites is consistent with the role of mu1 sites in beta-endorphin analgesia and catalepsy in vivo.

  7. THE EFFECT OF CHLORINATION OF NUCLEOTIDE BASES ON THE CONFORMATIONAL PROPERTIES OF THYMIDINE MONOPHOSPHATE.

    PubMed

    Mukhina, T M; Nikolaienko, T Yu

    2015-01-01

    Recent studies on Escherichia coli bacteria cultivation, in which DNA thymine was replaced with 5-chlorouracil have refreshed the problem of understanding the changes to physical properties of DNA monomers resultant from chemical modifications. These studies have shown that the replacement did not affect the normal activities and division of the bacteria, but has significantly reduced its life span. In this paper a comparative analysis was carried out by the methods of computational experiment of a set of 687 possible conformers of natural monomeric DNA unit (2'-deoxyribonucleotide thymidine monophosphate) and 660 conformers of 5-chloro-2'-deoxyuridine monophosphate - a similar molecules in which the natural nitrogenous base thymine is substituted with 5-chlorouracil. Structures of stable conformers of the modified deoxyribonucleotide have been obtained and physical factors, which determine their variation from the conformers of the unmodified molecule have been analyzed. A comparative analysis of the elastic properties of conformers of investigated molecules and non-covalent interactions present in them was conducted. The results can be usedfor planning experiments on synthesis of artficial DNA suitable for incorporation into living organisms. PMID:26255348

  8. Mitochondrial DNA depletion and thymidine phosphate pool dynamics in a cellular model of mitochondrial neurogastrointestinal encephalomyopathy.

    PubMed

    Pontarin, Giovanna; Ferraro, Paola; Valentino, Maria L; Hirano, Michio; Reichard, Peter; Bianchi, Vera

    2006-08-11

    Mitochondrial (mt) neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disease associated with depletion, deletions, and point mutations of mtDNA. Patients lack a functional thymidine phosphorylase and their plasma contains high concentrations of thymidine and deoxyuridine; elevation of the corresponding triphosphates probably impairs normal mtDNA replication and repair. To study metabolic events leading to MNGIE we used as model systems skin and lung fibroblasts cultured in the presence of thymidine and/or deoxyuridine at concentrations close to those in the plasma of the patients, a more than 100-fold excess relative to controls. The two deoxynucleosides increased the mt and cytosolic dTTP pools of skin fibroblasts almost 2-fold in cycling cells and 8-fold in quiescent cells. During up to a two-month incubation of quiescent fibroblasts with thymidine (but not with deoxyuridine), mtDNA decreased to approximately 50% without showing deletions or point mutations. When we removed thymidine, but maintained the quiescent state, mtDNA recovered rapidly. With thymidine in the medium, the dTTP pool of quiescent cells turned over rapidly at a rate depending on the concentration of thymidine, due to increased degradation and resynthesis of dTMP in a substrate (=futile) cycle between thymidine kinase and 5'-deoxyribonucleotidase. The cycle limited the expansion of the dTTP pool at the expense of ATP hydrolysis. We propose that the substrate cycle represents a regulatory mechanism to protect cells from harmful increases of dTTP. Thus MNGIE patients may increase their consumption of ATP to counteract an unlimited expansion of the dTTP pool caused by circulating thymidine. PMID:16774911

  9. Alterations of nuclear DNA synthesis after irradiation of the cellular slime mold Dictyostelium discoideum: studies performed in a mutant strain displaying enhanced thymidine uptake

    SciTech Connect

    Hurley, D.L.

    1986-01-01

    The auxotrophic Dictyostelium discoideum strain HPS 401 was studied. Thymidine at 8 ..mu..g/ml or thymidylate at 50 ..mu..g/ml supported growth to maximal cell densities. Thin layer chromatography of cell extracts showed rapid intracellular accumulation of thymidine in HPS 401 vs slightly detectable accumulation in wild-type cells. Measurements showed that methionine and thymidylate were taken into all strains at a low rate, but HPS 401 had enhanced uptake of thymidine and uridine compared to wild-type. The HPS 401 phenotype is due to the efficient utilization of thymidine as a result of increased nucleoside uptake. Rapid nuclear purification removed mitochondrial DNA without decreasing the single-strand molecular weight of the nuclear DNA. The nuclear DNA peaks on alkaline sucrose gradients were identified using filter hybridization to cloned probes. As measured by pulse-chase labelling, production of full-sized main band DNA required 45-50 minutes. Pulse labelling of the cells immediately after ultraviolet irradiation caused the single-strand molecular weight of the DNA synthesized to decrease from 8 x 10/sup 6/ daltons at O J/m/sup 2/ to 3.9 x 10/sup 6/ daltons at 50 J/m/sup 2/ to 2.6 x 10/sup 6/ daltons at 200 J/m/sup 2/. The time required for maturation into full-sized DNA increased from 1 hour at O J/m/sup 2/ to 4 hours at 20 J/m/sup 2/ and to 21 hours at 200 J/m/sup 2/. Measured amounts of DNA synthesis at times after ultraviolet irradiation showed a period of reduced incorporation, followed by the resumption of control levels. The lag period ended at the same time as the production of full-sized DNA resumed.

  10. Uptake of /sup 3/H-choline and synthesis of /sup 3/H-acetylcholine by human penile corpus cavernosum

    SciTech Connect

    Blanco, R.; Saenz de Tejada, I.; Azadzoi, K.; Goldstein, I.; Krane, R.J.; Wotiz, H.H.; Cohen, R.A.

    1986-03-05

    The neuroeffectors which relax penile smooth muscle and lead to erection are unknown; physiological studies of human corpus cavernosum, in vitro, have suggested a significant role of cholinergic neurotransmission. To further characterize the importance of cholinergic nerves, biopsies of human corpus cavernosum were obtained at the time of penile prosthesis implantation. Tissues were incubated in /sup 3/H-choline (10/sup -5/M, 80 Ci/mmol) in oxygenated physiological salt solution at 37/sup 0/C, pH 7.4 for 1 hour. Radiolabelled compounds were extracted with perchloric acid (0.4 M) and acetylcholine and choline were separated by HPLC; /sup 14/C-acetylcholine was used as internal standard. /sup 3/H-choline was accumulated by the tissues (20 +/- 1.9 fmol/mg), and /sup 3/H-acetylcholine was synthesized (4.0 +/- 1.1 fmol/mg). In control experiments, heating of the tissue blocked synthesis of /sup 3/H-acetylcholine. Inhibition of high affinity choline transport by hemicholinium-3 (10/sup -5/M) diminished tissue accumulation of /sup 3/H-choline and significantly reduced the synthesis of /sup 3/H-acetylcholine (0.5 +/ 0.2 fmol/mg, p < 0.05). These results provide direct evidence of neuronal accumulation of choline and enzymatic conversion to acetylcholine in human corpus cavernosum. Taken together with the physiological studies, it can be concluded that cholinergic neurotransmission in human corpus cavernosum plays a role in penile erection.

  11. Pharmacological studies on quaternized 4(3H)-quinazolinones.

    PubMed

    Buyuktimkin, S; Ekinci, A C; Buyuktimkin, N; Otuk, G

    1992-11-01

    Locomotor activity-inhibiting, anticonvulsant, muscle relaxant, analgesic, and antimicrobial properties of 2-methyl-3-pyridinium-acetylamino-4(3H)-quinazolinone chloride (1), 2-methyl-3-(4-methylpyridinium)acetylamino-4(3H)-quinazolinone chloride (2), 2-methyl-3-(4-ethylpyridinium)acetylamino-4(3H)-quinazolinone chloride (3), 2-methyl-3-(3-carboxamidopyridinium)acetylamino-4(3H)-quinazolinon e chloride (4), and 2-methyl-3-(4-carboxamidopyridinium)-acetylamino-4(3H)- quinazolinone chloride (5) were investigated. The locomotor activity-inhibiting properties and anticonvulsant activity of 2 were almost equal to those of methaqualone. The analgesic activities of 2 and 3 in the hot-plate test were equal to that of aspirin, whereas in the Koster test, the analgesic activity of 2 was higher. The compounds did not exhibit antimicrobial or muscle relaxant properties. Most active compounds had higher lipophilicity values than those of inactive compounds. PMID:1447711

  12. Late-onset MNGIE without peripheral neuropathy due to incomplete loss of thymidine phosphorylase activity.

    PubMed

    Massa, Roberto; Tessa, Alessandra; Margollicci, Maria; Micheli, Vanna; Romigi, Andrea; Tozzi, Giulia; Terracciano, Chiara; Piemonte, Fiorella; Bernardi, Giorgio; Santorelli, Filippo M

    2009-12-01

    Mitochondrial NeuroGastroIntestinal Encephalomyopathy (MNGIE) is an autosomal recessive disorder characterized by severe gastrointestinal dysmotility, cachexia, peripheral neuropathy, ptosis, ophthalmoplegia, and leukoencephalopathy with early onset and severe prognosis. Mutations in the TYMP/ECGF1 gene cause a loss of thymidine phosphorylase catalytic activity, disrupting the homeostasis of intramitochondrial nucleotide pool. We report a woman with a very late onset of MNGIE, lacking peripheral neuropathy. Thymidine phosphorylase activity was markedly reduced in cultured fibroblasts, but only mildly reduced in buffy coat, where the defect is usually detected, and plasma thymidine was mildly increased compared to typical MNGIE patients. TYMP/ECGF1 analysis detected two heterozygous mutations, including a novel missense mutation. These findings indicate that a partial loss of thymidine phosphorylase activity may induce a late-onset and incomplete MNGIE phenotype. PMID:19853446

  13. Multistep virtual screening for rapid and efficient identification of non-nucleoside bacterial thymidine kinase inhibitors.

    PubMed

    Zander, Johannes; Hartenfeller, Markus; Hähnke, Volker; Proschak, Ewgenij; Besier, Silke; Wichelhaus, Thomas A; Schneider, Gisbert

    2010-08-16

    Antimicrobial activity of trimethoprim/sulfamethoxazole (SXT) against Staphylococcus aureus (S. aureus) is antagonized by thymidine, which is abundant in infected or inflamed human tissue. To restore the antimicrobial activity of SXT in the presence of thymidine, we screened for small-molecule inhibitors of S. aureus thymidine kinase with non-nucleoside scaffolds. We present the successful application of an adaptive virtual screening protocol for novel antibiotics using a combination of ligand- and structure-based approaches. Two consecutive rounds of virtual screening and in vitro testing were performed that resulted in several non-nucleoside hits. The most potent compound exhibits substantial antimicrobial activity against both methicillin-resistant S. aureus strain ATCC 700699 and nonresistant strain ATCC 29213, when combined with SXT in the presence of thymidine. This study demonstrates how virtual screening can be used to guide hit finding in antibacterial screening campaigns with minimal experimental effort. PMID:20648496

  14. Thymidine kinase-deficient herpes simplex virus type 2 genital infection in guinea pigs.

    PubMed Central

    Stanberry, L R; Kit, S; Myers, M G

    1985-01-01

    In guinea pigs, thymidine kinase-producing strains of herpes simplex virus type 2 replicated to high titer in the vagina and spinal cord, and animals developed severe clinical disease. Infection with thymidine kinase-deficient virus resulted in similar vaginal virus titers; however, animals exhibited little or no clinical illness and only low titers of virus were detected in spinal cord homogenate cultures. Neural and extraneural latent infection as well as recurrent infection were noted in animals inoculated with either thymidine kinase-producing or -deficient viruses. These data suggest that neural pathways are important in the pathogenesis of genital herpes and that virus-coded thymidine kinase may influence virulence but is not required for latency. Images PMID:2991558

  15. [Synthesis and biological properties of α-thymidine 5'-aryl phosphonates].

    PubMed

    Ivanov, M A; Karpenko, I L; Chernousova, L N; Andreevskaia, S N; Smirnova, T G; Aleksandrova, L A

    2013-01-01

    The interaction of CDI-activated diethyl phosphonoacetate with methyl 4-aminobenzoat or 3,5-difluoromethylphenylamine followed by treatment with Me3SiBr in DMF led to N-aryl aminocarbonylmethyl phosphonates and their ethyl esters. Their coupling with 3'-acetyl-α-thymidine followed by removal of the acetyl groups gave (α-D-thymidine-5'-il) N-[4-(methoxycarbonyl-, aminocarbonyl- and carboxy)phenyl]-aminocarbonylmethyl phosphonates, (α-D-thymidine-5'-il)-[3,5-bis(trifluoromethyl)phenylaminocarbonyl]methyl phosphonate and their ethyl esters. The phosphonates were stable in different conditions, low cytotoxic (in Vero and K562 cells) and were able to penetrate into K562 cells. The only ethyl ester of (α-D-thymidine-5'-il) N-[4-(methoxycarbonyl)phenyl]-aminocarbonylmethyl phosphonate in high concentration (200 μg/mL) inhibited in vitro the growth of laboratory sensitive strain of Mycobacterium tuberculosis H37Rv. PMID:25696933

  16. Extending Thymidine Kinase Activity to the Catalytic Repertoire of Human Deoxycytidine Kinase

    SciTech Connect

    Hazra, Saugata; Sabini, Eliszbetta; Ort, Stephan; Konrad, Manfred; Lavie, Arnon

    2009-03-04

    Salvage of nucleosides in the cytosol of human cells is carried out by deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1). Whereas TK1 is only responsible for thymidine phosphorylation, dCK is capable of converting dC, dA, and dG into their monophosphate forms. Using structural data on dCK, we predicted that select mutations at the active site would, in addition to making the enzyme faster, expand the catalytic repertoire of dCK to include thymidine. Specifically, we hypothesized that steric repulsion between the methyl group of the thymine base and Arg104 is the main factor preventing the phosphorylation of thymidine by wild-type dCK. Here we present kinetic data on several dCK variants where Arg104 has been replaced by select residues, all performed in combination with the mutation of Asp133 to an alanine. We show that several hydrophobic residues at position 104 endow dCK with thymidine kinase activity. Depending on the exact nature of the mutations, the enzyme's substrate preference is modified. The R104M-D133A double mutant is a pyrimidine-specific enzyme due to large K{sub m} values with purines. The crystal structure of the double mutant R104M-D133A in complex with the L-form of thymidine supplies a structural explanation for the ability of this variant to phosphorylate thymidine and thymidine analogs. The replacement of Arg104 by a smaller residue allows L-dT to bind deeper into the active site, making space for the C5-methyl group of the thymine base. The unique catalytic properties of several of the mutants make them good candidates for suicide-gene/protein-therapy applications.

  17. Deoxypyrimidine monophosphate bypass therapy for thymidine kinase 2 deficiency

    PubMed Central

    Garone, Caterina; Garcia-Diaz, Beatriz; Emmanuele, Valentina; Lopez, Luis C; 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-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. Subject Categories Genetics, Gene Therapy & Genetic Disease; Metabolism PMID:24968719

  18. Sonochemical transformation of thymidine: A mass spectrometric study.

    PubMed

    Chandran, Jisha; Aravind, Usha K; Aravindakumar, C T

    2015-11-01

    Ultrasound is extensively used in medical field for a number of applications including targeted killing of cancer cells. DNA is one of the most susceptible entities in any kind of free radical induced reactions in living systems. In the present work, the transformation of thymidine (dT) induced by ultrasound (US) was investigated using high resolution mass spectrometry (LC-Q-ToF-MS). dT was subjected to sonolysis under four different frequencies (200, 350, 620 and 1000 kHz) and at three power densities (10.5, 24.5 and 42 W/mL) in aerated as well as argon saturated conditions. A total of twenty modified nucleosides including non-fully characterized dT dimeric compounds were detected by LC-Q-ToF-MS. Out of these products, seven were obtained only in the argon atmosphere and two only in the aerated conditions. Among the identified products, there were base modified products and sugar modified products. The products were formed by the reaction of hydroxyl radical and hydrogen atom. Under aerated conditions, the reactions proceed via the formation of hydroperoxides, while in argon atmosphere disproportionation and radical recombinations predominate. The study provides a complete picture of sonochemical transformation pathways of dT which has relevance in DNA damage under ultrasound exposure. PMID:26186835

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

    PubMed

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

    2012-12-01

    Thymidine kinase 1 (TK1) is a soluble biomarker associated with DNA synthesis. This prospective study evaluated serum TK1 activity in dogs presenting with hemoabdomen and a splenic mass. An ELISA using azidothymidine as a substrate was used to evaluate TK1 activity. Sixty-two dogs with hemoabdomen and 15 normal controls were studied. Serum TK1 activity was significantly higher in dogs with hemangiosarcoma (HSA) than in normal dogs (mean ± SEM = 17.0 ± 5.0 and 2.01 ± 0.6, respectively), but not dogs with benign disease (mean ± SEM = 10.0 ± 3.3). Using a cut-off of 6.55 U/L, TK activity demonstrated a sensitivity of 0.52, specificity of 0.93, positive predictive value of 0.94 and negative predictive value of 0.48 for distinguishing HSA versus normal. When interval thresholds of <1.55 and >7.95 U/L were used together, diagnostic utility was increased. Serum TK1 evaluation may help to discriminate between benign disease and HSA in dogs with hemoabdomen and a splenic mass. PMID:22236280

  20. Vorinostat synergises with capecitabine through upregulation of thymidine phosphorylase

    PubMed Central

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

    2010-01-01

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

  1. The proliferation marker thymidine kinase 1 in clinical use

    PubMed Central

    ZHOU, JI; HE, ELLEN; SKOG, SVEN

    2013-01-01

    Tumor-related biomarkers are used for the diagnosis, prognosis and monitoring of treatments and follow-up of cancer patients, although only a few are fully accepted for the detection of invisible/visible tumors in health screening. Thymidine kinase 1 (TK1), a cell cycle-dependent and thus a proliferation-related marker, has been extensively studied during the last decades, using both biochemical and immunological techniques. Therefore, TK1 is an emerging potential proliferating biomarker in oncology that may be used for the prognosis and monitoring of tumor therapy, relapse and survival. In addition, TK1 concentration in serum (STK1p) is a useful biomarker in healthy screening for the detection of potential malignancy development as well as the identification of early-stage tumors, with a few false-positive cases (ROC value, 0.96; tumor proliferation sensitivity, 0.80; specificity, 0.99). In this review, we examine results regarding the expression of STK1p and TK1 in relation to cancer patients and STK1p in health screening published between 2000 and 2012. The use of tumor-related markers recommended by international cancer organizations is also discussed. This review provides valuable information for applications in tumor patients, in health screening and for cancer research. PMID:24649117

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

    PubMed

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

    2005-08-01

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

  3. Development of a Novel Plasmid-Free Thymidine Producer by Reprogramming Nucleotide Metabolic Pathways

    PubMed Central

    Kim, Jin-Sook; Jeong, Min-Kyung; Koo, Bong-Seong

    2015-01-01

    A novel thymidine-producing strain of Escherichia coli was prepared by genome recombineering. Eleven genes were deleted by replacement with an expression cassette, and 7 genes were integrated into the genome. The resulting strain, E. coli HLT013, showed a high thymidine yield with a low deoxyuridine content. DNA microarrays were then used to compare the gene expression profiles of HLT013 and its isogenic parent strain. Based on microarray analysis, the pyr biosynthesis genes and 10 additional genes were selected and then expressed in HLT013 to find reasonable candidates for enhancing thymidine yield. Among these, phage shock protein A (PspA) showed positive effects on thymidine production by diminishing redox stress. Thus, we integrated pspA into the HLT013 genome, resulting in E. coli strain HLT026, which produced 13.2 g/liter thymidine for 120 h with fed-batch fermentation. Here, we also provide a basis for new testable hypotheses regarding the enhancement of thymidine productivity and the attainment of a more complete understanding of nucleotide metabolism in bacteria. PMID:26319873

  4. Comparison of the metabolism of [1,2,6,7-3H(N)]cholesteryl oleate, cholesteryl [9,10-3H]oleate, and cholesteryl [1-14C]oleate labeled lipoproteins in the rat.

    PubMed

    Terpstra, A H

    1994-04-01

    The intravascular metabolism of sterol labeled [1,2,6,7-3H(N)]cholesteryl oleate and acyl labeled cholesteryl [9,10-3H]oleate and cholesteryl [1-14C]oleate was compared in the rat, an animal species without plasma cholesteryl ester transfer activity (CETA). In a first series of studies, the metabolism of sterol labeled [1,2,6,7-3H(N)]cholesteryl oleate and acyl labeled cholesteryl [1-14C]oleate was compared, and the two tracers had identical plasma clearance rates when incorporated into human low density lipoproteins (LDL). The 3H sterol labeled cholesteryl ester (CE), however, had a plasma clearance rate lower than the 14C acyl labeled CE when incorporated into rat alpha- and beta-migrating LDL and human or rat high density lipoproteins (HDL). Unesterified 3H cholesterol reappeared in the plasma whereas the 14C radioactivity in the plasma remained associated with the CE. In a second set of studies, LDL and HDL were radiolabeled with cholesteryl [9,10-3H]oleate and cholesteryl [1-14C]oleate. Large amounts of 3H radioactivity that were dialyzable and not associated with the lipoprotein CE reappeared in the plasma during the kinetic studies. The two tracers had identical plasma disappearance rates when the plasma samples were dialyzed. The results of these studies indicate that the nature of the tracer used to trace lipoprotein CE can affect the estimated kinetic parameters of plasma CE. PMID:8060380

  5. R-matrix description of particle energy spectra produced by low-energy 3H + 3H reactions

    DOE PAGESBeta

    Brune, C. R.; Caggiano, J. A.; Sayre, D. B.; Bacher, A. D.; Hale, G. M.; Paris, M. W.

    2015-07-20

    An R-matrix model for three-body final states is presented and applied to a recent measurement of the neutron energy spectrum from the 3H + 3H→ 2n + α reaction. The calculation includes the n alpha and n n interactions in the final state, angular momentum conservation, antisymmetrization, and the interference between different channels. A good fit to the measured spectrum is obtained, where clear evidence for the 5He ground state is observed. The model is also used to predict the alpha-particle spectrum from 3H + 3H as well as particle spectra from 3He + 3He. The R-matrix approach presented heremore » is very general, and can be adapted to a wide variety of problems with three-body final states.« less

  6. (/sup 3/H)forskolin- and (/sup 3/H)dihydroalprenolol-binding sites and adenylate cyclase activity in heart of rats fed diets containing different oils

    SciTech Connect

    Alam, S.Q.; Ren, Y.F.; Alam, B.S.

    1988-03-01

    The characteristics of the cardiac adenylate cyclase system were studied in rats fed diets containing fish oil (menhaden oil) and other oils. Adenylate cyclase activity generally was higher in cardiac homogenates and membranes of rats fed diet containing 10% menhaden oil than in the other oils. The increase in enzyme activity, especially in forskolin-stimulated activity, was associated with an increase in the concentration of the (/sup 3/H) forskolin-binding sites in cardiac membranes of rats fed menhaden oil. The beta-adrenergic receptor concentration was not significantly altered although the affinity for (/sup 3/H)dihydroalprenolol-binding was lower in membranes of rats fed menhaden oil than those fed the other oils. omega-3 fatty acids from menhaden oil were incorporated into the cardiac membrane phospholipids. The results suggest that the observed increase in myocardial adenylate cyclase activity of rats fed menhaden oil may be due to an increase in the number of the catalytic subunits of the enzyme or due to a greater availability of the forskolin-binding sites.

  7. Photoaffinity labeling of opiate receptors with /sup 3/H-etorphine: possible species differences in glycosylation

    SciTech Connect

    Bowen, W.D.; Kooper, G.

    1986-01-01

    Opiate receptors from whole rat brain (minus cerebellum) and cow striatum were labeled irreversibly using the intrinsic photolability of /sup 3/H-etorphine. After incubation with 2 nM /sup 3/H-etorphine and centrifugal washing, membranes were irradiated with light of 254 nm. Non-specific binding was determined by carrying out incubations in presence and absence of 10 microM levallorphan. Specific binding in photolabeled membranes was 75-80%, with a photo-incorporation yield of approximately 50%. Photolabeled membranes were extracted with CHAPS/Lubrol and unbound /sup 3/H-etorphine was removed by dialysis and passage over Sephadex G-25. Solubilized proteins were then subjected to chromatography on wheat germ agglutinin, and retained proteins were eluted with N-acetyl D-glucosamine (NAG). Protein profiles from rat brain and cow striatum were identical, with 89% of the total protein flowing through unretained and 11% eluted by NAG. However, the profile of radioactivity was markedly different in the two species. With rat, the specific activity (cpm/A280) was the same for flow-through and NAG-eluate. With cow, the specific activity of the NAG-eluate was 17 times greater than the flow-through. These results indicate that cow striatum and rat whole brain contain populations of opiate receptors which are glycosylated differently.

  8. Thymidine Phosphorylase Participates in Platelet Signaling and Promotes Thrombosis

    PubMed Central

    Li, Wei; Gigante, Alba; Perez-Perez, Maria-Jesus; Yue, Hong; Hirano, Michio; McIntyre, Thomas; Silverstein, Roy L

    2014-01-01

    Rationale Platelets contain abundant thymidine phosphorylase (TYMP), which is highly expressed in diseases with high risk of thrombosis, such as atherosclerosis and type II diabetes. Objective Test the hypothesis that TYMP participates in platelet signaling and promotes thrombosis. Methods and Results By using a ferric chloride (FeCl3) induced carotid artery injury thrombosis model, we found time to blood flow cessation was significantly prolonged in Tymp−/− and Tymp+/− mice compared to wild type (WT) mice. Bone marrow transplantation and platelet transfusion studies demonstrated that platelet TYMP was responsible for the antithrombotic phenomenon in the TYMP deficient mice. Collagen-, collagen-related peptide (CRP)-, adenosine diphosphate-and/or thrombin-induced platelet aggregation were significantly attenuated in Tymp+/− and Tymp−/− platelets, and in WT or human platelets pretreated with TYMP inhibitor KIN59. Tymp deficiency also significantly decreased agonist-induced P-select in expression. TYMP contains an N-terminal SH3 domain binding proline-rich motif and forms a complex with the tyrosine kinases Lyn, Fyn and Yes in platelets. TYMP-associated Lyn was inactive in resting platelets, and TYMP trapped and diminished active Lyn after collagen stimulation. Tymp/Lyn double haploinsufficiency diminished the antithrombotic phenotype of Tymp+/− mice. TYMP deletion or inhibition of TYMP with KIN59 dramatically increased PECAM-1 tyrosine phosphorylation and diminished CRP or collagen induced AKT phosphorylation. In vivo administration of KIN59 significantly inhibited FeCl3 induced carotid artery thrombosis without affecting hemostasis. Conclusion TYMP participates in multiple platelet signaling pathways and regulates platelet activation and thrombosis. Targeting TYMP might be a novel anti-platelet and anti-thrombosis therapy. PMID:25287063

  9. The Use of 14C-FIAU to Predict Bacterial Thymidine Kinase Presence: Implications for Radiolabeled FIAU Bacterial Imaging

    PubMed Central

    Peterson Kristin, L; Reid William, C; Freeman Alexandra, F; Holland Steven, M; Roderic, Pettigrew; 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 124I-FIAU-PET. Methods We obtained representative strains of bacterial pathogens isolated from actual patients with genetic immunodeficiencies. We evaluated the bacterial susceptibility of different strains to the effect of incubation with FIAU, which would implicate the presence of the thymidine kinase (TK) enzyme. We also incubated the bacteria with 14C-FIAU and consequently measured its rate of incorporation in the bacterial DNA using a liquid scintillation counter. Results Unlike the other bacterial strains, the growth of Pseudomonas aeruginosa was not halted by FIAU at any concentration. All the tested clinical isolates demonstrated different levels of 14C-FIAU uptake, except for P. aeruginosa. Conclusion Radiolabeled FIAU has been successful in delineating bacterial infections, both in preclinical and pilot translational studies. In patients with immunodeficiencies, Pseudomonas infections are commonly encountered and are usually difficult to differentiate from fungal infections. The use of radiolabeled FIAU for in vivo imaging of those patients, however, would not be useful, considering the apparent lack of TK enzyme in Pseudomonas. One has to keep in mind that not all pathogenic bacteria possess the TK enzyme and as such will not all retain

  10. Identification of the herpes simplex virus DNA sequences present in six herpes simplex virus thymidine kinase-transformed mouse cell lines.

    PubMed Central

    Leiden, J M; Frenkel, N; Rapp, F

    1980-01-01

    We have used a novel filter hybridization approach to detect and map the herpes simplex virus (HSV) DNA sequences which are present in four HSV thymidine kinase (HSVtk+)-transformed cell lines which were derived by exposure of thymidine kinase negative (tk-) mouse cells to UV light-irradiated HSV type 2 (HSV-2). In addition, we have mapped the HSV-1 DNA sequences which are present in two HSV-1tk+-transformed cell lines produced by transfection of tk- mouse cells with sheared HSV-1 DNA. The results of these studies can be summarized as follows. (i) The only HSV DNA sequences which were common to all HSVtk+-transformed cells were those located between map coordinates 0.28 and 0.32. Thus, this region contains all of the viral DNA sequences which are necessary for the expression of HSV-mediated tk transformation. (ii) Many of the cell lines also contained variable amounts of non-tk gene viral DNA sequences located between map coordinates 0.11 to 0.57 and 0.82 to 1.00, suggesting that incorporation of the viral DNA sequences located between these map coordinates is a relatively random event. (iii) The viral DNA sequences located between map coordinates 0 to 0.11 and 0.57 to 0.82 were uniformly absent from all of the HSVtk+ cell lines tested, suggesting that there is a strong negative selective pressure against incorporation of these viral DNA sequences. Images PMID:6245232

  11. Preformance Analysis of NH3-H2O Absorption Cycle

    NASA Astrophysics Data System (ADS)

    Tsujimori, Atsushi; Ozaki, Eiichi

    Different from H2O-LiBr absorption cycle, it is necessary to have rectifier between generator and condenser in NH3-H2O absorption cycle, because there mixes some steam in refrigerant vapor in the process of regenerating refrigerant from the ammonia strong aqueous solution. And in some case ex. partial load or heating, the efficiency of rectifier might decrease, if the flow rate of refrigerant vapor and ammonia aqueous solution decrease. As a result, steam flow into condenser with ammonia refrigerant vapor, which reduces cycle COPs of cooling and heating. Accordingly in order to evaluate the effect of ammonia concentration in refrigerant for the performance of NH3-H2O absorption heat pump, the simple design approach of modeling condenser and evaporator is introduced in this paper. In the model, the calculation of heat rate in condenser and evaporator was simplified considering the characteristic of NH3-H2O liquid-vapor equilibrium. Then the simulation for cycle perforance based on GAX absorption cycle was made using the efficiency of rectifier that established the ammonia concentration in refrigerant and it was derived that 3 [%] decrease of ammonia concentration in refrigerant induced 15 [%] decrcase of cooling COP and 7 [%] decrease of heating COP and that there existed the most suitable circulation ratio for each ammonia concentration in refrigerant.

  12. (/sup 3/H)nitrendipine binding to adrenal capsular membranes

    SciTech Connect

    Finkel, M.S.; Aguilera, G.; Catt, K.J.; Keiser, H.R.

    1984-08-20

    The physiologic regulation of aldosterone secretion is dependent on extracellular calcium and appears to be mediated by increases in cytosolic free calcium concentration in the zona glomerulosa cell. A specific role for voltage-dependent calcium channels was suggested by previous studies with the calcium channel antagonist verapamil. The authors therefore studied the (/sup 3/H)nitrendipine calcium channel binding site in adrenal capsules. These studies revealed a single class of saturable, high affinity sites with K/sub D/ = .26 +/- .04 nM and B/sub max/ = 105 +/- 5.7 fmol/mg protein. Specific binding of (/sup 3/H)nitrendipine was inhibited by calcium channel antagonists with potencies nitrendipine = nifedipine >> verapamil, while diltiazem had no inhibitory effect. In the rat, binding sites for (/sup 3/H)nitrendipine were located in the adrenal capsule and medulla and were undetectable in the zona fasciculata. Physiologic studies with collagenase-dispersed adrenal glomerulosa cells demonstrated that nifedipine selectively inhibited angiotensin-II and potassium-stimulated steroidogenesis. These observations suggest both a pharmacologic and physiologic role for the nitrendipine binding site in aldosterone production. 17 references, 2 figures, 1 table.

  13. Astronomical identification of the C3H radical

    NASA Astrophysics Data System (ADS)

    Thaddeus, P.; Gottlieb, C. A.; Hjalmarson, A.; Johansson, L. E. B.; Irvine, W. M.; Friberg, P.; Linke, R. A.

    1985-07-01

    The C3H radical has been identified in the millimeter-wave spectra of IRC +10216 and TMC-1. In IRC +10216, four rotational transitions have been observed, three in the lower fine-structure ladder (2Pi1/2) and one in the upper (2Pi3/2), each a resolved or partially resolved lambda-doublet. In TMC-1, both lambda components of the lowest lying 3/2-1/2 transition of the 2Pi1/2 ladder have been observed, each with well-resolved hfs. In IRC +10216, the excitation of C3H is similar to that of SiCC: the rotational temperature Trot within the 2Pi1/2 ladder is low (8.5 K), because of rapid radiative decay, while Trot across the ladders is high (about 52 K), because interconnecting far-IR radiative transitions are only weakly permitted. The column density of C3H in IRC +10216 averaged over the estimated source diameter of 84 arcsec is 2.8 x 10 to the 13th/sq cm, an order of magnitude less than that of C2H and C4H.

  14. Isolation, identification, and assay of [3H]-porfiromycin adducts of EMT6 mouse mammary tumor cell DNA: effects of hypoxia and dicumarol on adduct patterns.

    PubMed

    Tomasz, M; Hughes, C S; Chowdary, D; Keyes, S R; Lipman, R; Sartorelli, A C; Rockwell, S

    1991-07-01

    [3H]-(N-la-methyl) Porfiromycin (POR) was employed to detect and identify the radiolabeled mono- and bis-adducts formed in living EMT6 mouse mammary tumor cells under different conditions. To provide authentic standard adducts, calf-thymus DNA was treated with POR under reductive activation, then digested to nucleosides and POR-nucleoside adducts. The three major adducts formed were isolated by HPLC and authenticated. Two were mono-adducts, composed of deoxyguanosine linked at its N2-position to C-1 of POR and of 10-decarbamoyl POR. The third was a bis-adduct, in which POR was crosslinked to two deoxyguanosines at their N2-positions. DNA from [3H]-POR treated EMT6 cells was digested an analyzed by HPLC. DNA-associated label was located in thymidine and in two mono-adducts and one bis-adduct identical to those described above. Label in thymidine resulted from N-demethylation of POR and reincorporation of label into new thymidylate residues. Adducts were formed more abundantly in hypoxia than in air. In addition, the mono-adduct to crosslink ratios were different, approximately 1:1 and 2:1 for hypoxic and aerobic cells, respectively. The different patterns of alkylation in air and hypoxia may be related to the greater toxicity of POR in hypoxia. When cells were treated simultaneously with POR and dicumarol, adduct levels were lower, and a new, unknown adduct was observed primarily under hypoxia; these changes may be related to the altered toxicity of POR in the presence of dicumarol. The HPLC assay detected simultaneously the full array of stable mono- and bis-adducts in DNA with good sensitivity (greater than or equal to 2 x 10(6) adducts/nucleotide) and excellent reproducibility. This assay should be generally applicable to all cells and tissues when MC or POR with high specific radioactivity can be employed. PMID:1714285

  15. Determination of the amino acid residue involved in [3H]beta-funaltrexamine covalent binding in the cloned rat mu-opioid receptor.

    PubMed

    Chen, C; Yin, J; Riel, J K; DesJarlais, R L; Raveglia, L F; Zhu, J; Liu-Chen, L Y

    1996-08-30

    We previously demonstrated that [3H]beta-funaltrexamine ([3H]beta-FNA) labeled the rat mu opioid receptor expressed in Chinese hamster ovary cells with high specificity, and [3H]beta-FNA-labeled receptors migrated as one broad band with a mass of 80 kDa. In this study, we determined the region and then the amino acid residue of the mu receptor involved in the covalent binding of [3H]beta-FNA. [3H]beta-FNA-labeled receptors were solubilized and purified to approximately 10% purity by immunoaffinity chromatography with antibodies against a C-terminal domain peptide. The site of covalent bond formation was determined to be within Ala206-Met243 by CNBr cleavage of partially purified labeled mu receptors and determinations of sizes of labeled receptor fragments. The amino acid residue of beta-FNA covalent incorporation was then determined by site-directed mutagenesis studies within this region. Mutation of Lys233 to Ala, Arg, His, and Leu completely eliminated covalent binding of [3H]beta-FNA, although these mutants bound beta-FNA with high affinity. Mutations of other amino acid residues did not affect covalent binding of [3H]beta-FNA. These results indicate that [3H]beta-FNA binds covalently to Lys233. Since [3H]beta-FNA is a rigid molecule, the information will be very useful for molecular modeling of interaction between morphinans and the mu receptor. PMID:8702924

  16. Interaction of [3H] estradiol - and [3H] monohydroxytamoxifen-estrogen receptor complexes with a monoclonal antibody.

    PubMed

    Tate, A C; DeSombre, E R; Greene, G L; Jensen, E V; Jordan, V C

    1983-01-01

    The aim of this study was to compare and contrast the interaction of estrogen [( 3H]17 beta-estradiol)- or antiestrogen [( 3H]monohydroxytamoxifen)-receptor complexes from human breast tumor cytosols with monoclonal antibodies raised to the human breast tumor estrogen receptor. Breast tumor cytosols containing estrogen receptor which sedimented as radiolabeled peaks in either the 8S, 8S and 4S, or 4S regions of sucrose density gradients, interacted with the monoclonal antibody D547 to produce a broad 9-10S peak, a broad 8S-10S peak, or a more discrete 8S peak, respectively. On high salt (0.4M KC1) sucrose density gradients the 4S ligand-receptor complex plus antibody produced a binding peak at approximately the 8S region of the gradient. These sedimentation studies with the monoclonal antibody D547, and similar studies with the monoclonal antibody D58, could detect no differences in the cytosolic estrogen receptor whether complexed with [3H]estradiol or with [3H]monohydroxytamoxifen. These observations were confirmed by Scatchard equilibrium saturation analysis and sucrose density gradient analysis of cytosols from the MCF-7 human breast cancer cell line. The antibody D547 interacted with 8S ER from these cytosols to produce a broad 8S-10S peak, but the antibody produced no change in the affinity or number of binding sites present in these cytosols. It seems, therefore, that the antigenic determinants recognized by these particular antibodies on the breast tumor cytosolic receptor are not significantly altered by the binding of either an estrogen or an antiestrogen to the receptor. PMID:6671136

  17. Measurement of local rates of brain protein synthesis by quantitative autoradiography: validation with L-(/sup 3/H)valine

    SciTech Connect

    Dwyer, B.E.; Donatoni, P.; Wasterlain, C.G.

    1982-12-01

    Following the injection of 4-day old rats with 150 mM L-(3,4-/sup 3/H)valine (10 mumol/g, IP) the incorporation of /sup 3/H into protein was linear 2 hours. Valine specific activity in the brain acid-soluble fraction was constant between 30 and 120 min after injection with a mean value of 82.3% of the injectate. Significant amounts of tritated metabolites accumulated in the brain acid-soluble fraction (41.4% of radioactivity at 120 min) but do not prove an impediment to measuring rates of protein synthesis. The rate of protein synthesis in cerebral cortex of the 4-day old rat was measured by quantitative autoradiography using (/sup 3/H)valine and /sup 3/H-sensitive film. The measured rate shows excellent agreement with that found previously using L-(1-/sup 14/C)valine. Our results suggest that (/sup 3/H)valine can be a useful precursor to measure local rates of brain protein synthesis by quantitative autoradiography.

  18. Proton transfer dynamics in the hydrogen bond. Inelastic neutron scattering, infrared and Raman spectra of Na 3H(SO 4) 2, K 3H(SO 4) 2 and Rb 3H(SO 4) 2

    NASA Astrophysics Data System (ADS)

    Fillaux, F.; Lautié, A.; Tomkinson, J.; Kearley, G. J.

    1991-06-01

    Na 3H(SO 4) 2, K 3H(SO 4) 2 and Rb 3H(SO 4) 2 crystals are composed of (SO 4HSO 4) -3 dimers linked by rather strong hydrogen bonds ( RO…O=2.43 Å for Na 3H(SO 4) 2, RO…O=2.48 Å for Rb 3H(SO 4) 2 and RO…O=2.49 Å for K 3H(SO 4) 2). Crystallographic data of the salts at room temperature indicate either asymmetric (Na 3H(SO 4) 2) or symmetric (K 3H(SO 4) 2 and Rb 3H(SO 4) 2) hydrogen bonds. Inelastic neutron scattering (INS), infrared and Raman spectra of crystal powders at 20 K are reported for these three compounds. The OH bending modes, which give large INS intensities, appear only weakly in the infrared. The two bending modes are degenerate in Na 3H(SO 4) 2 which has the shortest hydrogen bond but are well separated in K 3H(SO 4) 2 and Rb 3H(SO 4) 2. The OH stretching band profiles in INS are also quite different from those in the infrared. Strong INS bands at 57 and 44 cm -1 for K 3H(SO 4) 2 and Rb 3H(SO 4) 2, respectively, are assigned to 0→1 transitions in quasi-symmetric double-minimum potentials for the OH stretching coordinates. For K 3H(SO 4) 2 the frequency is unaffected by temperature between 2 and 100 K. Potential functions are calculated and the dynamics of the proton transfer are discussed. Infrared spectra are thus dominated by OH stretching transitions in asymmetric double-minimum potentials with low barriers, with relative intensities indicating a large electrical anharmonicity.

  19. Comparative study on pharmacokinetics and in vivo alpha1-adrenoceptor binding of [3H]tamsulosin and [3H]prazosin in rats.

    PubMed

    Ohkura, T; Yamada, S; Deguchi, Y; Kimura, R

    1999-04-01

    The plasma concentration, total radioactivity and in vivo alpha1-adrenoceptor binding in rat tissues after intravenous (i.v.) injection of [3H]tamsulosin were measured and they were compared with those obtained after the injection of [3H]prazosin. The plasma concentration of [3H]tamsulosin was consistently higher than that of [3H]prazosin, with 1.4 times greater areas under the curve (AUC(0-infinity)) of plasma concentration. As there was a significantly lower value of apparent volume of central compartment (Vd(c)) and distribution volume at steady state (Vd(ss)) for [3H]tamsulosin than [3H]prazosin with little difference in elimination rate constant (beta), the higher concentration of [3H]tamsulosin in plasma might be associated mainly with the smaller volume of distribution. The ratio of total radioactivity in tissues to the plasma unbound concentration of [3H]tamsulosin after i.v. injection of the ligand was consistently lower than that of [3H]prazosin. These observations suggest that [3H]tamsulosin is distributed in rat tissues in a more limited manner than [3H]prazosin. A significantly lower level of in vivo specific binding of [3H]tamsulosin than [3H]prazosin was observed in the spleen, heart and liver. Further, the apparent dissociation constant (Kd) and maximal number of binding sites (Bmax) for in vivo specific [3H]tamsulosin binding were considerably lower than those for [3H]prazosin binding. Therefore, these findings suggest that [3H]tamsulosin labels preferentially a subpopulation of the alpha1-adrenoceptor sites in rat tissues labeled by [3H]prazosin. In conclusion, the present study has shown that there is a significant difference in the pharmacokinetics and in vivo alpha1-adrenoceptor binding characteristics between tamsulosin and prazosin. PMID:10328564

  20. Rotational spectroscopy of 2H,3H-perfluoropentane

    NASA Astrophysics Data System (ADS)

    Duong, Chinh H.; Obenchain, Daniel A.; Cooke, S. A.; Novick, Stewart E.

    2016-06-01

    The structure of 2H,3H-perfluoropentane, CF3CHFCHFCF2CF3, has been determine by a combination of Chirp-pulsed Fourier transform microwave (CP-FTMW) spectroscopy and cavity FTMW spectroscopy. Of the four possible stereoisomers, only the enantiomeric pair (R,R)/(S,S) were observed experimentally; there was no spectroscopic evidence for the enantiomeric pair (R,S)/(S,R). The conformeric structure of the (R,R)/(S,S) stereoisomer(s) was that of partial helices with C-C-C-C dihedral angles of 12° (helical) and 1° (staggered).

  1. Thymidine and deoxyuridine accumulate in tissues of patients with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE).

    PubMed

    Valentino, Maria Lucia; Martí, Ramon; Tadesse, Saba; López, Luis Carlos; Manes, Jose L; Lyzak, Judy; Hahn, Angelika; Carelli, Valerio; Hirano, Michio

    2007-07-24

    Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disease due to ECGF1 gene mutations causing thymidine phosphorylase (TP) deficiency. Analysis of post-mortem samples of five MNGIE patients and two controls, revealed TP activity in all control tissues, but not in MNGIE samples. Converse to TP activity, thymidine and deoxyuridine were absent in control samples, but present in all tissues of MNGIE patients. Concentrations of both nucleosides in the tissues were generally higher than those observed in plasma of MNGIE patients. Our observations indicate that in the absence of TP activity, tissues accumulate nucleosides, which are excreted into plasma. PMID:17612528

  2. Late-onset MNGIE due to partial loss of thymidine phosphorylase activity.

    PubMed

    Martí, Ramon; Verschuuren, Jan J G M; Buchman, Alan; Hirano, Ikuo; Tadesse, Saba; van Kuilenburg, André B P; van Gennip, Albert H; Poorthuis, Ben J H M; Hirano, Michio

    2005-10-01

    Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is caused by mutations in the gene encoding thymidine phosphorylase (TP). All MNGIE patients have had severe loss of TP function and prominent plasma accumulations of the TP substrates thymidine (dThd) and deoxyuridine (dUrd). Here, we report for the first time to our knowledge three MNGIE patients with later onset, milder phenotype, and less severe TP dysfunction, compared with typical MNGIE patients. This report demonstrates a direct relationship between the biochemical defects and clinical phenotypes in MNGIE and supports the notion that reduction of dThd and dUrd accumulation or TP replacement could be useful therapy for MNGIE. PMID:16178026

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

    PubMed

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

    2016-05-01

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

  4. Mouse thymidine kinase: the promoter sequence and the gene and pseudogene structures in normal cells and in thymidine kinase deficient mutants.

    PubMed Central

    Seiser, C; Knöfler, M; Rudelstorfer, I; Haas, R; Wintersberger, E

    1989-01-01

    The mouse genome carries one gene and two pseudogenes for cytoplasmic thymidine kinase. The overall structure of these genes was determined with the help of cosmids and lambda phage clones and the upstream sequence containing the promoter was determined. The data allow an allocation of bands seen in the complex patterns of genomic Southern blots obtained from the DNA of wild type cells and of thymidine kinase deficient mutants to the gene as well as to the two pseudogenes. The much used LTK cell line was found to lack the entire gene but to retain the pseudogenes. Two other TK cell lines had DNA patterns indistinguishable from the wild type. Whereas the LTK line did not produce any TKmRNA, the two other mutants had normal amounts of TKmRNA but no cytoplasmic TK activity. Images PMID:2911464

  5. Kinetic studies with N2-phenylguanines and with L-thymidine indicate that herpes simplex virus type-1 thymidine kinase and thymidylate kinase share a common active site.

    PubMed

    Maga, G; Focher, F; Wright, G E; Capobianco, M; Garbesi, A; Bendiscioli, A; Spadari, S

    1994-08-15

    It is known that the Herpes simplex virus type 1 (HSV-1)-encoded thymidine kinase (TK) co-purifies with an associated thymidylate kinase (TMPK) activity and that thymidylate (TMP) inhibits the phosphorylation of thymidine by the HSV-1 TK. Here we demonstrate that: (i) TMP phosphorylation catalysed by the viral TMPK is competitively inhibited by thymidine (TdR) with a Ki equal to its Km as substrate for the viral TK; (ii) L-thymidine (L-TdR), the enantiomer of the naturally occurring D-TdR and a substrate for the HSV-1 TK [Spadari, Maga, Focher, Ciarrocchi, Manservigi, Arcamone, Capobianco, Caruso, Colonna, Iotti and Garbesi (1992) J. Med. Chem. 35, 4214-4220], is a powerful inhibitor of the HSV-1 TMPK activity with a Ki value identical with its Km as a substrate for the viral TK; (iii) both viral TK and TMPK activities are inhibited, in a competitive way and with identical Ki values, by novel, non-substrate inhibitors of HSV-1 TK, N2-phenylguanines; (iv) L-TdR is phosphorylated to L-TMP by the viral TK, but L-TMP is not phosphorylated to L-TDP by the viral TMPK activity; and (v) L-TMP inhibits competitively and with identical potencies the phosphorylation of TdR and TMP catalysed respectively by the HSV-1 TK and TMPK activities. In conclusion, our data demonstrate that both TK and TMPK activities encoded by HSV-1 share a common active site which is very tolerant in accepting modified nucleosides, but cannot readily accommodate modified nucleoside monophosphates. PMID:8068016

  6. Kinetic studies with N2-phenylguanines and with L-thymidine indicate that herpes simplex virus type-1 thymidine kinase and thymidylate kinase share a common active site.

    PubMed Central

    Maga, G; Focher, F; Wright, G E; Capobianco, M; Garbesi, A; Bendiscioli, A; Spadari, S

    1994-01-01

    It is known that the Herpes simplex virus type 1 (HSV-1)-encoded thymidine kinase (TK) co-purifies with an associated thymidylate kinase (TMPK) activity and that thymidylate (TMP) inhibits the phosphorylation of thymidine by the HSV-1 TK. Here we demonstrate that: (i) TMP phosphorylation catalysed by the viral TMPK is competitively inhibited by thymidine (TdR) with a Ki equal to its Km as substrate for the viral TK; (ii) L-thymidine (L-TdR), the enantiomer of the naturally occurring D-TdR and a substrate for the HSV-1 TK [Spadari, Maga, Focher, Ciarrocchi, Manservigi, Arcamone, Capobianco, Caruso, Colonna, Iotti and Garbesi (1992) J. Med. Chem. 35, 4214-4220], is a powerful inhibitor of the HSV-1 TMPK activity with a Ki value identical with its Km as a substrate for the viral TK; (iii) both viral TK and TMPK activities are inhibited, in a competitive way and with identical Ki values, by novel, non-substrate inhibitors of HSV-1 TK, N2-phenylguanines; (iv) L-TdR is phosphorylated to L-TMP by the viral TK, but L-TMP is not phosphorylated to L-TDP by the viral TMPK activity; and (v) L-TMP inhibits competitively and with identical potencies the phosphorylation of TdR and TMP catalysed respectively by the HSV-1 TK and TMPK activities. In conclusion, our data demonstrate that both TK and TMPK activities encoded by HSV-1 share a common active site which is very tolerant in accepting modified nucleosides, but cannot readily accommodate modified nucleoside monophosphates. PMID:8068016

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

    PubMed

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

    2008-11-01

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

  8. A Search for Interstellar Oxiranecarbonitrile (C3H3NO)

    NASA Technical Reports Server (NTRS)

    Dicken, J. E.; Irvine, W. M.; Ohishi, M.; Arrhenius, G.; Bauder, A.; Mueller, F.; Eschenmoser, A.

    1996-01-01

    We report a search in cold, quiescent and in 'hot core' type interstellar molecular clouds for the small cyclic molecule oxiranecarbonitrile (C3H3NO), which has been suggested as a precursor of important prebiotic molecules. We have determined upper limits to the column density and fractional abundance for the observed sources and find that, typically, the fractional abundance by number relative to molecular hydrogen Of C3H3NO is less than a few times 10(exp -10). This limit is one to two orders of magnitude less than the measured abundance of such similarly complex species as CH3CH2CN and HCOOCH3 in well-studied hot cores. A number of astrochemical discoveries were made, including the first detection of the species CH3CH2CN in the massive star-forming clouds G34.3+0.2 and W51M and the first astronomical detections of some eight rotational transitions of CH3CH2CN, CH3CCH, and HCOOCH3. In addition, we found 8 emission lines in the 89 GHz region and 18 in the 102 GHz region which we were unable to assign.

  9. Effective theory of 3H and 3He

    NASA Astrophysics Data System (ADS)

    König, Sebastian; Grießhammer, Harald W.; Hammer, H.-W.; van Kolck, U.

    2016-06-01

    We present a new perturbative expansion for pionless effective field theory with Coulomb interactions in which at leading order (LO) the spin-singlet nucleon–nucleon channels are taken in the unitarity limit. Presenting results up to next-to-leading order for the Phillips line and the neutron–deuteron doublet-channel phase shift, we find that a perturbative expansion in the inverse {}1{S}0 scattering lengths converges rapidly. Using a new systematic treatment of the proton–proton sector that isolates the divergence due to one-photon exchange, we renormalize the corresponding contribution to the {}3{{H}} –{}3{He} binding energy splitting and demonstrate that the Coulomb force in pionless EFT is a completely perturbative effect in the trinucleon bound-state regime. In our new expansion, the LO is exactly isospin-symmetric. At next-to-leading order, we include isospin breaking via the Coulomb force and two-body scattering lengths, and find for the energy splitting {({E}B{(}3{He})-{E}B{(}3{{H}}))}{NLO}\\quad =(-0.86+/- 0.17)\\quad {MeV}.

  10. A search for interstellar oxiranecarbonitrile (C3H3NO).

    PubMed

    Dickens, J E; Irvine, W M; Ohishi, M; Arrhenius, G; Pitsch, S; Bauder, A; Muller, F; Eschenmoser, A

    1996-04-01

    We report a search in cold, quiescent and in 'hot core' type interstellar molecular clouds for the small cyclic molecule oxiranecarbonitrile (C3H3NO), which has been suggested as a precursor of important prebiotic molecules. We have determined upper limits to the column density and fractional abundance for the observed sources and find that, typically, the fractional abundance by number relative to molecular hydrogen of C3H3NO is less than a few times 10(-10). This limit is one to two orders of magnitude less than the measured abundance of such similarly complex species as CH3CH2CN and HCOOCH3 in well-studied hot cores. A number of astrochemical discoveries were made, including the first detection of the species CH3CH2CN in the massive star-forming clouds G34.3+0.2 and W51M and the first astronomical detections of some eight rotational transitions of CH3CH2CN, CH3CCH, and HCOOCH3. In addition, we found 8 emission lines in the 89 GHz region and 18 in the 102 GHz region which we were unable to assign. PMID:11536752

  11. The 3H-3He Charge Radii Difference

    NASA Astrophysics Data System (ADS)

    Myers, L. S.; Arrington, J. R.; Higinbotham, D. W.

    2016-03-01

    The upcoming E12-14-009 [1] experiment at Jefferson Lab will determine the ratio of the electric form factors for the A=3 mirror nuclei 3He and 3H. The measurement will use a 1.1 GeV electron beam, a special collimator plate to allow for simultaneous optics measurements, and the low-activity tritium target being prepared for Jefferson Lab. By observing the dependence of the form factor ratio as a function of Q2 over 0.05-0.09 GeV2, the dependence of the radii extraction on the shape of the form factors is minimized. As a result, we anticipate the uncertainty of the extracted charge radii difference to be 0.03 fm, a reduction of 70% from the current measurement. Using precise measurements of the 3He charge radius from isotopic shift or μHe measurements [2-4], we can deduce the absolute 3H charge radius. The results will provide a direct comparison to recent calculations of the charge radii.

  12. Evidence for triangular D3h symmetry in 12C.

    PubMed

    Marín-Lámbarri, D J; Bijker, R; Freer, M; Gai, M; Kokalova, Tz; Parker, D J; Wheldon, C

    2014-07-01

    We report a measurement of a new high spin Jπ=5- state at 22.4(2) MeV in 12C which fits very well to the predicted (ground state) rotational band of an oblate equilateral triangular spinning top with a D3h symmetry characterized by the sequence 0+, 2+, 3-, 4±, 5- with almost degenerate 4+ and 4- (parity doublet) states. Such a D3h symmetry was observed in triatomic molecules, and it is observed here for the first time in nuclear physics. We discuss a classification of other rotation-vibration bands in 12C such as the (0+) Hoyle band and the (1-) bending mode band and suggest measurements in search of the predicted ("missing") states that may shed new light on clustering in 12C and light nuclei. In particular, the observation (or nonobservation) of the predicted ("missing") states in the Hoyle band will allow us to conclude the geometrical arrangement of the three alpha particles composing the Hoyle state at 7.654 MeV in 12C. PMID:25032922

  13. Beryllium-induced immune response in C3H mice

    SciTech Connect

    Benson, J.M.; Bice, D.E.; Nikula, K.J.

    1995-12-01

    Studies conducted at ITRI over the past several years have investigated whether Beagle dogs, monkeys, and mice are suitable models for human chronic beryllium-induced lung disease (CBD). Recent studies have focused on the histopathological and immunopathological changes occurring in A/J and C3H/HeJ mice acutely exposed by inhalation to Be metal. Lung lesions in both strains of mice included focal lymphocyte aggregates comprised primarily of B lymphocytes and lesser amounts of T-helper lymphocytes and microgranulomas consisting chiefly of macrophages and T-helper lymphocytes. The distribution of proliferating cells within the microgranulomas was similar to the distribution of T-helper cells. These results strongly suggested that A/J and C3H/HeJ mice responded to inhaled Be metal in a fashion similar to humans in terms of pulmonary lesions and the apparent in situ proliferation of T-helper cells. Results of these studies confirm lymphocyte involvement in the pulmonary response to inhaled Be metal.

  14. Dating degassed groundwater with 3H/3He

    NASA Astrophysics Data System (ADS)

    Visser, Ate; Broers, Hans Peter; Bierkens, Marc F. P.

    2007-10-01

    The production of gases in groundwater under contaminated locations by geochemical and biological processes is not uncommon. Degassing of these gases from groundwater and repartitioning of noble gases between water and gas phase distorts groundwater dating by 3H/3He. We observed noble gas concentrations below atmospheric equilibrium in 20 out of 34 groundwater samples from agriculturally polluted sandy areas in the Netherlands. From the absence of nitrate in degassed samples, we conclude that denitrification causes degassing. The 22Ne/20Ne ratios show that degassing had attained solubility equilibrium and had not caused isotopic fractionation by diffusion. To correct for the loss of tritiogenic 3He due to degassing, we present a single-step equilibrium degassing model. We use the total dissolved gas pressure at the monitoring screen to estimate the depth and timing of degassing, which is essential to estimate travel times from degassed samples. By propagating the uncertainties in the underlying measurements and assumptions through the travel time calculations, we found a travel time uncertainty of 3 years (a). We therefore conclude that 3H/3He dating can produce valuable information on groundwater flow even at sites with strong degassing.

  15. Bindings of /sup 3/H-prazosin and /sup 3/H-yohimbine to alpha adrenoceptors in the guinea-pig stomach

    SciTech Connect

    Taniguchi, T.; Nishikawa, H.

    1988-01-01

    Alpha adrenoceptor subtypes have been investigated by radioligand binding study in guinea-pig stomach using /sup 3/H-prazosin and /sup 3/H-yohimbine. The specific /sup 3/H-prazosin binding to guinea-pig stomach was saturable and of high affinity with a Bmax of 33 fmol/mg protein. Specific /sup 3/H-yohimbine binding to the tissue was also saturable and of high affinity with a Bmax of 150 fmol/mg protein. Adrenergic drugs competed for /sup 3/H-prazosin binding in order of prazosin > phentolamine > methoxamine > norepinephrine > clonidine > epinephrine > yohimbine. These drugs competed for /sup 3/H-yohimbine binding in order of yohimbine > phentolamine > clonidine > epinephrine > norepinephrine > prazosin > methoxamine. They also examined whether dopamine receptors exist in guinea-pig stomach, using radioligand binding study. Specific binding of /sup 3/H-spiperone, /sup 3/H-apomorphine, /sup 3/H-dopamine and /sup 3/H-domperidone was not detectable in the stomach. Dopaminergic drugs such as dopamine, haloperidol, domperidone and sulpiride competed for /sup 3/H-prazosin binding in order of haloperidol > domperidone > dopamine > sulpiride. Metoclopramide, sulpiride and dopamine competed for /sup 3/H-yohimbine binding in order of metoclopramide > sulpiride > dopamine.

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

    PubMed

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

    2006-01-01

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

  17. C3H2 observations as a diagnostic probe for molecular clouds

    NASA Technical Reports Server (NTRS)

    Avery, L. W.

    1986-01-01

    Recently the three-membered ring molecule, cyclopropenylidene, C3H2, has been identified in the laboratory and detected in molecular clouds by Thaddeus, Vrtilek and Gottlieb (1985). This molecule is wide-spread throughout the Galaxy and has been detected in 25 separate sources including cold dust clouds, circumstellar envelopes, HII regions, and the spiral arms observed against the Cas supernova remnant. In order to evaluate the potential of C3H2 as a diagnostic probe for molecular clouds, and to attempt to identify the most useful transitions, statistical equilibrium calculations were carried out for the lowest 24 levels of the ortho species and the lowest 10 levels of the para species. Many of the sources observed by Matthews and Irvine (1985) show evidence of being optically thick in the 1(10)-1(01) line. Consequently, the effects of radiative trapping should be incorporated into the equilibrium calculations. This was done using the Large Velocity Gradient approximation for a spherical cloud of uniform density. Some results of the calculations for T(K)=10K are given. Figures are presented which show contours of the logarithm of the ratio of peak line brightness temperatures for ortho-para pairs of lines at similar frequencies. It appears that the widespread nature of C3H2, the relatively large strength of its spectral lines, and their sensitivity to density and molecular abundance combine to make this a useful molecule for probing physical conditions in molecular clouds. The 1(10)-1(01) and 2(20)-2(11) K-band lines may be especially useful in this regard because of the ease with which they are observed and their unusual density-dependent emission/absorption properties.

  18. Pressure Effects on Product Channels of the Allyl Radical Reactions; C3H5+C3H5 and C3H5+CH3

    NASA Astrophysics Data System (ADS)

    Halpern, J. B.; N'Doumi, M.; Fahr, A.

    2011-12-01

    Relatively large hydrocarbon molecules (C4, C6 and larger) have been detected in several planetary environments. The mechanism for the formation of such large molecular species and detailed mechanism for their potential destruction are not well understood and are of considerable current interest. Previously we have studied the kinetics and product channels of small unsaturated hydrocarbon radical (C2 and C3s) reactions relevant to planetary atmospheric modeling. Reactions of C2 radicals (such as vinyl, H2CCH and ethynyl C2H) and C3 radicals (such as propargyl, HCCCH2) can affect the abundances of a large number of stable observable C3, C4, C5, C6 and larger molecules, including linear, aromatic and even poly aromatic molecules. Pressure-dependent product yields have been determined experimentally for the self- and cross-radical reactions performed at 298 K and at pressures between ~4 Torr (0.5 kPa) and 760 Torr (101 kPa). Final reaction products were quantitatively determined using a gas chromatograph with mass spectrometry/flame ionization detection (GC/MS/FID). In some cases complementary computational studies extended the pressure and temperature range of the experiments and provided valuable information on the complex reaction mechanisms. Theses studies provide a systematic framework so that important energetic and structural parameters for radical-radical reactions can be assessed. Here we report recent results for the allyl radical reactions H2CCCH3+ H2CCCH3 and H2CCCH3+CH3. For the allyl radical self-reaction, at high pressures the "head -to-head", combination channel forming 1,5-hexadiene is dominant with a combination/disproportionation = 1,5-hexadiene/propyne ratio of about 24 at 500 Torr (67 kPa, T=298K). At low pressures the ratio is substantially reduced to about 1.2 (at 0.3 kPa) and other major products are observed including allene, propene, 1-butene and propyne.

  19. One dimensional 1H, 2H and 3H

    NASA Astrophysics Data System (ADS)

    Vidal, A. J.; Astrakharchik, G. E.; Vranješ Markić, L.; Boronat, J.

    2016-05-01

    The ground-state properties of one-dimensional electron-spin-polarized hydrogen 1H, deuterium 2H, and tritium 3H are obtained by means of quantum Monte Carlo methods. The equations of state of the three isotopes are calculated for a wide range of linear densities. The pair correlation function and the static structure factor are obtained and interpreted within the framework of the Luttinger liquid theory. We report the density dependence of the Luttinger parameter and use it to identify different physical regimes: Bogoliubov Bose gas, super-Tonks–Girardeau gas, and quasi-crystal regimes for bosons; repulsive, attractive Fermi gas, and quasi-crystal regimes for fermions. We find that the tritium isotope is the one with the richest behavior. Our results show unambiguously the relevant role of the isotope mass in the properties of this quantum system.

  20. Encapsulated scintillators monitor /sup 3/H-solute concentrations

    SciTech Connect

    Kirk, G.; Gruner, S.

    1982-02-01

    The short range of the /sup 3/H beta allows shielding of microbeds of scintillator by a several um thick coating of a water based gel. Gels may be used which are permeable to a wide variety of tritiated molecules. Thus, the light output of a mixture of the coated beads and a solution of the tritiated compound is proportional to the solution concentration of the tritiated substance. The mixture may also contain particles to which the gel is impermeable, such as cells, vesicles, large proteins, etc., but which can alter the concentration of the tritiated compound by uptake or release. In this case, the light output monitors the fractional uptake of the tritiated material. The design criteria for encapsulating the scintillators and dynamically monitoring the scintillation output are discussed. A simple method for encapsulating plastic scintillator microbeads, suitable for monitoring slow concentration changes, is described and tested.

  1. Study of the $\\tau^- to 3h^- 2h^+ \

    SciTech Connect

    Aubert, Bernard; Barate, R.; Boutigny, D.; Couderc, F.; Karyotakis, Y.; Lees, J.P.; Poireau, V.; Tisserand, V.; Zghiche, A.; Grauges, E.; Palano, A.; Pappagallo, M.; Pompili, A.; Chen, J.C.; Qi, N.D.; Rong, G.; Wang, P.; Zhu, Y.S.; Eigen, G.; Ofte, I.; Stugu, B. /more authors..

    2005-05-04

    The branching fraction of the {tau}{sup -} {yields} 3h{sup -} 2h{sup +} {nu}{sub {tau}} decay (h = {pi}, K) is measured with the BABAR detector to be (8.56 {+-} 0.05 {+-} 0.42) x 10{sup -4}, where the first error is statistical and the second systematic. The observed structure of this decay is significantly different from the phase space prediction, with the {rho} resonance playing a strong role. The decay {tau}{sup -} {yields} f{sub 1}(1285){pi}{sup -}{nu}{sub {tau}}, with the f{sub 1}(1285) meson decaying to four charged pions, is observed and the branching fraction is measured to be (3.9 {+-} 0.7 {+-} 0.5) x 10{sup -4}.

  2. A thymidine-terminated molecular beacon for selective Hg 2+ or sequence-specific DNA assay

    NASA Astrophysics Data System (ADS)

    Hou, Peng; Long, Yunfei; Zhao, Jin; Wang, Jianxiu; Zhou, Feimeng

    2012-02-01

    A new molecular beacon (MB) in which fluorescein (FAM) attached to its 3' end acts as a fluorophore and a bridged thymidine-Hg-thymidine (T-Hg-T) complex acts as a quencher is designed. The fluorescence resonance energy transfer (FRET) between the fluorophore and the quencher results in annihilation of the FAM fluorescence. Experimental conditions that govern the fluorescence quenching, such as number of thymidine bases, pH value, and salt concentration, have been optimized. The MB was found to be highly selective for Hg 2+ among a number of metal ions investigated. In the presence of single-stranded (ss-) target oligonucleotides (ODNs) with bases complementary to those in the loop of MB, the FAM fluorescence can be largely restored due to DNA duplex formation. The present method for DNA assay is also sequence-specific and can determine target ODN concentration at a nanomolar level. The substitution of the quencher group in a conventional MB molecule with simple thymidine bases affords an inexpensive ODN that retains the unique property of the MB molecule.

  3. MOLECULAR DISSECTION OF MUTATIONS AT THE HETEROZYGOUS THYMIDINE KINASE LOCUS IN MOUSE LYMPHOMA CELLS

    EPA Science Inventory

    The mouse lymphoma L5178Y TK+/- 3.7.2C cell line allows quantitation of induced TK+/- -> TK-/- mutations at the heterozygous thymidine kinase (tk) locus. K-/- mutant colonies show a bimodal size distribution, reflecting a difference in the growth rates of the two size classes tha...

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

    SciTech Connect

    Franzolin, Elisa; Rampazzo, Chiara; Perez-Perez, Maria-Jesus; Hernandez, Ana-Isabel; Balzarini, Jan; Bianchi, Vera . E-mail: vbianchi@mail.bio.unipd.it

    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{sup -} 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.

  5. Expression of complete chicken thymidine kinase gene inserted in a retrovirus vector.

    PubMed Central

    Bandyopadhyay, P K; Temin, H M

    1984-01-01

    The chicken thymidine kinase (tk) gene was inserted into spleen necrosis virus. Thymidine kinase activity was expressed even when the promoter and terminator sequences for tk RNA synthesis were retained. When the promoter was present in the same orientation as the promoter in the long terminal repeat of the virus, deletions occurred both in the virus and in the tk gene, and the thymidine kinase-transforming activity of the recovered virus was low. Splicing of apparent intervening sequences in the tk gene was also observed. When the orientation of the tk promoter was opposite to the promoter in the long terminal repeat, virus synthesis was diminished, whereas thymidine kinase activity was expressed at an elevated level compared with virus in which the promoter was in the same orientation. However, when the apparent tk promoter was deleted from virus with the tk gene in the opposite orientation, a high level of virus synthesis was observed, probably as a result of absence of interference of RNA synthesis from converging promoters. The intervening sequences in the virus in which the promoters were in opposite orientation were not spliced. Images PMID:6325895

  6. LOCALIZATION OF THE MOUSE THYMIDINE KINASE GENE TO THE DISTAL PORTION OF CHROMOSOME 11

    EPA Science Inventory

    We report the regional mapping of the thymidine kinase (tk-1) gene in the mouse using two complementary analyses: 1) investigation of chromosome aberrations associated with tx-1 gene inactivation in the L5178Y TX+/-3.7.2c cell line and (2) in situ molecular hybridization of a clo...

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

    SciTech Connect

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

    1988-01-05

    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.

  8. Ascorbic acid and striatal transport of (/sup 3/H)1-methyl-4-phenylpyridine (MPP/sup +/) and (/sup 3/H)dopamine

    SciTech Connect

    Debler, E.A.; Hashim, A.; Lajtha, A.; Sershen, H.

    1988-01-01

    The inhibition of uptake of (/sup 3/H)dopamine and (/sup 3/H)1-methyl-4-phenylpyridine (MPP/sup +/) was examined in mouse striatal synaptosomal preparations. Kinetic analysis indicated that ascorbic acid is a noncompetitive inhibitor of (/sup 3/H)MPP/sup +/ uptake. No inhibition of (/sup 3/H)dopamine uptake is observed. The dopamine uptake blockers, GBR-12909, cocaine, and mazindol strongly inhibit (IC/sub 50/ < 1 ..mu..M) both (/sup 3/H)dopamine and (/sup 3/H)MPP/sup +/ transport. Nicotine, its metabolites, and other tobacco alkaloids are weak inhibitors except 4-phenylpyridine and lobeline, which are moderate inhibitors of both (/sup 3/H)dopamine and (/sup 3/H)MPP/sup +/ uptake. These similarities in potencies are in agreement with the suggestion that (/sup 3/H)MPP/sup +/ and (/sup 3/H) are transported by the same carrier. The differences observed in the alteration of dopaminergic transport and mazindol binding by ascorbic acid suggest that ascorbic acid's effects on (/sup 3/H)MPP/sup +/ transport are related to translocation and/or dissociation processes occurring subsequent to the initial binding event.

  9. ( sup 3 H)phenamil binding protein of the renal epithelium Na+ channel. Purification, affinity labeling, and functional reconstitution

    SciTech Connect

    Barbry, P.; Chassande, O.; Marsault, R.; Lazdunski, M.; Frelin, C. )

    1990-01-30

    This paper describes a large-scale purification procedure of the amiloride binding component of the epithelium Na+ channel. (3H)Phenamil was used as a labeled ligand to follow the purification. The first two steps are identical with those previously described. A third step was a hydroxyapatite column. The purified material consisted of a homodimer of two 88-kDa proteins that migrated anomalously in SDS-PAGE to give an apparent Mr of 105,000. Deglycosylation by treatment with neuraminidase and endoglycosidase F or with neuraminidase and glycopeptidase F indicated that less than 5% of the mass of the native receptor was carbohydrate. Sedimentation analysis of the purified Na+ channel in H2O and D2O sucrose gradients and gel filtration experiments led to an estimated molecular weight of the (3H)phenamil receptor protein-detergent-phospholipid complex of 288,000 and of the native (3H)phenamil receptor protein of 158,000. (3H)Br-benzamil is another labeled derivative of amiloride that recognized binding sites that had the same pharmacological properties as (3H)phenamil binding sites and that copurified with them. Upon irradiation of kidney membranes, (3H)Br-benzamil incorporated specifically into a 185-kDa polypeptide chain under nonreducing electrophoretic conditions and a 105-kDa protein under reducing conditions. The same labeling pattern was observed at the different steps of the purification. Reconstitution of the purified phenamil receptor into large unilamellar vesicles was carried out. A low but significant phenamil- and amiloride-sensitive electrogenic Na+ transport was observed.

  10. 3,4-Methylenedioxyamphetamine (MDA) analogues exhibit differential effects on synaptosomal release of 3H-dopamine and 3H-5-hydroxytryptamine

    SciTech Connect

    McKenna, D.J.; Guan, X.M.; Shulgin, A.T. )

    1991-03-01

    The effect of various analogues of the neurotoxic amphetamine derivative, MDA (3,4-methylenedioxyamphetamine) on carrier-mediated, calcium-independent release of 3H-5-HT and 3H-DA from rat brain synaptosomes was investigated. Both enantiomers of the neurotoxic analogues MDA and MDMA (3,4-methylenedioxymethamphetamine) induce synaptosomal release of 3H-5-HT and 3H-DA in vitro. The release of 3H-5-HT induced by MDMA is partially blocked by 10(-6) M fluoxetine. The (+) enantiomers of both MDA and MDMA are more potent than the (-) enantiomers as releasers of both 3H-5-HT and 3H-DA. Eleven analogues, differing from MDA with respect to the nature and number of ring and/or side chain substituents, also show some activity in the release experiments, and are more potent as releasers of 3H-5-HT than of 3H-DA. The amphetamine derivatives {plus minus}fenfluramine, {plus minus}norfenfluramine, {plus minus}MDE, {plus minus}PCA, and d-methamphetamine are all potent releasers of 3H-5-HT and show varying degrees of activity as 3H-DA releasers. The hallucinogen DOM does not cause significant release of either 3H-monoamine. Possible long-term serotonergic neurotoxicity was assessed by quantifying the density of 5-HT uptake sites in rats treated with multiple doses of selected analogues using 3H-paroxetine to label 5-HT uptake sites. In the neurotoxicity study of the compounds investigated, only (+)MDA caused a significant loss of 5-HT uptake sites in comparison to saline-treated controls. These results are discussed in terms of the apparent structure-activity properties affecting 3H-monoamine release and their possible relevance to neurotoxicity in this series of MDA congeners.

  11. 3,4-Methylenedioxyamphetamine (MDA) analogues exhibit differential effects on synaptosomal release of 3H-dopamine and 3H-5-hydroxytryptamine.

    PubMed

    McKenna, D J; Guan, X M; Shulgin, A T

    1991-03-01

    The effect of various analogues of the neurotoxic amphetamine derivative, MDA (3,4-methylenedioxyamphetamine) on carrier-mediated, calcium-independent release of 3H-5-HT and 3H-DA from rat brain synaptosomes was investigated. Both enantiomers of the neurotoxic analogues MDA and MDMA (3,4-methylenedioxymethamphetamine) induce synaptosomal release of 3H-5-HT and 3H-DA in vitro. The release of 3H-5-HT induced by MDMA is partially blocked by 10(-6) M fluoxetine. The (+) enantiomers of both MDA and MDMA are more potent than the (-) enantiomers as releasers of both 3H-5-HT and 3H-DA. Eleven analogues, differing from MDA with respect to the nature and number of ring and/or side chain substituents, also show some activity in the release experiments, and are more potent as releasers of 3H-5-HT than of 3H-DA. The amphetamine derivatives (+/-)fenfluramine, (+/-)norfenfluramine, (+/-)MDE, (+/-)PCA, and d-methamphetamine are all potent releasers of 3H-5-HT and show varying degrees of activity as 3H-DA releasers. The hallucinogen DOM does not cause significant release of either 3H-monoamine. Possible long-term serotonergic neurotoxicity was assessed by quantifying the density of 5-HT uptake sites in rats treated with multiple doses of selected analogues using 3H-paroxetine to label 5-HT uptake sites. In the neurotoxicity study of the compounds investigated, only (+)MDA caused a significant loss of 5-HT uptake sites in comparison to saline-treated controls. These results are discussed in terms of the apparent structure-activity properties affecting 3H-monoamine release and their possible relevance to neurotoxicity in this series of MDA congeners. PMID:1829838

  12. Selective labeling of serotonin uptake sites in rat brain by (/sup 3/H)citalopram contrasted to labeling of multiple sites by (/sup 3/H)imipramine

    SciTech Connect

    D'Amato, R.J.; Largent, B.L.; Snowman, A.M.; Snyder, S.H.

    1987-07-01

    Citalopram is a potent and selective inhibitor of neuronal serotonin uptake. In rat brain membranes (/sup 3/H)citalopram demonstrates saturable and reversible binding with a KD of 0.8 nM and a maximal number of binding sites (Bmax) of 570 fmol/mg of protein. The drug specificity for (/sup 3/H)citalopram binding and synaptosomal serotonin uptake are closely correlated. Inhibition of (/sup 3/H)citalopram binding by both serotonin and imipramine is consistent with a competitive interaction in both equilibrium and kinetic analyses. The autoradiographic pattern of (/sup 3/H)citalopram binding sites closely resembles the distribution of serotonin. By contrast, detailed equilibrium-saturation analysis of (/sup 3/H)imipramine binding reveals two binding components, i.e., high affinity (KD = 9 nM, Bmax = 420 fmol/mg of protein) and low affinity (KD = 553 nM, Bmax = 8560 fmol/mg of protein) sites. Specific (/sup 3/H)imipramine binding, defined as the binding inhibited by 100 microM desipramine, is displaced only partially by serotonin. Various studies reveal that the serotonin-sensitive portion of binding corresponds to the high affinity sites of (/sup 3/H)imipramine binding whereas the serotonin-insensitive binding corresponds to the low affinity sites. Lesioning of serotonin neurons with p-chloroamphetamine causes a large decrease in (/sup 3/H)citalopram and serotonin-sensitive (/sup 3/H)imipramine binding with only a small effect on serotonin-insensitive (/sup 3/H)imipramine binding. The dissociation rate of (/sup 3/H)imipramine or (/sup 3/H)citalopram is not altered by citalopram, imipramine or serotonin up to concentrations of 10 microM. The regional distribution of serotonin sensitive (/sup 3/H)imipramine high affinity binding sites closely resembles that of (/sup 3/H)citalopram binding.

  13. Preservation of "peripheral" benzodiazepine receptors: differential effects of freezing on [3H]Ro 5-4864 and [3H]PK 11195 binding.

    PubMed

    Basile, A S; Skolnick, P

    1987-04-01

    The equilibrium binding constants of [3H]Ro 5-4864 (a "peripheral" benzodiazepine receptor ligand) to renal membranes preserved by various freezing techniques were investigated. The Bmax for [3H]Ro 5-4864 binding to membranes from kidneys preserved as unwashed homogenates stored at -80, -20, or 5 degrees C, whole kidneys stores at -20 or 5 degrees C or as washed homogenate stored at -20 degrees C was significantly decreased (approximately 35%). Only when kidneys were frozen intact (using a dry-ice/acetone slurry) and stored at -80 degrees C was the density of [3H]Ro 5-4864 binding unchanged. However, the Bmax of [3H]PK 11195 (a putative "peripheral" benzodiazepine receptor antagonist) binding to renal membranes was unchanged following storage techniques that reduced the density of [3H]Ro 5-4864 binding 38%. No change was observed in the Kd values for [3H]Ro 5-4864 and [3H]PK 11195 binding to renal membranes preserved under any condition tested. These results demonstrate a method for the preservation of [3H]Ro 5-4864 binding to renal membranes, and suggests that [3H]Ro 5-4864 and [3H]PK 11195 bind to unique sites on or near the "peripheral" benzodiazepine receptor. [corrected] PMID:3035290

  14. (/sup 3/H)Ouabain binding and Na+, K+-ATPase in resealed human red cell ghosts

    SciTech Connect

    Shoemaker, D.G.; Lauf, P.K.

    1983-03-01

    The interaction of the cardiac glycoside (/sup 3/H)ouabain with the Na+, K+ pump of resealed human erythrocyte ghosts was investigated. Binding of (/sup 3/H)ouabain to high intracellular Na+ ghosts was studied in high extracellular Na+ media, a condition determined to produce maximal ouabain binding rates. Simultaneous examination of both the number of ouabain molecules bound per ghost and the corresponding inhibition of the Na+, K+-ATPase revealed that one molecule of (/sup 3/H)ouabain inhibited one Na+, K+-ATPase complex. Intracellular magnesium or magnesium plus inorganic phosphate produced the lowest ouabain binding rate. Support of ouabain binding by adenosine diphosphate (ADP) was negligible, provided synthesis of adenosine triphosphate (ATP) through the residual adenylate kinase activity was prevented by the adenylate kinase inhibitor Ap5A. Uridine 5'-triphosphate (UTP) alone did not support ouabain binding after inhibition of the endogenous nucleoside diphosphokinase by trypan blue and depletion of residual ATP by the incorporation of hexokinase and glucose. ATP acting solely at the high-affinity binding site of the Na+, K+ pump (Km approximately 1 microM) promoted maximal (/sup 3/H)ouabain binding rates. Failure of 5'-adenylyl-beta-gamma-imidophosphate (AMP-PNP) to stimulate significantly the rate of ouabain binding suggests that phosphorylation of the pump was required to expose the ouabain receptor.

  15. The 3H(d,γ)5He Reaction for Ec.m. ≤ 300 keV

    NASA Astrophysics Data System (ADS)

    Parker, C. E.; Brune, C. R.; Massey, T. N.; O'Donnell, J. E.; Richard, A. L.; Sayre, D. B.

    2016-03-01

    The 3H(d, γ)5He reaction has been measured using a 500-keV pulsed deuteron beam incident on a stopping titanium tritide target at Ohio University's Edwards Accelerator Laboratory. The time-of-flight (TOF) technique has been used to distinguish the γ-rays from neutrons detected in the bismuth germinate (BGO) γ-ray detector. A stilbene scintillator and an NE-213 scintillator have been used to detect the neutrons from the 3H(d, n)4He reaction using both the pulse-shape discrimination and TOF techniques. A newly-designed target holder with a silicon surface barrier detector to simultaneously measure α-particles to normalize the neutron count was incorporated for subsequent measurements. The γ-rays have been measured at laboratory angles of 0°, 45°, 90°, and 135°. Information about the γ-ray energy distribution for the unbound ground state and first excited state of 5He can be obtained experimentally by comparing the BGO data to Monte Carlo simulations. The 3H(d, γ)/3H(d, n) branching ratio has also been determined.

  16. Alpha-adrenoceptors in dog mesenteric vessels--subcellular distribution and number of ( sup 3 H)prazosin and ( sup 3 H)rauwolscine binding sites

    SciTech Connect

    Shi, A.G.; Ahmad, S.; Kwan, C.Y.; Daniel, E.E. )

    1990-04-01

    Binding of the alpha-adrenergic antagonists ({sup 3}H)prazosin and ({sup 3}H)rauwolscine to well-characterized subcellular membrane fractions isolated from dog mesenteric arteries and veins was studied. Binding of both ligands was saturable with Kd values of 0.5 +/- 0.1 nM for ({sup 3}H)prazosin and 5.85 +/- 0.85 nM for ({sup 3}H)rauwolscine in arteries, and 0.87 +/- 0.4 nM for ({sup 3}H)prazosin and 6.6 +/- 1.5 nM for ({sup 3}H)rauwolscine in veins. In veins, the maximum number of binding sites for ({sup 3}H)rauwolscine was higher than that for ({sup 3}H)prazosin, whereas in arteries the maximum number of binding sites for each ligand was similar. In microsomes from dog aorta, the maximum number of bindings sites for ({sup 3}H)prazosin was higher than that for ({sup 3}H)rauwolscine. Neuronal membrane contamination in these studies was minimized by dissection procedures and evaluated by the comparison of ({sup 3}H)saxitoxin binding in various preparations. Only mesenteric veins responded functionally to agonists acting on alpha 2 adrenoceptors. This study thus identified two distinct populations of ({sup 3}H)prazosin and ({sup 3}H)rauwolscine binding sites in the plasma membranes of dog mesenteric vessels and suggests that a much higher density of alpha 2-compared to alpha 1-adrenoceptor binding sites is required for a contractile response.

  17. In vivo labeling of cocaine receptors with sup 3 H-(-) cocaine, sup 3 H-WIN 35,065-2 and sup 3 H-WIN 35,428

    SciTech Connect

    Scheffel, U.; Boja, J.W.; Stathis, M.; Kuhar, M.J. )

    1990-02-26

    {sup 11}C-(-)cocaine (-COC) has recently been employed to image -COC binding sites in vivo using PET. Two analogs of -COC, WIN 35,065-2 (WIN-2) and WIN 35,428 (CFT), have been shown in vitro to exhibit higher affinity for the -COC receptor than -COC. The present study evaluates {sup 3}H-WIN-2 and {sup 3}H-CFT as in vivo receptor labels in mice with a view towards the use of these compounds as PET ligands for -COC receptors in the living human brain. {sup 3}H-labeled -COC, WIN-2 and CFT were injected i.v. into mice and their specific binding in the CNS determined. Peak striatal/cerebellar (S/C) ratios were reached at 5 minutes post injection with -COC (1.56), at 45 minutes with {sup 3}H-WIN-2 (3.30) and 60 minutes with {sup 3}H-CFT (4.0). The specificity of in vivo binding of {sup 3}H-WIN-2 and {sup 3}H-CFT was tested by pre-injection of various drugs. Binding of {sup 3}H-WIN-2 and {sup 3}H-CFT was dose-dependently blocked by cold WIN-2 and CFT, and by dopamine uptake site inhibitors (mazindol, GBR 12,909, nomifensine), but not by (+)COC, paroxetine and desipramine. The data indicate that {sup 3}H-WIN-2 and {sup 3}H-CFT exhibit improved in vivo binding (higher S/C ratios, longer retention time at the -COC receptor/dopamine transporter) compared to -COC and support their testing in PET studies.

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

    SciTech Connect

    Zhang, Jufeng; Wang, Zhanli; Wei, Fang; Qiu, Wei; Zhang, Liangren; Huang, Qian . E-mail: qhuang@sjtu.edu.cn

    2007-08-17

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

  19. Oxygen Replacement with Selenium at the Thymidine 4-Position for the Se Base Pairing and Crystal Structure Studies

    SciTech Connect

    Salon,J.; Sheng, J.; Jiang, J.; Chen, G.; Caton-Williams, J.; Huang, Z.

    2007-01-01

    The T-A and C-G base pairing and stacking allow the formation of the stable DNA duplex structure for genetic information storage, transcription, and replication. To replace the oxygen of the nucleotide nucleobases with selenium for the studies of the base-pair recognition, the duplex stability, and the nuclei acid crystal structures, we have synthesized for the first time the 4-Se thymidine phosphoramidite and incorporated it into oligonucleotides via solid-phase synthesis with high coupling yield (99%). The Se modification on the nucleobase is relatively stable under the elevated temperature. Using the dU{sub Se} (2'-Se-dU) to facilitate the crystallization, we have successfully crystallized the DNA containing the 4-Se-T substitution and determined its structure at 1.50 {angstrom} resolution. The UV-melting and X-ray crystal structure studies have indicated that the Se substitution on the nucleobase does not cause a significant structure perturbation, the large Se atom on the thymine can be successfully accommodated by the DNA duplex, and the Se-mediated hydrogen bond (longer than the usual hydrogen bond) is formed within the modified T-A base pair. In addition, the Se derivatization on the nucleobases further facilitates X-ray crystal structure determination of nucleic acids and their protein complexes via Se MAD phasing.

  20. Oncolytic Adenoviruses Armed with Thymidine Kinase Can Be Traced by PET Imaging and Show Potent Antitumoural Effects by Ganciclovir Dosing

    PubMed Central

    Abate-Daga, Daniel; Andreu, Nuria; Camacho-Sánchez, Juan; Alemany, Ramon; Herance, Raúl; Millán, Olga; Fillat, Cristina

    2011-01-01

    Replication-competent adenoviruses armed with thymidine kinase (TK) combine the concepts of virotherapy and suicide gene therapy. Moreover TK-activity can be detected by noninvasive positron emission-computed tomography (PET) imaging, what could potentially facilitate virus monitoring in vivo. Here, we report the generation of a novel oncolytic adenovirus that incorporates the Tat8-TK gene under the control of the Major Late Promoter in a highly selective backbone thus providing selectivity by targeting the retinoblastoma pathway. The selective oncolytic TK virus, termed ICOVIR5-TK-L, showed reduced potency compared to a non-selective counterpart. However the combination of ICOVIR5-TK-L with ganciclovir (GCV) induced a potent antitumoural effect similar to that of wild type adenovirus in a preclinical model of pancreatic cancer. Although the treatment with GCV provoked a reduction in the viral yield, both in vitro and in vivo, a two-cycle treatment of virus and GCV resulted in an enhanced antitumoral response that correlated with high TK-activity, based on microPET measurements. Thus, TK-expressing oncolytic adenoviruses can be traced by PET imaging providing real time information on the activity of the virus and its antitumoral potency can be optimized by GCV dosing. PMID:22028820

  1. Translational Compensation of a Frameshift Mutation Affecting Herpes Simplex Virus Thymidine Kinase Is Sufficient To Permit Reactivation from Latency

    PubMed Central

    Griffiths, Anthony; Chen, Shun-Hua; Horsburgh, Brian C.; Coen, Donald M.

    2003-01-01

    Herpes simplex virus thymidine kinase is important for reactivation of virus from its latent state and is a target for the antiviral drug acyclovir. Most acyclovir-resistant isolates have mutations in the thymidine kinase gene; however, how these mutations confer clinically relevant resistance is unclear. Reactivation from explanted mouse ganglia was previously observed with a patient-derived drug-resistant isolate carrying a single guanine insertion within a run of guanines in the thymidine kinase gene. Despite this mutation, low levels of active enzyme were synthesized following an unusual ribosomal frameshift. Here we report that a virus, generated from a pretherapy isolate from the same patient, engineered to lack thymidine kinase activity, was competent for reactivation. This suggested that the clinical isolate contains alleles of other genes that permit reactivation in the absence of thymidine kinase. Therefore, to establish whether thymidine kinase synthesized via a ribosomal frameshift was sufficient for reactivation under conditions where reactivation requires this enzyme, we introduced the mutation into the well-characterized strain KOS. This mutant virus reactivated from latency, albeit less efficiently than KOS. Plaque autoradiography revealed three phenotypes of reactivating viruses: uniformly low thymidine kinase activity, mixed high and low activity, and uniformly high activity. We generated a recombinant thymidine kinase-null virus from a reactivating virus expressing uniformly low activity. This virus did not reactivate, confirming that mutations in other genes that would influence reactivation had not arisen. Therefore, in strains that require thymidine kinase for reactivation from latency, low levels of enzyme synthesized via a ribosomal frameshift can suffice. PMID:12663777

  2. Binding of dexetimide and levetimide to [3H](+)pentazocine- and [3H]1,3-di(2-tolyl)guanidine-defined sigma recognition sites.

    PubMed

    DeHaven-Hudkins, D L; Hudkins, R L

    1991-01-01

    The potent antimuscarinic benzetimide and its resolved stereoisomers dexetimide and levetimide were tested for their affinities at sigma sites labelled by [3H](+)pentazocine or [3H]1,3-di(2-tolyl)guanidine. Levetimide was a potent and stereoselective inhibitor of [3H](+)pentazocine binding, with a Ki of 2.2 nM, while dexetimide was nine-fold less potent (Ki = 19 nM). Dexetimide and levetimide potently inhibited [3H]DTG binding although without stereoselectivity (Ki values of 65 and 103 nM, respectively). Levetimide may be a useful tool with which to investigate sigma recognition sites and sigma subtypes. PMID:1656155

  3. Ethanol intake and sup 3 H-serotonin uptake II: A study in alcoholic patients using platelets sup 3 H-paroxetine binding

    SciTech Connect

    Daoust, M.; Boucly, P. ); Ernouf, D. ); Breton, P. ); Lhuintre, J.P.

    1991-01-01

    The kinetic parameters of {sup 3}H-paroxetine binding and {sup 3}H-serotonin uptake were studied in platelets of alcoholic patients. There was no difference between alcoholic and non alcoholic subjects in {sup 3}H-paroxetine binding. When binding and {sup 3}H-serotonin uptake were studied, in the same plasma of the same subjects, the Vmax of serotonin uptake was increased in alcoholics. The data confirm the involvement of serotonin uptake system in alcohol dependance and suggest that serotonin uptake and paroxetine binding sites may be regulated independently in this pathology.

  4. Phosphorus balance and mineral metabolism with 3 h daily hemodialysis.

    PubMed

    Ayus, J C; Achinger, S G; Mizani, M R; Chertow, G M; Furmaga, W; Lee, S; Rodriguez, F

    2007-02-01

    Poor control of mineral metabolism is independently associated with mortality in patients receiving hemodialysis. We analyzed data from a 12-month, prospective, non-randomized, controlled study of daily hemodialysis (DHD) (six sessions/week 3 h each) (n=26) vs conventional hemodialysis (CHD) (three sessions/week 4 h each) (n=51) for achievement of mineral metabolism goals and we performed a substudy of weekly dialytic phosphorus removal in DHD vs CHD. Phosphorus control was superior in the DHD group (% change from baseline to end-of-study -27+/-30% vs +7%+/-35% in the CHD group, P=0.0001). Percentage of patients using phosphate binders decreased from 77 to 40% among subjects on DHD, whereas these parameters did not change (76 vs 77%) in the CHD group (P=0.03 by Breslow-Day test for homogeneity of the odds ratios). Weekly mean phosphorus removal was higher in the DHD group (2452+/-720 mg/week vs 1572+/-366 mg/week, P=0.04). Mean normalized protein catabolic rate increased (0.90+/-0.43-1.22+/-0.26 g/kg/day, P=0.0013). DHD was also associated with an increase in the percent of subjects achieving three or more mineral metabolism goals (for phosphorus, calcium x phosphorus and parathyroid hormone) (15 vs 46%, P=0.046). In conclusion, DHD improves phosphorus control by increasing dialytic phosphorus removal while maintaining nutritional status and reducing the use of phosphate binders. The net effect allows for improved achievement of mineral metabolism goals. PMID:17191084

  5. Electron Scattering and Doping Mechanisms in Solid-Phase-Crystallized In2O3:H Prepared by Atomic Layer Deposition.

    PubMed

    Macco, Bart; Knoops, Harm C M; Kessels, Wilhelmus M M

    2015-08-01

    Hydrogen-doped indium oxide (In2O3:H) has recently emerged as an enabling transparent conductive oxide for solar cells, in particular for silicon heterojunction solar cells because its high electron mobility (>100 cm(2)/(V s)) allows for a simultaneously high electrical conductivity and optical transparency. Here, we report on high-quality In2O3:H prepared by a low-temperature atomic layer deposition (ALD) process and present insights into the doping mechanism and the electron scattering processes that limit the carrier mobility in such films. The process consists of ALD of amorphous In2O3:H at 100 °C and subsequent solid-phase crystallization at 150-200 °C to obtain large-grained polycrystalline In2O3:H films. The changes in optoelectronic properties upon crystallization have been monitored both electrically by Hall measurements and optically by analysis of the Drude response. After crystallization, an excellent carrier mobility of 128 ± 4 cm(2)/(V s) can be obtained at a carrier density of 1.8 × 10(20) cm(-3), irrespective of the annealing temperature. Temperature-dependent Hall measurements have revealed that electron scattering is dominated by unavoidable phonon and ionized impurity scattering from singly charged H-donors. Extrinsic defect scattering related to material quality such as grain boundary and neutral impurity scattering was found to be negligible in crystallized films indicating that the carrier mobility is maximized. Furthermore, by comparison of the absolute H-concentration and the carrier density in crystallized films, it is deduced that <4% of the incorporated H is an active dopant in crystallized films. Therefore, it can be concluded that inactive H atoms do not (significantly) contribute to defect scattering, which potentially explains why In2O3:H films are capable of achieving a much higher carrier mobility than conventional In2O3:Sn (ITO). PMID:26168056

  6. Uptake and metabolism of L-(/sup 3/H)glutamate and L-(/sup 3/H)glutamine in adult rat cerebellar slices

    SciTech Connect

    de Barry, J.; Vincendon, G.; Gombos, G.

    1983-10-01

    Using very low concentrations (1 mumol range) of L-2-3-(/sup 3/H)glutamate, (/sup 3/H-Glu) or L-2-3-(/sup 3/H)glutamine (/sup 3/H-Gln), the authors have previously shown by autoradiography that these amino acids were preferentially taken up in the molecular layer of the cerebellar cortex. Furthermore, the accumulation of /sup 3/H-Glu was essentially glial in these conditions. Uptake and metabolism of either (/sup 3/H-Glu) or (/sup 3/H-Gln) were studied in adult rat cerebellar slices. Both amino acids were rapidly converted into other metabolic compounds: after seven minutes of incubation in the presence of exogenous /sup 3/H-Glu, 70% of the tissue accumulated radioactivity was found to be in compounds other than glutamate. The main metabolites were Gln (42%), alpha-ketoglutarate (25%) and GABA (1,4%). In the presence of exogenous /sup 3/H-Gln the rate of metabolism was slightly slower (50% after seven minutes of incubation) and the metabolites were also Glu (29%), alpha-ketoglutarate (15%) and GABA (5%). Using depolarizing conditions (56 mM KCl) with either exogenous /sup 3/H-Glu or /sup 3/H-Gln, the radioactivity was preferentially accumulated in glutamate compared to control. From these results we conclude: i) there are two cellular compartments for the neurotransmission-glutamate-glutamine cycle; one is glial, the other neuronal; ii) these two cellular compartments contain both Gln and Glu; iii) transmitter glutamate is always in equilibrium with the so-called ''metabolic'' pool of glutamate; iv) the regulation of the glutamate-glutamine cycle occurs at least at two different levels: the uptake of glutamate and the enzymatic activity of the neuronal glutaminase.

  7. DNA Mismatch Repair Interacts with CAF-1- and ASF1A-H3-H4-dependent Histone (H3-H4)2 Tetramer Deposition.

    PubMed

    Rodriges Blanko, Elena; Kadyrova, Lyudmila Y; Kadyrov, Farid A

    2016-04-22

    DNA mismatch repair (MMR) is required for the maintenance of genome stability and protection of humans from several types of cancer. Human MMR occurs in the chromatin environment, but little is known about the interactions between MMR and the chromatin environment. Previous research has suggested that MMR coincides with replication-coupled assembly of the newly synthesized DNA into nucleosomes. The first step in replication-coupled nucleosome assembly is CAF-1-dependent histone (H3-H4)2 tetramer deposition, a process that involves ASF1A-H3-H4 complex. In this work we used reconstituted human systems to investigate interactions between MMR and CAF-1- and ASF1A-H3-H4-dependent histone (H3-H4)2 tetramer deposition. We have found that MutSα inhibits CAF-1- and ASF1A-H3-H4-dependent packaging of a DNA mismatch into a tetrasome. This finding supports the idea that MMR occurs before the DNA mismatch is packaged into the tetrasome. Our experiments have also revealed that CAF-1- and ASF1A-H3-H4-dependent deposition of the histone (H3-H4)2 tetramers does not interfere with MMR reactions. In addition, we have established that unnecessary degradation of the discontinuous strand that takes place in both DNA polymerase δ (Pol δ)- and DNA polymerase ϵ (Pol ϵ)-dependent MMR reactions is suppressed by CAF-1- and ASF1A-H3-H4-dependent deposition of the histone (H3-H4)2 tetramers. These data suggest that CAF-1- and ASF1A-H3-H4-dependent deposition of the histone (H3-H4)2 tetramers is compatible with MMR and protects the discontinuous daughter strand from unnecessary degradation by MMR machinery. PMID:26945061

  8. Direct assay of thymidine kinase bound to ion-exchange paper for dot spotting and enzyme blotting analysis

    SciTech Connect

    van den Berg, K.J.

    1986-05-15

    The direct assay of thymidine kinase (Tk) bound to ion-exchange paper was investigated as a means to further simplify the analytical procedure. Thymidine kinase bound firmly and quantitatively to ion-exchange paper at near neutral pH. The enzymatic properties of Tk did not change while bound to the ion-exchange paper. The amount of phosphorylated /sup 12//sub 5/IdU or /sup 125/IdC formed on ion-exchange paper was proportional to the amount of applied Tk. Enzymatic activity could be determined visually by autoradiography or by gamma counting. This method was relatively independent of the protein concentration or volume of the sample and which allows the assay from dilute solutions. A simplified dot spot method that can be used for the assay of thymidine kinase activity in cell extracts is described. Thymidine kinase could also be visualized after electrophoresis and blotting on ion-exchange paper.

  9. Influence of structural and electronic properties of uranyl derivatives on the inhibition of thymidine phosphorylase

    SciTech Connect

    Dimoglo, A.S.; Bersuker, I.B.; Gorbachev, M.Yu.

    1986-07-01

    The inhibition of enzymes by definite compounds lies at the basis of the mechanism of the action of most drugs. Uracil and its derivatives are effective inhibitors of thymidine phosphorylase and other related enzymes. Cancer cells are especially sensitive to the absence of thymidine. The study of the inhibiting action of uracil derivatives has been conducted previously. In this article, the authors used the hydrophobicity constants of the substituents, directly bonded to the uracil framework in the 1- and 3-positions and the 6-position as well as the constants of ortho- and meta-substituents in benzene rings bonded to uracil in the investigated compounds for the derivation of correlation equations relating the inhibiting activity to the physicochemical parameters. A table is presented of the 142 uracil derivatives taken for logical-structural analysis.

  10. Unbalanced deoxynucleotide pools cause mitochondrial DNA instability in thymidine phosphorylase-deficient mice.

    PubMed

    López, Luis C; Akman, Hasan O; García-Cazorla, Angeles; Dorado, Beatriz; Martí, Ramón; Nishino, Ichizo; Tadesse, Saba; Pizzorno, Giuseppe; Shungu, Dikoma; Bonilla, Eduardo; Tanji, Kurenai; Hirano, Michio

    2009-02-15

    Replication and repair of DNA require equilibrated pools of deoxynucleoside triphosphate precursors. This concept has been proven by in vitro studies over many years, but in vivo models are required to demonstrate its relevance to multicellular organisms and to human diseases. Accordingly, we have generated thymidine phosphorylase (TP) and uridine phosphorylase (UP) double knockout (TP(-/-)UP(-/-)) mice, which show severe TP deficiency, increased thymidine and deoxyuridine in tissues and elevated mitochondrial deoxythymidine triphosphate. As consequences of the nucleotide pool imbalances, brains of mutant mice developed partial depletion of mtDNA, deficiencies of respiratory chain complexes and encephalopathy. These findings largely account for the pathogenesis of mitochondrial neurogastrointestinal encephalopathy (MNGIE), the first inherited human disorder of nucleoside metabolism associated with somatic DNA instability. PMID:19028666

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

    PubMed

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

    2007-01-01

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

  12. A new approach to the synthesis of the 5'-deoxy-5'-methylphosphonate linked thymidine oligonucleotide analogues.

    PubMed Central

    Szabó, T; Kers, A; Stawinski, J

    1995-01-01

    A new synthetic method for the preparation of the 5'-deoxy-5'-methylphosphonate linked thymidine oligonucleotides (5'-methylenephosphonate analogues) was developed. The method is based on the use of a phosphonate protecting group, 4-methoxy-1-oxido-2-picolyl, enabling intramolecular nucleophilic catalysis which together with the condensing agent, 2,4,6-triisopropylbenzenesulfonyl chloride, secures fast and efficient formation of the 5'-methylenephosphonate internucleosidic bonds. The produced protected oligomers were treated with thiophenol and triethylamine to remove the phosphonate protecting groups, cleaved from the solid support using concentrated aqueous ammonia, and purified by HPLC. Several thymidine oligonucleotide analogues with the chain length of up to 20 nucleotidic units, in which all internal 5'-oxygen atoms have been replaced by methylene groups directly bound to phosphorus, were synthesised using this methodology. PMID:7731801

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

    SciTech Connect

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

    1982-06-01

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

  14. Novel ribofuranosylnucleoside lead compounds for potent and selective inhibitors of mitochondrial thymidine kinase-2.

    PubMed Central

    Balzarini, J; Zhu, C; De Clercq , E; Pérez-Pérez, M J; Chamorro, C; Camarasa, M J; Karlsson, A

    2000-01-01

    The ribonucleoside analogues (E)-5-(2-bromovinyl)uridine (5-BV-Urd) and 3'-spiro-(4'-amino-1',2'-oxathiole-2',2'-dioxide)-5-methyluridine (3'-AOD-5-MeUrd) emerged as potent and selective competitive inhibitors of mitochondrial thymidine kinase (TK)-2 with respect to thymidine (K(i)/K(m) values of 9.0 and 1.2 respectively). Cytosolic TK-1 did not show measurable affinity for these compounds. [(32)P]Phosphate transfer studies from [gamma-(32)P]ATP to 5-BV-Urd and 3'-AOD-5-MeUrd revealed extremely poor substrate activity but potent inhibitory potential of the compounds. It was concluded that the ribonucleosides 5-BV-Urd and 3'-AOD-5-MeUrd represent two new lead compounds for potent and selective inhibitors of mitochondrial TK-2. PMID:10998359

  15. Synthesis of [1,2-3H2]cholecalciferol and metabolism of [4-14C,1,2-3H2]- and [4-14C,1-3H]-cholecalciferol in rachitic rats and chicks

    PubMed Central

    Lawson, D. E. M.; Pelc, B.; Bell, P. A.; Wilson, P. W.; Kodicek, E.

    1971-01-01

    [1,2-3H2]Cholecalciferol has been synthesized with a specific radioactivity of 508mCi/mmol by using tristriphenylphosphinerhodium chloride, the homogeneous hydrogen catalyst. With doses of 125ng (5i.u.) of [4-14C,1-3H2]cholecalciferol the tissue distribution in rachitic rats of cholecalciferol and its metabolites (25-hydroxycholecalciferol and peak P material) was similar to that found in chicken with 500ng doses of the double-labelled vitamin. The only exceptions were rat kidney, with a very high concentration of vitamin D, and rat blood, with a higher proportion of peak P material, containing a substance formed from vitamin D with the loss of hydrogen from C-1. Substance P formed from [4-14C,1,2-3H2]cholecalciferol retained 36% of 3H, the amount expected from its distribution between C-1 and C-2, the 3H at C-1 being lost. 25-Hydroxycholecalciferol does not seem to have any specific intracellular localization within the intestine of rachitic chicks. The 3H-deficient substance P was present in the intestine and bone 1h after a dose of vitamin D and 30min after 25-hydroxycholecalciferol. There was very little 25-hydroxycholecalciferol in intestine at any time-interval, but bone and blood continued to take it up over the 8h experimental period. It is suggested that the intestinal 3H-deficient substance P originates from outside this tissue. The polar metabolite found in blood and which has retained its 3H at C-1 is not a precursor of the intestinal 3H-deficient substance P. PMID:4329870

  16. Cutaneous absorption and decontamination of ( sup 3 H)T-2 toxin in the rat model

    SciTech Connect

    Bunner, B.L.; Wannemacher, R.W. Jr.; Dinterman, R.E.; Broski, F.H. )

    1989-01-01

    Cutaneous absorption and decontamination of ({sup 3}H)T-2 mycotoxin using various treatment modalities incorporating water, detergent, sprays, and scrubbing of application sites were examined in the rat model at 5, 30, 60, and 1440 min (24 h) postexposure. Rats were killed immediately after treatment and radiolabeled T-2 remaining in full-thickness skin samples was determined. Absorption and decontamination were followed over time, and decontaminating treatment modalities were evaluated for efficacy. Less than 1% of the applied dose was absorbed in 5 min, and 50% was absorbed in 24 h. At 5 min, 99.5 {plus minus} 0.05% of nonabsorbed (residual) ({sup 3}H)T-2 was removed, and 58 {plus minus} 5.2% of residual toxin was removed at 24 h with a 2.5% detergent/water spray. When treatment modalities were evaluated at 60 min, a 2.5% detergent/water scrub followed by a detergent/water spray produced optimal decontamination by removing 81 {plus minus} 2.2% of residual toxin. All treatment modalities using detergent and/or water removed significant amounts of toxin, a dry scrub was not efficacious. Treatment should be initiated as soon as possible after exposure for best results. However, the stratum corneum acts as a reservoir for the toxin, and decontamination should be carried out even if delayed several hours or days after exposure. Dermal absorption pharmacokinetics found in these studies are similar to those described for other low-molecular-weight compounds, and the decontamination results from T-2 toxin should be applicable to other, similar toxic substances.

  17. SAMHD1 Specifically Affects the Antiviral Potency of Thymidine Analog HIV Reverse Transcriptase Inhibitors

    PubMed Central

    Ballana, Ester; Badia, Roger; Terradas, Gerard; Torres-Torronteras, Javier; Ruiz, Alba; Pauls, Eduardo; Riveira-Muñoz, Eva; Clotet, Bonaventura; Martí, Ramon

    2014-01-01

    Sterile alpha motif and histidine-aspartic domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase recently recognized as an antiviral factor that acts by depleting dNTP availability for viral reverse transcriptase (RT). SAMHD1 restriction is counteracted by the human immunodeficiency virus type 2 (HIV-2) accessory protein Vpx, which targets SAMHD1 for proteosomal degradation, resulting in an increased availability of dNTPs and consequently enhanced viral replication. Nucleoside reverse transcriptase inhibitors (NRTI), one of the most common agents used in antiretroviral therapy, compete with intracellular dNTPs as the substrate for viral RT. Consequently, SAMHD1 activity may be influencing NRTI efficacy in inhibiting viral replication. Here, a panel of different RT inhibitors was analyzed for their different antiviral efficacy depending on SAMHD1. Antiviral potency was measured for all the inhibitors in transformed cell lines and primary monocyte-derived macrophages and CD4+ T cells infected with HIV-1 with or without Vpx. No changes in sensitivity to non-NRTI or the integrase inhibitor raltegravir were observed, but for NRTI, sensitivity significantly changed only in the case of the thymidine analogs (AZT and d4T). The addition of exogenous thymidine mimicked the change in viral sensitivity observed after Vpx-mediated SAMHD1 degradation, pointing toward a differential effect of SAMHD1 activity on thymidine. Accordingly, sensitivity to AZT was also reduced in CD4+ T cells infected with HIV-2 compared to infection with the HIV-2ΔVpx strain. In conclusion, reduction of SAMHD1 levels significantly decreases HIV sensitivity to thymidine but not other nucleotide RT analog inhibitors in both macrophages and lymphocytes. PMID:24913159

  18. Thymidine phosphorylase gene mutations in patients with mitochondrial neurogastrointestinal encephalomyopathy syndrome.

    PubMed

    Slama, A; Lacroix, C; Plante-Bordeneuve, V; Lombès, A; Conti, M; Reimund, J M; Auxenfants, E; Crenn, P; Laforêt, P; Joannard, A; Seguy, D; Pillant, H; Joly, P; Haut, S; Messing, B; Said, G; Legrand, A; Guiochon-Mantel, A

    2005-04-01

    The mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) syndrome is characterized by the association of gastrointestinal and neurological symptoms. It is a rare autosomal recessive mitochondrial disorder with multiple mitochondrial DNA deletions and/or depletion. It is caused by thymidine phosphorylase (TP) gene mutations resulting in a complete abolition of TP activity. We tested 31 unrelated patients presenting either with a complete MNGIE syndrome (8 patients), a severe intestinal pseudo-obstruction (10 patients), and multiple deletions and/or depletion of mitochondrial DNA (13 patients). All the tested patients presenting with a complete MNGIE had increased thymidine levels in plasma and urine, and no TP activity. The group with pseudo-obstruction syndrome had normal or partial reduction of TP activity. We found pathogenic mutations on TP gene only in the MNGIE syndrome group: all the MNGIE patients were compound heterozygous or homozygous for mutations in the TP gene. Eight of these mutations are yet unreported, confirming the lack of genotype/phenotype correlation in this syndrome. Enzymatic activity and thymidine level are thus rapid diagnosis tests to detect MNGIE affected patients prior to genetic testing for patients with gastrointestinal symptoms. PMID:15781193

  19. Relaxed enantioselectivity of human mitochondrial thymidine kinase and chemotherapeutic uses of L-nucleoside analogues.

    PubMed Central

    Verri, A; Priori, G; Spadari, S; Tondelli, L; Focher, F

    1997-01-01

    Our discovery that Herpes virus thymidine kinase (TK) and cellular deoxycytidine kinase lack enantioselectivity, being able to phosphorylate both D- and L-enantiomers of the substrate, suggested the use of unnatural L-nucleoside analogues as antiviral drugs (Herpes, hepatitis and immunodeficiency viruses). Several L-nucleoside analogues have displayed a short-term cytotoxicity much lower than their corresponding D-counterpart. Since the delayed cytotoxicity of a drug often depends on its effects on mitochondrial metabolism, we have investigated the degree of enantioselectivity of human mitochondrial thymidine kinase (mt-TK). We demonstrate that mt-TK does not show an absolute enantioselectivity, being able to recognize, although with lower efficiency, the L-enantiomers of thymidine, deoxycytidine and modified deoxyuridines, such as (E)-5-(2-bromovinyl)-2'-deoxyuridine and 5-iodo-2'-deoxyuridine. Interestingly, the reported negative co-operativity of mt-TK phosphorylating beta-D-2'-deoxythymidine (D-Thd), disappears when the deoxyribose moiety has the inverted configuration, resulting in the preferential phosphorylation of d-Thd even in the presence of high concentrations of the L-enantiomer. This, coupled with the higher Km for beta-L-2'-deoxythymidine (L-Thd), makes mt-TK resistant to high concentrations of L-Thd and L-Thd analogues, minimizing the mitochondria-dependent delayed cytotoxicity that might be caused by the administration of L-nucleoside analogues as antivirals. PMID:9359870

  20. A system for assaying homologous recombination at the endogenous human thymidine kinase gene

    SciTech Connect

    Benjamin, M.B.; Little, J.B. ); Potter, H. ); Yandell, D.W. Massachusetts Eye and Ear Infirmary, Boston Harvard Medical School, Boston, MA )

    1991-08-01

    A system for assaying human interchromosomal recombination in vitro was developed, using a cell line containing two different mutant thymidine kinase genes (TK) on chromosomes 17. Heteroalleles were generated in the TK{sup +/+} parent B-lymphoblast cell line WIL-2 by repeated exposure to the alkylating nitrogen mustard ICR-191, which preferentially causes +1 or {minus}1 frameshifts. Resulting TK{sup {minus}/{minus}} mutants were selected in medium containing the toxic thymidine analog trifluorothymidine. In two lines, heterozygous frameshifts were located in exons 4 and 7 of the TK gene separated by {approx}8 kilobases. These lines undergo spontaneous reversion to TK{sup +} at a frequency of < 10{sup {minus}7}, and revertants can be selected in cytidine/hypoxanthine/aminopterin/thymidine medium. The nature and location of these heteroallelic mutations make large deletions, rearrangements, nondisjunction, and reduplication unlikely mechanisms for reversion to TK{sup +}. The mode of reversion to TK{sup +} was specifically assessed by DNA sequencing, use of single-strand conformation polymorphisms, and analysis of various restriction fragment length polymorphisms (RFLPs) linked to the TK gene on chromosome 17. The data suggest that a proportion of revertants has undergone recombination and gene conversion at the TK locus, with concomitant loss of frameshifts and allele loss at linked RFLPs. Models are presented for the origin of two recombinants.

  1. Enzymatic synthesis and RNA interference of nucleosides incorporating stable isotopes into a base moiety.

    PubMed

    Hatano, Akihiko; Shiraishi, Mitsuya; Terado, Nanae; Tanabe, Atsuhiro; Fukuda, Kenji

    2015-10-15

    Thymidine phosphorylase was used to catalyze the conversion of thymidine (or methyluridine) and uracil incorporating stable isotopes to deoxyuridine (or uridine) with the uracil base incorporating the stable isotope. These base-exchange reactions proceeded with high conversion rates (75-96%), and the isolated yields were also good (64-87%). The masses of all synthetic compounds incorporating stable isotopes were identical to the theoretical molecular weights via EIMS. (13)C NMR spectra showed spin-spin coupling between (13)C and (15)N in the synthetic compounds, and the signals were split, further proving incorporation of the isotopes into the compounds. The RNA interference effects of this siRNA with uridine incorporating stable isotopes were also investigated. A 25mer siRNA had a strong knockdown effect on the MARCKS protein. The insertion position and number of uridine moieties incorporating stable isotopes introduced into the siRNA had no influence on the silencing of the target protein. This incorporation of stable isotopes into RNA and DNA has the potential to function as a chemically benign tracer in cells. PMID:26404411

  2. Reaction channels and spectroscopic constants of astrophysical relevant Silicon bearing molecules SiC3H,+ and SiC3H

    NASA Astrophysics Data System (ADS)

    Inostroza Pino, N.; Cardenas, C.; Fuentealba, P.

    2014-10-01

    Reaction channels and spectroscopic properties of a series of silicon-carbon-bearing isomers of SiC3H+ and SiC3H, which are suitable species for astrophysical detection in carbon-rich sources, are calculated with correlated ab initio CCSD(T) and density functional theory methods. We present four isomers of SiC3H+ for which the electronic ground states have closed-shell configurations. For SiC3H, we considered the same structures in order to present a complete study. The global minimum among the SiC3H+ isomers corresponds to the rhomboidal structure with a transannular bond in a 1A1 electronic state (rb3-SiC3H+ C2v X1A1). The next minima correspond to a second rhomboid 1A1 isomer and a linear isomer (X1Σ+) with relative energies 0.86 and 0.93 eV, respectively at the CCSD(T)/cc-pvTZ level of theory. The most stable mono-hydrogenated silicon carbon isomer is linear, followed by two rhomboidal isomers, rb2-SiC3H and rb3-SiC3H (0.23 and 0.31 eV). For each structure, a set of spectroscopic parameters including their equilibrium structures, rotational constants, harmonic frequencies and dipole moment is presented. Furthermore, we discuss plausible formation pathways of SiC3H+ isomers which are classified as charge-exchange, ion-neutral and dissociative recombination reactions. These results show one favourable pathway to produce rb3-SiC3H+ from rb-SiC3-3s. The formation energy of the cation's isomers coming from neutral isomers as linear l1-SiC3H, rb3-SiC3H and rb2-SiC3H plus H+ as reactants (charge-exchange reaction) are 203.8 kcal mol-1 (8.84eV), 175.4 kcal mol-1 (7.60 eV) and 195.2 kcal mol-1 (8.46 eV), which provides us with evidence of the endergonic character of these reactions. As a consequence, it does not seem to be feasible to produce a cation from neutral reactant plus H+ by a charge-exchange reaction that was proposed by UMIST.

  3. Autoradiographic analysis of the in vivo distribution of 3H-imipramine and 3H-desipramine in brain: Comparison to in vitro binding patterns

    SciTech Connect

    Duncan, G.E.; Paul, I.A.; Fassberg, J.B.; Powell, K.R.; Stumpf, W.E.; Breese, G.R. )

    1991-03-01

    Using high resolution autoradiographic techniques, the distribution of radioactivity in forebrain and brainstem was assessed after 4 injection of 3H-impramine or 3H-desipramine. Results were compared with regional binding of the drugs to brain sections in vitro. Similar topographic binding of 3H-imipramine and 3H-desipramine was observed in vitro among brain regions, except in the paraventricular nucleus of the hypothalamus and locus coeruleus, where binding was greater for 3H-desipramine. For both 3H-desipramine and 3H-imipramine, some brain regions that exhibited high binding in vitro also showed high accumulation after in vivo injection. However, certain regions that contained high densities of binding sites for the antidepressant drugs as measured by in vitro binding showed very low accumulation of radioactivity after in vivo treatment. Such regions included the dentate gyrus of the hippocampus, layer 1 of piriform cortex, caudate-putamen, pontine and midbrain central gray, and cerebellar granular layer. Compared to in vitro binding of the drugs, the distribution of imipramine and desipramine in vivo appears more anatomically selective. For imipramine, primary sites of action in vivo, as indicated by the topographic distribution in brain, appear to be the locus coeruleus, hippocampus, lateral septal nucleus, and amygdala. For desipramine, the greatest accumulation in vivo was found in the locus coeruleus, paraventricular nucleus of the hypothalamus, and anterior thalamic nuclei.

  4. Rate constant measurement of the recombination reaction C[sub 3]H[sub 3] + C[sub 3]H[sub 3

    SciTech Connect

    Morter, C.L.; Farhat, S.K.; Adamson, J.D.; Glass, G.P.; Curl, R.F. )

    1994-07-14

    Using the technique of infrared kinetic absorption spectroscopy, the second-order rate constant for the recombination reaction of the propargyl radical (C[sub 3]H[sub 3] + C[sub 3]H[sub 3]) has been measured and found to have the value (1.2 [+-] 0.2) x 10[sup [minus]10] cm[sup 3] molecule[sup [minus]1] s[sup [minus]1] at 295 K. The radical was produced in a flow cell by excimer laser flash photolysis ([lambda] = 193 nm) of the precursors C[sub 3]H[sub 3]Cl or C[sub 3]H[sub 3]Br and detected using time-resolved IR absorption. Absolute concentrations of C[sub 3]H[sub 3] were determined by comparing the C[sub 3]H[sub 3] absorption intensity with that of the Br atom. This calibration scheme was checked by producing methyl radicals by photolysis of methyl bromide and comparing the rate constant for methyl recombination thus obtained with literature values. The quantum yield for HCl production from the photodissociation of C[sub 3]H[sub 3]Cl at 193 nm was determined to be 0.07 [+-] 0.01. 47 refs., 10 figs., 1 tab.

  5. An observational investigation of the identity of B11244 (l-C{sub 3}H{sup +}/C{sub 3}H{sup -})

    SciTech Connect

    McGuire, Brett A.; Carroll, P. Brandon; Gratier, Pierre; Guzmán, Viviana; Pety, Jerome; Roueff, Evelyne; Gerin, Maryvonne; Blake, Geoffrey A.; Remijan, Anthony J.

    2014-03-01

    Pety et al. have reported the detection of eight transitions of a closed-shell, linear molecule (B11244) in observations toward the Horsehead photodissociation region (PDR), which they attribute to the l-C{sub 3}H{sup +} cation. Recent high-level ab initio calculations have called this assignment into question; the anionic C{sub 3}H{sup –} molecule has been suggested as a more likely candidate. Here, we examine observations of the Horsehead PDR, Sgr B2(N), TMC-1, and IRC+10216 in the context of both l-C{sub 3}H{sup +} and C{sub 3}H{sup –}. We find no observational evidence of K{sub a} = 1 lines, which should be present were the carrier indeed C{sub 3}H{sup –}. Additionally, we find a strong anticorrelation between the presence of known molecular anions and B11244 in these regions. Finally, we discuss the formation and destruction chemistry of C{sub 3}H{sup –} in the context of the physical conditions in the regions. Based on these results, we conclude there is little evidence to support the claim that the carrier is C{sub 3}H{sup –}.

  6. Nicotinic binding in rat brain: autoradiographic comparison of (/sup 3/H)acetylcholine, (/sup 3/H)nicotine, and (/sup 125/I)-alpha-bungarotoxin

    SciTech Connect

    Clarke, P.B.; Schwartz, R.D.; Paul, S.M.; Pert, C.B.; Pert, A.

    1985-05-01

    Three radioligands have been commonly used to label putative nicotinic cholinoceptors in the mammalian central nervous system: the agonists (/sup 3/H)nicotine and (/sup 3/H)acetylcholine ((/sup 3/H)ACh--in the presence of atropine to block muscarinic receptors), and the snake venom extract, (/sup 125/I)-alpha-bungarotoxin((/sup 125/I)BTX), which acts as a nicotinic antagonist at the neuromuscular junction. Binding studies employing brain homogenates indicate that the regional distributions of both (/sup 3/H)nicotine and (/sup 3/H)ACh differ from that of (/sup 125/I)BTX. The possible relationship between brain sites bound by (/sup 3/H)nicotine and (/sup 3/H)ACh has not been examined directly. The authors have used the technique of autoradiography to produce detailed maps of (/sup 3/H)nicotine, (/sup 3/H)ACh, and (/sup 125/I)BTX labeling; near-adjacent tissue sections were compared at many levels of the rat brain. The maps of high affinity agonist labeling are strikingly concordant, with highest densities in the interpeduncular nucleus, most thalamic nuclei, superior colliculus, medial habenula, presubiculum, cerebral cortex (layers I and III/IV), and the substantia nigra pars compacta/ventral tegmental area. The pattern of (/sup 125/I)BTX binding is strikingly different, the only notable overlap with agonist binding being the cerebral cortex (layer I) and superior colliculus. (/sup 125/I)BTX binding is also dense in the inferior colliculus, cerebral cortex (layer VI), hypothalamus, and hippocampus, but is virtually absent in thalamus. Various lines of evidence suggest that the high affinity agonist-binding sites in brain correspond to nicotinic cholinergic receptors similar to those found at autonomic ganglia; BTX-binding sites may also serve as receptors for nicotine and are possibly related to neuromuscular nicotinic cholinoceptors.

  7. High affinity binding of [3H]-tyramine in the central nervous system.

    PubMed Central

    Vaccari, A.

    1986-01-01

    Optimum assay conditions for the association of [3H]-para-tyramine [( 3H]-pTA) with rat brain membranes were characterized, and a saturable, reversible, drug-specific, and high affinity binding mechanism for this trace amine was revealed. The binding capacity (Bmax) for [3H]-pTA in the corpus striatum was approximately 30 times higher than that in the cerebellum, with similar dissociation constants (KD). The binding process of [3H]-pTA involved the dopamine system, inasmuch as (a) highest binding capacity was associated with dopamine-rich regions; (b) dopamine and pTA equally displaced specifically bound [3H]-pTA; (c) there was a severe loss in striatal binding capacity for [3H]-pTA and, reportedly, for [3H]-dopamine, following unilateral nigrostriatal lesion; (d) acute in vivo reserpine treatment markedly decreased the density of [3H]-pTA and, reportedly, of [3H]-dopamine binding sites. In competition experiments [3H]-pTA binding sites, though displaying nanomolar affinity for dopamine, revealed micromolar affinities for the dopamine agonists apomorphine and pergolide, and for several dopamine antagonists, while having very high affinity for reserpine, a marker for the catecholamine transporter in synaptic vesicles. The binding process of [3H]-pTA was both energy-dependent (ouabain-sensitive), and ATP-Mg2+-insensitive; furthermore, the potencies of various drugs in competing for [3H]-pTA binding to rat striatal membranes correlated well (r = 0.96) with their reported potencies in inhibiting [3H]-dopamine uptake into striatal synaptosomes. It is concluded that [3H]-pTA binds at a site located on/within synaptic vesicles where it is involved in the transport mechanism of dopamine. PMID:3801770

  8. Regiospecific transfer of tritium into 3H2O from labeled estrogens by mushroom tyrosinase.

    PubMed

    Jellinck, P H; Norton, B; Fishman, J

    1984-10-01

    The specificity of mushroom tyrosinase in displacing 3H from estradiol and catechol estrogens labeled at C-1, C-2, C-4 or C-6,7 was investigated under various conditions. [2-3H]E2 Yielded significant amounts of 3H2O, in the presence of NADH, and the rate of 3H loss from the steroid paralleled that of the radioactivity remaining in the aqueous fraction after extraction with organic solvents. NADH had little effect on the release of 3H from [1-3H]E2 or [4-3H]E2 but glutathione was highly active in this respect, with considerable differences being observed between lyophilizable 3H2O and yields of water-soluble products. It is proposed that 3H losses from C-2 of estradiol reflects oxidative displacement of this isotope by tyrosinase while the loss observed from C-1 and C-4 is the result of non-enzymatic conjugation with glutathione after the formation of the catechol estrogen. The difference between lyophilizable 3H2O and the yield of water-soluble products obtained with [1-3H]E2 and [4-3H]E2 provided a measure of the relative amount of conjugation occurring at C-1 and C-4. These findings were confirmed by double label experiments with 3H- and 14C-labeled estrogens and the isolation of the glutathionyl derivatives. The catechol estrogens did not serve as substrates for further hydroxylation by the enzyme even when C-2 was available for this reaction. These experiments give further information about the specificity of tyrosinase in its reaction with aromatic steroids and provide a simple and rapid method for confirming the distribution of 3H at C-2 or C-4 of estradiol. PMID:6092783

  9. Hydrocalcite (CaCO3 * H2O) and Nesquehonite (MgCO3 * 3H2O) in Carbonate Scales.

    PubMed

    Marschner, H

    1969-09-12

    Hydrocalcite (CaCO(3) * H(2)O) with exactly one molecule of hydrate water is the main component of carbonate scales deposited from cold water in contact with air. When the magnesium content of the water is high, the hydrocalcite occurs together with MgCO(3) * 3H(2)O (nesquehonite). From the conditions under which hydrocalcite is transformed into calcite and aragonite, it appears that in some cases aragonite in nature may be formed by way of an intermediary of CaCO(3) * H(2)O. PMID:17779803

  10. Distribution of /sup 3/H-proline in alveolar bone of the mouse as seen by radioautography

    SciTech Connect

    Johnson, R.B.

    1986-11-01

    Previous studies of the turnover of alveolar bone collagenous proteins have devoted little attention to the variable patterns in this process caused by bone remodeling. The present study seeks to document changes resulting from physiologic tooth movements in the incorporation and removal of the /sup 3/H-proline label within the interdental septum of alveolar bone. One week following /sup 3/H-proline injection, three zones could be distinguished: the appositional band, new bone, and old bone. Radioautography demonstrated that formation of new bone on the distal wall of the septum entrapped fibers of the periodontal ligament to create Sharpey's fibers. At the alveolar crest, new bone entrapped transseptal fibers to form transalveolar Sharpey's fibers. Grain counts were made within each area and over the total septum and were compared statistically. The data strongly suggested regional variations in protein remodeling. Counts from old and new bone were significantly different from the total septum or the appositional band (P less than .001). Regression lines were drawn to represent incorporation and removal of the isotope; slopes were calculated and compared statistically. The rate of incorporation and removal was significantly greater in the appositional band and in the total septum in comparison to old bone (P less than .001). The rates of incorporation and removal in the appositional band, old bone, and total septum were significantly different (P less than .001). Half-life of the labeled protein of old bone was 16.78 weeks; in the appositional band, 7.66 weeks; and in the total septum, 7.64 weeks. These data suggest that regional variations in collagen remodeling must be considered in a study of interdental bone and that the total septal grain counts are not indicative of the remodeling in the component zones.

  11. Binding of dexetimide and levetimide to ( sup 3 H)(+)pentazocine- and ( sup 3 H)1,3-Di(2-tolyl)guanidine-defined. sigma. recognition sites

    SciTech Connect

    Dehaven-Hudkins, D.L.; Hudkins, R.L. Virginia Commonwealth Univ., Richmond, VA )

    1991-01-01

    The potent antimuscarinic benzetimide and its resolved stereoisomers dexetimide and levetimide were tested for their affinities at {sigma} sites labelled by ({sup 3}H)(+)pentazocine or ({sup 3}H)1,3-di(2-tolyl)guanidine. Levetimide was a potent and stereoselective inhibitor of ({sup 3}H)(+)pentazocine binding, with a K{sub i} of 2.2 nM, while dexetimide was nine-fold less potent (K{sub i} = 19 nM). Dexetimide and levetimide potently inhibited ({sup 3}H)DTG binding although without stereoselectivity (K{sub i} values of 65 and 103 nM, respectively). Levetimide may be a useful tool with which to investigate {sigma} recognition sites and {sigma} subtypes.

  12. Compounds extracted from Phyllantus and Jatropha elliptica inhibit the binding of [3H]glutamate and [3H]GMP-PNP in rat cerebral cortex membrane.

    PubMed

    Martini, L H; Souza, C R; Marques, P B; Calixto, J B; Yunes, R A; Souza, D O

    2000-02-01

    Glutamate is to be considered a nociceptive neurotransmitter and glutamatergic antagonists present antinoceptive activity. In this study we investigated the effects of the naturally occurring antinociceptive compounds rutin, geraniin and quercetine extracted from Phyllanthus, as well as the diterpene jatrophone, extracted from Jatropha elliptica on the binding of [3H]glutamate and [3H]GMP-PNP [a GTP analogue which binds to extracellular site(s), modulating the glutamatergic transmission] in rat brain membrane. Jatrophone inhibited [3H]glutamate binding and geraniin inhibited [3H]GMP-PNP binding. Quercetine inhibited the binding of both ligands. These results may indicate a neurochemical parameter possibly related to the antinoceptive activity of these natural compounds. PMID:10786704

  13. Synthesis ofN-(2-chloro-5-methylthiophenyl)-N'-(3-methyl-thiophenyl)-N'-[3H3]methylguanidine, l brace [3H3]CNS-5161 r brace

    SciTech Connect

    Gibbs, Andrew R.; Morimoto, Hiromi; VanBrocklin, Henry F.; Williams, Philip G.; Biegon, Anat

    2001-09-28

    The preparation of the title compound, [{sup 3}H{sub 3}]CNS-5161, was accomplished in three steps starting with the production of [{sup 3}H{sub 3}]iodomethane (CT{sub 3}I). The intermediate N-[{sup 3}H{sub 3}]methyl-3-(thiomethylphenyl)cyanamide was prepared in 77% yield by the addition of CT{sub 3}I to 3-(thiomethylphenyl)cyanamide, previously treated with sodium hydride. Reaction of this tritiated intermediate with 2-chloro-5-thiomethylaniline hydrochloride formed the guanidine compound [{sup 3}H{sub 3}]CNS-5161. Purification by HPLC gave the desired labeled product in an overall yield of 9% with greater than 96% radiochemical purity and a final specific activity of 66 Ci mmol{sup -1}.

  14. Human platelet dense granules: Improved isolation preliminary characterization of ( sup 3 H)-serotonin uptake and tetrabanazine-displaceable ( sup 3 H)-ketanserin binding

    SciTech Connect

    Chatterjee, D.; Anderson, G.M.; Chakraborty, M.; Cohen, D.J. )

    1990-01-01

    An improved method for the isolation of human platelet dense granules was developed. A good yield of highly enriched dense granules was obtained after mild sonication and Percoll gradient centrifugation. The method has facilitated characterization of the granule, permitting the first report of K{sub m} and V{sub max} values for ({sup 3}H)-serotonin uptake, as well as the first determination of K{sub d} and B{sub max} values for tetrabenazine-displaceable ({sup 3}H)-ketanserin binding, in the human platelet dense granule. The rates and affinities of ({sup 3}H)-serotonin uptake were similar to those previously reported for porcine dense granules. Tetrabenazine-displaceable ({sup 3}H)-ketanserin binding was observed with a K{sub d} similar to, and a B{sub max} approximately 10-fold lower than, that previously seen in bovine chromaffin granules.

  15. Synthesis of C-11-{beta}-aminoisobutyric acid (C-11-{beta}-AlB): A major in vivo catabolite of [methyl-C-11]thymidine

    SciTech Connect

    Alauddin, M.M.; Conti, P.S.; Fissekis, J.D.

    1995-05-01

    Carbon-11 labeled thymidine (TdR) is being used for brain tumor imaging in patients with PET. Following clearance of 5-methyl C-11 TdR from plasma in humans, there is a progressive increase of C-11 activity in normal brain and tumor presumably secondary to accumulation of C-11 beta-AIB, a major by-product of thymidine catabolism in vivo. Canine studies have demonstrated that the major radiolabeled species in acid soluble extracts of brain and tumor tissues during C-14 TdR studies is beta-AIB. The previously reported synthesis of beta-AIB is not suitable for incorporation of carbon-11. A convenient method of synthesis of C-11 beta-AIB was developed where commercially available beta-alanine ethyl ester was converted to the cold precursor reagent, benzaldimine-beta-alanine ethyl ester, in 87% yield. Treatment of the imine derivative with LDA (1.1 eq) in THF at -78{degrees} C, followed by addition of iodomethane (1.1 eq) produced the alpha-methylated benzaldimine-beta-alanine ethyl ester in 73% chemical yield. Deprotection of the amino group by acidic hydrolysis followed by basic hydrolysis of the ester group produced the desired product in 50% chemical yield. Chemical structures of unlabeled intermediates and product were confirmed by H-1 NMR and CI mass spectrometry. Labeling was accomplished using C-11-methyl iodide prepared from C-11-CO{sub 2} according to literature methods. After removal of protecting groups and neutralization, the enatiomeric mixture was purified by HPLC using a semipreparative reverse phase C-18 column and PBS as eluent. The desired compound was eluted at 8.26 minutes. In preliminary runs, the synthesis time was 39 minutes including HPLC purification, with radiochemical yields of 5-6% (EOB). Radiochemical purity was >99%

  16. Further study on fallout sup 3 H ingestion in Akita, Japan

    SciTech Connect

    Hisamatsu, S.; Takizawa, Y.; Katsumata, T.; Itoh, M.; Ueno, K.; Sakanoue, M. )

    1989-10-01

    To study fallout {sup 3}H ingestion in Japan, 16 separate food-group samples were collected from Akita City in northern Japan during early summer and late autumn in 1986. Furthermore, total diet samples which are duplicate composite food samples consumed by five or six persons for a period of 1 d were also obtained in Akita City. The {sup 3}H concentration in free water and that in a tissue-bound form were determined separately. Seasonal changes of {sup 3}H concentration in the food samples and the total diet samples were not found clearly. The average {sup 3}H concentration in the free water including tap water was 1.6 Bq L-1. The mean ratio of specific activity of tissue-bound {sup 3}H to that of {sup 3}H in free water was found to be 1.2. The average total {sup 3}H ingestion was estimated to be 4.0 Bq d-1, while the proportion of tissue-bound form {sup 3}H ingestion to total {sup 3}H ingestion was 11%. Cereal was the greatest contributing food group to ingestion of tissue-bound {sup 3}H. These findings were consistent with our previous results for food samples collected in 1985.

  17. (3H)bunazosin, a novel selective radioligand of alpha 1 adrenoceptors in human prostates

    SciTech Connect

    Yamada, S.; Suzuki, M.; Matsuoka, Y.; Kato, Y.; Kimura, R.; Maruyama, M.; Kawabe, K. )

    1991-09-01

    The binding properties of a new radioligand, (3H)bunazosin, were studied in membranes of human prostates with benign prostatic hypertrophy (BPH). Specific binding of (3H)bunazosin was saturable, reversible, and of high affinity (Kd = 0.55 {plus minus} 0.04 nM). The density of (3H)bunazosin binding sites (Bmax) was 676 {plus minus} 33 fmol/mg. protein. (3H)Bunazosin rapidly associated with its binding sites in membranes of human prostates and reached steady state by 20 min. at 25C. The rate constants for association and dissociation of (3H)bunazosin binding were calculated to be 0.11 {plus minus} 0.01/nM/min. and 0.05 {plus minus} 0.02/min. (n = 4), respectively. Seven alpha 1 adrenoceptor antagonists competed with (3H)bunazosin for the binding sites in the rank order: R-(-)-YM-12617 greater than prazosin greater than SGB-1534 greater than bunazosin greater than terazosin greater than naftopidil greater than urapidil. In parallel studies with (3H)bunazosin, the Kd and Bmax values for (3H)prazosin binding in human prostates were slightly lower. There was a similarity in the potency and rank order of seven alpha 1, adrenoceptor antagonists for the inhibition of (3H) bunazosin and (3H)prazosin binding in human prostates. The new (3H)bunazosin binding assay in human prostates is remarkable for its low degree of nonspecific binding as compared to (3H)prazosin, especially at high ligand concentrations. Thus, (3H)bunazosin may become a useful radioligand for the further analysis of the alph 1 adrenoceptor binding sites in human prostates.

  18. Characterization of radiation-induced products of thymidine 3{prime}-monophosphate and thymidylyl (3{prime}{yields}5{prime}) thymidine by high-performance liquid chromatography and laser-desorption fourier-transform mass spectrometry

    SciTech Connect

    Yoshida, H.; Hettich, R.L.

    1994-09-01

    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{prime}{yields}5{prime}) thymidine (TpT) were isolated by reverse-phase HPLC and identified by LD FTMS. Thymine and thymidine 3{prime}-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. In vivo formation of tritium-labeled lactic acid from (2-/sup 3/H)mannose or (15-/sup 3/H)retinol by hamster intestinal epithelial cells

    SciTech Connect

    Creek, K.E.; Shankar, S.; De Luca, L.M.

    1987-05-01

    In studies to reexamine the in vivo occurrence of retinyl phosphate mannose we injected hamsters with either (2-/sup 3/H)mannose or (15-/sup 3/H)retinol. The small intestine was removed, the epithelial cells were scraped, and a methanolic extract of the labeled cells was prepared and chromatographed on a Mono Q anion-exchange column. Intraperitoneal administration of either (2-/sup 3/H)mannose or (15-/sup 3/H)retinol lead to the formation of a tritium-labeled anionic compound with a retention time on the Mono Q column similar to that of standard retinyl phosphate mannose. However, the biochemical properties of this labeled anionic compound were those expected of an organic acid and not retinyl phosphate mannose. The compound was resistant to both strong acid hydrolysis and mild base hydrolysis, as well as digestion with alpha- or beta-mannosidase, phosphodiesterase I, nucleotide pyrophosphatase, or beta-glucuronidase. When chromatographed on an Aminex HPX-87H organic acid analysis column or a silicic acid column the labeled anionic compound derived from either (2-/sup 3/H)mannose or (15-/sup 3/H)retinol comigrated with standard lactic acid. Treatment of the anionic compound derived from (2-/sup 3/H)mannose with lactate oxidase or L-lactate 2-monooxygenase resulted in the formation of a tritium-labeled product that cochromatographed, respectively, with pyruvate or acetate on the Aminex HPX-87H column. However, treatment of the anionic compound derived from (15-/sup 3/H)retinol with these same two enzymes resulted in a labeled product that migrated on the Aminex column at the same position as tritiated water. This result demonstrated that the labeled hydrogen was removed during enzymatic digestion and suggested that it was present on the second carbon of lactic acid. During the course of these studies no evidence for the in vivo labeling of a compound with the properties of retinyl phosphate mannose was found.

  20. Insect Ryanodine Receptor: Distinct But Coupled Insecticide Binding Sites for [N-C3H3]Chlorantraniliprole, Flubendiamide, and [3H]Ryanodine

    PubMed Central

    Isaacs, André K.; Qi, Suzhen; Sarpong, Richmond; Casida, John E.

    2015-01-01

    Radiolabeled anthranilic diamide insecticide [N-C3H3]chlorantraniliprole was synthesized at high specific activity and compared with phthalic diamide insecticide flubendiamide and [3H]ryanodine in radioligand binding studies with house fly muscle membranes to provide the first direct evidence with a native insect ryanodine receptor that the major anthranilic and phthalic diamide insecticides bind at different allosterically coupled sites, i.e. there are three distinct Ca2+-release channel targets for insecticide action. PMID:22856329

  1. Changes in [3H]-PK 11195 and [3H]-8-OH-DPAT binding following forebrain ischaemia in the gerbil.

    PubMed Central

    Kenny, B. A.; MacKinnon, A. C.; Spedding, M.; Brown, C. M.

    1993-01-01

    1. A high density of [3H]-PK 11195 binding sites was present in gerbil cortical membranes (Bmax [3H]-PK 11195 1360 +/- 71 fmol mg-1 protein) in comparison to rat cortical membranes (254 +/- 21 fmol mg-1 protein). This effect was species-specific as similar findings were obtained with hippocampal membranes (Bmax 1430 +/- 111 fmol mg-1 protein in gerbil, compared to 196 +/- 31 in rat). 2. RO 5-4864, also a peripheral type benzodiazepine compound, displayed low affinity for the [3H]-PK 11195 site in the gerbil (pKi 6.57 +/- 0.02 and 6.70 +/- 0.12 in hippocampus and cortex respectively) compared to rat (pKi 8.16 +/- 0.07 and 8.48 +/- 0.02). Central benzodiazepine compounds, diazepam and flunitrazepam, also displayed this trend. 3. RO 5-4864 displaced [3H]-PK 11195 binding from gerbil and rat cortical membranes through a competitive interaction with Hill slopes close to unity. In both tissues, saturation isotherms of [3H]-PK 11195 binding indicated that the presence of RO 5-4864 caused changes in Kd without any effect on Bmax. In kinetic experiments, the presence of RO 5-4864 failed to modify the rate of dissociation of [3H]-PK 11195 from equilibrium in both rat and gerbil cortical membranes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8395288

  2. Kinetics of killing Listeria monocytogenes by macrophages: correlation of /sup 3/H-DNA release from labeled bacteria and changes in numbers of viable organisms by mathematical model

    SciTech Connect

    Davies, W.A.

    1982-12-01

    Conventional methods of assessing antibacterial activities of macrophages by viable counting are limited by the precision of the statistics and are difficult to interpret quantitatively because of unrestrained extracellular growth of bacteria. An alternative technique based on the release of radioactive DNA from labeled bacteria has been offered as overcoming these drawbacks. To assess it for use with macrophages I have made a correlation with the conventional viable counting method using a mathematical model. Opsonized Listeria monocytogenes labeled with /sup 3/H-thymidine were exposed to rat macrophages for periods up to 4 hr. Numbers of viable bacteria determined after sonication increased exponentially in the absence of live cells and this growth rate was progressively inhibited by increasing numbers of macrophages. After a lag period of 30-60 min soluble /sup 3/H appeared in the supernatant, the amount increasing with time and numbers of macrophages. To correlate these data I developed a mathematical model that considered that changes in numbers of viable organisms were due to the difference between rates of 1) growth of extracellular bacteria and 2) killing within the macrophage. On the basis of this model curves of best fit to the viable counts data were used to predict the release of radioactivity, assuming that death of a bacterium led to the total release of its label. These predictions and the experimental data agreed well, the lag period of 30-60 min between death of the bacterium and release of radioactivity being consistent with intracellular digestion. Release of soluble radioactivity appears to be an accurate reflection of the number of bacteria killed within the macrophage.

  3. Thymidine analog methods for studies of adult neurogenesis are not equally sensitive

    PubMed Central

    Leuner, Benedetta; Glasper, Erica R.; Gould, Elizabeth

    2009-01-01

    Adult neurogenesis is often studied by labeling new cells with the thymidine analog bromodeoxyuridine (BrdU) and using immunohistochemical methods for their visualization. Using this approach, considerable variability has been reported in the number of new cells produced in the dentate gyrus of adult rodents. We examined whether immunohistochemical methods, including BrdU antibodies from different vendors (Vector, BD, Roche, Dako, Novocastra, Accurate) and DNA denaturation pretreatments, alter the quantitative and qualitative patterns of BrdU labeling. We also compared the sensitivity and specificity of BrdU with two other thymidine analogs, iododeoxyuridine (IdU) and chlorodeoxyuridine (CldU). We found that the number of BrdU-labeled cells in the dentate gyrus of adult rats was dependent on the BrdU antibody used but was unrelated to differences in antibody penetration. Even at a higher concentration, some antibodies stained fewer cells (Vector, Novocastra). A sensitive BrdU antibody (BD) was specific for dividing cells; all BrdU-labeled cells stained for Ki67, an endogenous marker of cell proliferation. We also observed that DNA denaturation pretreatments affected the number of BrdU-labeled cells and staining intensity for a marker of neuronal differentiation, NeuN. Finally, we found that IdU and CldU, when used at molarities comparable to those that label the maximal number of cells with BrdU, are less sensitive. These data suggest that antibody and thymidine analog selection, as well as the staining procedure employed, can affect the number of newly generated neurons detected in the adult brain thus providing a potential explanation for some of the variability in the adult neurogenesis literature. PMID:19731267

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

    PubMed Central

    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 specific stage in viral pathogenesis at which this enzyme is required, we constructed virus deletion mutants that were acyclovir resistant and exhibited no detectable thymidine kinase activity. After corneal inoculation of mice, the mutants replicated to high titers in the eye but were severely impaired for acute replication in trigeminal ganglia and failed to reactivate from ganglia upon cocultivation with permissive cells. Nevertheless, latency-associated transcripts were expressed in neuronal nuclei of ganglia from mutant-infected mice and superinfection of the ganglia with a second virus rescued the latent mutant virus. Thus, contrary to a widely accepted hypothesis, the thymidine kinase-negative mutants established latent infections, implying that neither thymidine kinase activity nor ganglionic replication is necessary for establishment of latency. Rather, thymidine kinase appears to be necessary for reactivation from latency. These results suggest that acyclovir-resistant viruses could establish latent infections in clinical settings and have implications for the use of genetically engineered herpesviruses to deliver foreign genes to neurons. Images PMID:2543985

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

    SciTech Connect

    Rodriguez, R.; Ritter, M.A.; Fowler, J.F.; Kinsella, T.J. )

    1994-04-30

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

  6. Mitochondrial neurogastrointestinal encephalomyopathy: novel pathogenic mutations in thymidine phosphorylase gene in two Italian brothers.

    PubMed

    Libernini, Laura; Lupis, Chiara; Mastrangelo, Mario; Carrozzo, Rosalba; Santorelli, Filippo Maria; Inghilleri, Maurizio; Leuzzi, Vincenzo

    2012-08-01

    Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE, MIM 603041) is an autosomal recessive multisystem disorder occurring due to mutations in a nuclear gene coding for the enzyme thymidine phosphorylase (TYMP). Clinical features of MNGIE include gastrointestinal dysmotility, cachexia, ptosis or ophthalmoparesis, peripheral neuropathy, diffuse leukoencephalopathy, and signs of mitochondrial dysfunction in tissues. We report the clinical and molecular findings in two brothers in whom novel TYMP gene mutations (c.215-13_215delinsGCGTGA; c.1159 + 2T > A) were associated with different clinical presentations and outcomes. PMID:22618301

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

    SciTech Connect

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

    1988-06-01

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

  8. Understanding the C3H2 cyclic-to-linear ratio in L1544

    NASA Astrophysics Data System (ADS)

    Sipilä, O.; Spezzano, S.; Caselli, P.

    2016-06-01

    Aims: We aim to understand the high cyclic-to-linear C3H2 ratio (32 ± 4) that has been observed toward L1544. Methods: We combined a gas-grain chemical model with a physical model for L1544 to simulate the column densities of cyclic and linear C3H2 observed toward L1544. The most important reactions for the formation and destruction of both forms of C3H2 were identified, and their relative rate coefficients were varied to find the best match to the observations. Results: We find that the ratio of the rate coefficients of C3H3+ + e- ➝ C3H2 + H for cyclic and linear C3H2 must be ~ 20 to reproduce the observations, depending on the branching ratios assumed for the C3H3+ + e- ➝ C3H + H2 reaction. In current astrochemical networks it is assumed that cyclic and linear C3H2 are formed in a 1:1 ratio in the aforementioned reactions. Laboratory studies and/or theoretical calculations are needed to confirm the results of our chemical modeling, which is based on observational constraints.

  9. Survey of the nob5 mutation in C3H substrains

    PubMed Central

    2015-01-01

    Purpose A no b-wave (nob) electroretinography (ERG) phenotype arose spontaneously in a colony of C3H mice and was named nob5. A mutation was identified in the Gpr179 gene in homozygous nob5 mice. There is a concern that this mutation is also present in additional C3H sublines and may compromise retinal research performed using these lines. In this report, therefore, we provide a phenotype and genotype survey of nob5 in six C3H substrains present at the Jackson Laboratory. Methods Fundus changes were evaluated in the six C3H substrains with image-guided optical coherence tomography (OCT), and retinal function was assessed with ERG. The substrains were genotyped with PCR using appropriate primers for the nob5 mutation. Additionally, the genomic sequences of C3H/HeJ, available from the Jackson Laboratory, and C3H/HeH, available from the Wellcome Trust Sanger Institute, were examined for the Gpr179nob5 mutation. Results Two C3H congenic strains, C3Sn.BLiA-Pde6b+/DnJ and C3A.BLiA-Pde6b+/J, wild-type for Pde6b, used as the sighted control strains and had normal fundi, OCT, and ERG responses. Four C3H strains C3H/HeJ, C3HeB/FeJ, C3H/HeOuJ, and C3H/HeSnJ bearing the Pde6brd1 allele exhibited a grainy fundus appearance, retinal degeneration on OCT, and no rod and cone ERG responses. The nob5 mutation was not observed in the six C3H strains assessed with PCR genotyping. Further, the genomic sequences of C3H/HeJ and C3H/HeH did not contain the nob5 mutation. Conclusions The Gpr179nob5 allele is not present in C3H substrains at the Jackson Laboratory. Therefore, the usefulness of these C3H strains as commonly used models to study the effects of photoreceptor degeneration is not compromised. PMID:26396487

  10. [3H]benzo[a]pyrene utilization in rats following tracheal implant exposure.

    PubMed

    Marchok, A C; Fleming, G S; Tomkins, B A; Griest, W H

    1989-05-31

    Open-ended rat tracheal implants (OETI) were exposed to 40 micrograms [3H]benzo[a]pyrene (B[a]P)-gelatin pellets and the 3H activity in the OETI, the host's tissues and excretia was determined 3-96 h after insertion of the pellets. The radioactivity in the OETI reached near peak activity by 3 h, and decreased almost 10-fold by 24 h. Most of the activity was associated with parent B[a]P throughout the 95 h. The 3H activity in the surrounding tissue also was mostly associated with B[a]P, but the 3H activity in the liver, kidney, blood and urine was mostly associated with water-soluble plus conjugated metabolites. In the feces, 68% of the 3H activity was in B[a]P at 3 h, but mostly organic as well as water-soluble plus conjugated metabolites were extracted from it throughout the remaining 96 h. Forty-eight hours after insertion of the B[a]P pellets, the feces contained almost 16% of the total 3H activity. Pre-exposure of the OETI to B[a]P for 4 days before insertion of the [3H]B[a]P pellets stimulated metabolism of B[a]P in the tracheas approximately 2-fold, but had no significant effect on the host tissues. PMID:2728007

  11. [3H]-tetracaine binding on rat synaptosomes and sodium channels.

    PubMed Central

    Grima, M.; Schwartz, J.; Spach, M. O.; Velly, J.

    1985-01-01

    [3H]-tetracaine binding was studied in a rat synaptosomal preparation. [3H]-tetracaine bound to a single class of binding sites with a mean KD of 188 +/- 28 nM and a mean maximal binding capacity of 13 +/- 0.7 pmol mg-1 protein. [3H]-tetracaine binding was inhibited by tetracaine, procaine and by beta-adrenoceptor blocking agents which possess local anaesthetic properties. [3H]-tetracaine binding was not modified by neurotoxins interacting specifically with the sodium channels. PMID:2413934

  12. Synthesis of thiol-reactive lipopeptide adjuvants. Incorporation into liposomes and study of their mitogenic effect on mouse splenocytes.

    PubMed

    Roth, Audrey; Espuelas, Socorro; Thumann, Christine; Frisch, Benoît; Schuber, Francis

    2004-01-01

    Synthetic analogues of triacylated and diacylated lipopeptides derived from the N-terminal domain of respectively bacterial and mycoplasmal lipoproteins are highly potent immunoadjuvants when administered either in combination with protein antigens or covalently linked to small peptide epitopes. Because of their amphipathic properties, lipopeptides, such as S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-cysteinyl-alanyl-glycine (Pam(3)CAG), can be conveniently incorporated into liposomes and serve as anchors for antigens that are linked to them. To design vaccination constructs based on synthetic peptides and liposomes as vectors. we have accordingly synthesized a series of lipopeptides that differ by the number (Pam(3)C vs Pam(2)C) and nature of the acyl chains (palmitoyl vs oleoyl) and by the presence at their C-terminus of thiol-reactive functions, such as maleimide or bromoacetyl. When incorporated into liposomes, these latter functionalized lipopeptides allow, in aqueous media, a well controlled chemoselective conjugation of HS-peptides to the surface of the vesicles. Using a BALB/c mice splenocyte proliferation assay ([(3)H]thymidine incorporation), we have measured the lymphocyte activation potency of the different lipopeptides. We found that, compared to their free (emulsified) forms, the liposomal lipopeptides were endowed with enhanced mitogenic activities; i.e., up to 2 orders of magnitude for Pam(3)CAG which was more potent than Pam(2)CAG. The impact of functionalization on the cellular activity of Pam(3)CAG was dependent on the thiol-reactive group introduced: whereas the bromoacetyl derivative retained its full activity, the presence of a maleimide group virtually abolished the lymphocyte activation of the lipopeptide. Finally, the substitution of saturated palmitoyl chains by unsaturated oleoyl chains was inhibitory. Thus, thiol-reactive Ol(3)CAG derivatives were the least active mitogens in our assay. Taken together, our findings are of

  13. Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor: ( sup 3 H)nicotine as an agonist photoaffinity label

    SciTech Connect

    Middleton, R.E.; Cohen, J.B. )

    1991-07-16

    The agonist ({sup 3}H)nicotine was used as a photoaffinity label for the acetylcholine binding sties on the Torpedo nicotinic acetylcholine receptor (AChR). ({sup 3}H)Nicotine binds at equilibrium with K{sub eq} = 0.6 {mu}M to the agonist binding sites. Irradiation with 254-nm light of AChR-rich membranes equilibrated with ({sup 3}H)nicotine resulted in covalent incorporation into the {alpha}- and {gamma}-subunits, which was inhibited by agonists and competitive antagonists but not by noncompetitive antagonists. Inhibition of labeling by d-tubocurarine demonstrated that the {alpha}-subunit was labeled via both agonist sites but the {gamma}-subunit was labeled only via the site that binds d-tubocurarine with high affinity. Chymotryptic digestion of the {alpha}-subunit confirmed that Try-198 was the principal amino acid labeled by ({sup 3}H)nicotine. This confirmation required a novel radiosequencing strategy employing o-phthalaldehyde ({sup 3}H)Nicotine, which is the first photoaffinity agonist used, labels primarily Tyr-198 in contrast to competitive antagonist affinity labels, which label primarily Tyr-190 and Cys-192/Cys-193.

  14. Thymine glycol and thymidine glycol in human and rat urine: a possible assay for oxidative DNA damage

    SciTech Connect

    Cathcart, R.; Schwiers, E.; Saul, R.L.; Ames, B.N.

    1984-09-01

    Thymine glycol is a DNA damage product of ionizing radiation and other oxidative mutagens. In an attempt to find a noninvasive assay for oxidative DNA damage in individuals, we have developed an HPLC assay for free thymine glycol and thymidine glycol in urine. Our results indicate that humans excrete about 32 nmol of the two glycols per day. Rats, which have a higher specific metabolic rate and a shorter life span, excrete about 15 times more thymine glycol plus thymidine glycol per kg of body weight than do humans. We present evidence that thymine glycol and thymidine glycol are likely to be derived from repair of oxidized DNA, rather than from alternative sources such as the diet or bacterial flora. This noninvasive assay of DNA oxidation products may allow the direct testing of current theories which relate oxidative metabolism to the processes of aging and cancer in man. 33 references, 2 figures, 3 tables.

  15. Coordinating activation strategy for C(sp(3))-H/C(sp(3))-H cross-coupling to access β-aromatic α-amino acids.

    PubMed

    Li, Kaizhi; Wu, Qian; Lan, Jingbo; You, Jingsong

    2015-01-01

    The past decade has witnessed significant advances in C-H bond functionalizations with the discovery of new mechanisms. Non-precious transition-metal-catalysed radical oxidative coupling for C(sp(3))-H bond transformations is an appealing strategy for C-C bond formations. The radical oxidative C(sp(3))-H/C(sp(3))-H cross-coupling reactions of α-C(sp(3))-H bonds of amines with free radicals represent a conceptual and practical challenge. We herein develop the coordinating activation strategy to illustrate the nickel-catalysed radical oxidative cross-coupling between C(sp(3))-H bonds and (hetero)arylmethyl free radicals. The protocol can tolerate a rich variety of α-amino acids and (hetero)arylmethanes as well as arylmethylenes and arylmethines, affording a large library of α-tertiary and α-quaternary β-aromatic α-amino acids. This process also features low-cost metal catalyst, readily handled and easily removable coordinating group, synthetic simplicity and gram-scale production, which would enable the potential for economical production at commercial scale in the future. PMID:26415985

  16. Chemical destruction of CH3I, C2H5I, 1-C3H7I, and 2-C3H7I in saltwater

    NASA Astrophysics Data System (ADS)

    Jones, Charlotte E.; Carpenter, Lucy J.

    2007-07-01

    Destruction of volatile iodocarbons in the oceans can potentially play an important role in determining the predominant chemical forms of iodine emitted to the atmosphere. Here we report chlorination and hydrolysis removal rates for CH3I, C2H5I, 1-C3H7I, and 2-C3H7I relevant to oceanic conditions. We have used these rates to calculate oceanic lifetimes for each iodocarbon with respect to total chemical destruction, as a function of seawater temperature. The resulting lifetimes are compared to typical iodocarbon oceanic residence times with respect to volatilization to the MBL. The rate of destruction of 2-C3H7I is much more rapid than chemical removal of the primary alkyl iodides, potentially explaining previous observations of lower 2-C3H7I concentrations in seawater compared to 1-C3H7I. Finally, in light of these results, we briefly discuss the potential impact of rising global seawater temperatures on oceanic iodocarbon concentrations.

  17. Comparing the Assembly and Handedness Dynamics of (H3.3-H4)2 Tetrasomes to Canonical Tetrasomes

    PubMed Central

    Vlijm, Rifka; Lee, Mina; Ordu, Orkide; Boltengagen, Anastasiya; Lusser, Alexandra; Dekker, Nynke H.; Dekker, Cees

    2015-01-01

    Eukaryotic nucleosomes consists of an (H3-H4)2 tetramer and two H2A-H2B dimers, around which 147 bp of DNA are wrapped in 1.7 left-handed helical turns. During chromatin assembly, the (H3-H4)2 tetramer binds first, forming a tetrasome that likely constitutes an important intermediate during ongoing transcription. We recently showed that (H3-H4)2 tetrasomes spontaneously switch between a left- and right-handed wrapped state of the DNA, a phenomenon that may serve to buffer changes in DNA torque induced by RNA polymerase in transcription. Within nucleosomes of actively transcribed genes, however, canonical H3 is progressively replaced by its variant H3.3. Consequently, one may ask if and how the DNA chirality dynamics of tetrasomes is altered by H3.3. Recent findings that H3.3-containing nucleosomes result in less stable and less condensed chromatin further underline the need to study the microscopic underpinnings of H3.3-containing tetrasomes and nucleosomes. Here we report real-time single-molecule studies of (H3.3-H4)2 tetrasome dynamics using Freely Orbiting Magnetic Tweezers and Electromagnetic Torque Tweezers. We find that the assembly of H3.3-containing tetrasomes and nucleosomes by the histone chaperone Nucleosome Assembly Protein 1 (NAP1) occurs in an identical manner to that of H3-containing tetrasomes and nucleosomes. Likewise, the flipping behavior of DNA handedness in tetrasomes is not impacted by the presence of H3.3. We also examine the effect of free NAP1, H3.3, and H4 in solution on flipping behavior and conclude that the probability for a tetrasome to occupy the left-handed state is only slightly enhanced by the presence of free protein. These data demonstrate that the incorporation of H3.3 does not alter the structural dynamics of tetrasomes, and hence that the preferred incorporation of this histone variant in transcriptionally active regions does not result from its enhanced ability to accommodate torsional stress, but rather may be linked to

  18. Depth distribution of bacterial production in a stratified lake with an anoxic hypolimnion

    SciTech Connect

    McDonough, R.J.; Sanders, R.W.; Porter, K.G.; Kirchman, D.L.

    1986-11-01

    The purpose of this study was to determine the depth distribution of bacterial biomass and production in a stratified lake and to test techniques to measure bacterial production in anaerobic waters. Bacterial abundance and incorporation of both (/sup 3/H)thymidine and (/sup 3/H)leucine into protein were highest in the metalimnion, at the depth at which oxygen first became unmeasurable. In contrast, (/sup 3/H)thymidine incorporation into DNA was highest in the epilimnion. Recovery of added (/sup 3/H)DNA was about 90% in waters in which the portion of (/sup 3/H)thymidine incorporation into DNA was about 40%. At least some obligate anaerobic bacteria were capable of assimilating thymidine since aeration of anaerobic hypolimnion waters substantially inhibited thymidine incorporation. The depth profile of bacterial production estimated from total thymidine and leucine incorporation and the frequency of dividing cells were all similar, with maximal rates in the metalimnion. However, estimates of bacterial production based on frequency of dividing cells and leucine incorporation were usually significantly higher than estimates based on thymidine incorporation (using conversion factors from the literature), especially in anaerobic hypolimnion waters. These data indicate that the thymidine approach must be examined carefully if it is to be applied to aquatic systems with low oxygen concentrations. The results also indicate that the interface between the aerobic epilimnion and anaerobic hypolimnion is the site of intense bacterial mineralization and biomass production which deserves further study.

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

    PubMed Central

    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

  20. Site-specific somatic mitochondrial DNA point mutations in patients with thymidine phosphorylase deficiency.

    PubMed

    Nishigaki, Yutaka; Martí, Ramon; Copeland, William C; Hirano, Michio

    2003-06-01

    Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by loss-of-function mutations in the gene encoding thymidine phosphorylase (TP). This deficiency of TP leads to increased circulating levels of thymidine (deoxythymidine, dThd) and deoxyuridine (dUrd) and has been associated with multiple deletions and depletion of mitochondrial DNA (mtDNA). Here we describe 36 point mutations in mtDNA of tissues and cultured cells from MNGIE patients. Thirty-one mtDNA point mutations (86%) were T-to-C transitions, and of these, 25 were preceded by 5'-AA sequences. In addition, we identified a single base-pair mtDNA deletion and a TT-to-AA mutation. Next-nucleotide effects and dislocation mutagenesis may contribute to the formation of these mutations. These results provide the first demonstration that alterations of nucleoside metabolism can induce multiple sequence-specific point mutations in humans. We hypothesize that, in patients with TP deficiency, increased levels of dThd and dUrd cause mitochondrial nucleotide pool imbalances, which, in turn, lead to mtDNA abnormalities including site-specific point mutations. PMID:12813027

  1. Biochemical abnormalities in a patient with thymidine phosphorylase deficiency with fatal outcome.

    PubMed

    Bakker, Jaap A; Schlesser, Patrick; Smeets, Hubert J M; Francois, Baudouin; Bierau, Jörgen

    2010-12-01

    Deficiency of the cytosolic enzyme thymidine phosphorylase (TP) causes a multisystem disorder called mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) syndrome. Clinical symptoms are gastrointestinal dysfunction, muscle involvement and neurological deterioration. TP deficiency is biochemically characterised by accumulation of thymidine and deoxyuridine in body fluids and compromised mitochondrial deoxyribose nucleic acid (mtDNA) integrity (depletion and multiple deletions). In this report we describe a patient with the clinical and biochemical features related to the end stage of the disease. Home parenteral nutrition had started to improve the clinical condition and preparations were initiated for stem cell transplantation (SCT) as a last resort treatment. Unfortunately, the patient died during the induction phase of SCT. This report shows that TP deficiency is a severe clinical condition with a broad spectrum of affected tissues. TP deficiency can be easily determined by the measurement of pyrimidine metabolites in body fluids and TP activity in peripheral blood leucocytes. Early detection and treatment may prevent the progress of the clinical symptoms and, therefore, should be considered for inclusion in newborn screening programmes. PMID:20151198

  2. Adaptation of a retrovirus as a eucaryotic vector transmitting the herpes simplex virus thymidine kinase gene

    SciTech Connect

    Tabin, C.J.; Hoffman, J.W.; Goff, S.P.; Weinberg, R.A.

    1982-04-01

    The authors investigated the feasibility of using retroviruses as vectors for transferring DNA sequences into animal cells. The thymidine kinase (tk) gene of herpes simplex virus was chosen as a convenient model. The internal BamHI fragments of a DNA clone of Moloney leukemia virus (MLV) were replaced with a purified BamHI DNA segment containing the tk gene. Chimeric genomes were created carrying the tk insert on both orientations relative to the MLV sequence. Each was transfected into TK/sup -/ cells along with MLV helper virus, and TK/sup +/ colonies were obtained by selection in the presence of hypoxanthine, aminopterin, and thymidine (HAT). Virus collected from TK/sup +/-transformed, MLV producer cells passed the TK/sup +/ phenotype to TK/sup -/ cells. Nonproducer cells were isolated, and TK/sup +/ transducing virus was subsequently rescued from them. The chimeric virus showed single-hit kinetics in infections. Virion and cellular RNA and cellular DNA from infected cells were all shown to contain sequences which hybridized to both MLV- and tk-specific probes. The sizes of these sequences were consistent with those predicted for the chimeric virus. In all respects studied, the chimeric MLV-tk virus behaved like known replication-defective retroviruses. These experiments suggest great general applicability of retroviruses as eucaryotic vectors.

  3. The kinetic mechanism of Human Thymidine Phosphorylase - a molecular target for cancer drug development.

    PubMed

    Deves, Candida; Rostirolla, Diana Carolina; Martinelli, Leonardo Kras Borges; Bizarro, Cristiano Valim; Santos, Diogenes Santiago; Basso, Luiz Augusto

    2014-03-01

    Human Thymidine Phosphorylase (HTP), also known as the platelet-derived endothelial cell growth factor (PD-ECGF) or gliostatin, catalyzes the reversible phosphorolysis of thymidine (dThd) to thymine and 2-deoxy-α-d-ribose-1-phosphate (2dR1P). HTP is a key enzyme in the pyrimidine salvage pathway involved in dThd homeostasis in cells. HTP is a target for anticancer drug development as its enzymatic activity promotes angiogenesis. Here, we describe cloning, expression, and purification to homogeneity of recombinant TYMP-encoded HTP. Peptide fingerprinting and the molecular mass value of the homogenous protein confirmed its identity as HTP assessed by mass spectrometry. Size exclusion chromatography showed that HTP is a dimer in solution. Kinetic studies revealed that HTP displayed substrate inhibition for dThd. Initial velocity and isothermal titration calorimetry (ITC) studies suggest that HTP catalysis follows a rapid-equilibrium random bi-bi kinetic mechanism. ITC measurements also showed that dThd and Pi binding are favorable processes. The pH-rate profiles indicated that maximal enzyme activity was achieved at low pH values. Functional groups with apparent pK values of 5.2 and 9.0 are involved in dThd binding and groups with pK values of 6.1 and 7.8 are involved in phosphate binding. PMID:24407036

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

    PubMed

    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. PMID:27559096

  5. Site-specific somatic mitochondrial DNA point mutations in patients with thymidine phosphorylase deficiency

    PubMed Central

    Nishigaki, Yutaka; Martí, Ramon; Copeland, William C.; Hirano, Michio

    2003-01-01

    Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by loss-of-function mutations in the gene encoding thymidine phosphorylase (TP). This deficiency of TP leads to increased circulating levels of thymidine (deoxythymidine, dThd) and deoxyuridine (dUrd) and has been associated with multiple deletions and depletion of mitochondrial DNA (mtDNA). Here we describe 36 point mutations in mtDNA of tissues and cultured cells from MNGIE patients. Thirty-one mtDNA point mutations (86%) were T-to-C transitions, and of these, 25 were preceded by 5′-AA sequences. In addition, we identified a single base-pair mtDNA deletion and a TT-to-AA mutation. Next-nucleotide effects and dislocation mutagenesis may contribute to the formation of these mutations. These results provide the first demonstration that alterations of nucleoside metabolism can induce multiple sequence-specific point mutations in humans. We hypothesize that, in patients with TP deficiency, increased levels of dThd and dUrd cause mitochondrial nucleotide pool imbalances, which, in turn, lead to mtDNA abnormalities including site-specific point mutations. PMID:12813027

  6. The role of herpes simplex virus-1 thymidine kinase alanine 168 in substrate specificity.

    PubMed

    Candice L, Willmon; Django, Sussman; Margaret E, Black

    2008-01-01

    Herpes simplex virus type 1 (HSV) thymidine kinase (TK) has been widely used in suicide gene therapy for the treatment of cancer due to its broad substrate specificity and the inability of the endogenous human TK to phosphorylate guanosine analogs such as ganciclovir (GCV). The basis of suicide gene therapy is the introduction of a gene that encodes a prodrug-activating enzyme into tumor cells. After administration, the prodrug is selectively converted to a toxic drug by the suicide gene product thereby bringing about the eradication of the cancer cells. A major drawback to this therapy is the low activity the enzyme displays towards the prodrugs, requiring high prodrug doses that result in adverse side effects. Earlier studies revealed two HSV TK variants (SR39 and mutant 30) derived by random mutagenesis with enhanced activities towards GCV in vitro and in vivo. While these mutants contain multiple amino acid substitutions, molecular modeling suggests that substitutions at alanine 168 (A168) may be responsible for the observed increase in prodrug sensitivity. To evaluate this, site-directed mutagenesis was used to individually substitute A168 with phenylalanine or tyrosine to reflect the mutations found in SR39 and mutant 30, respectively. Additionally, kinetic parameters and the ability of these mutants to sensitize tumor cells to GCV in comparison to wild-type thymidine kinase were determined. PMID:18949076

  7. Binding of (/sup 3/H)forskolin to solubilized preparations of adenylate cyclase

    SciTech Connect

    Nelson, C.A.; Seamon, K.B.

    1988-01-01

    The binding of (/sup 3/H)forskolin to proteins solubilized from bovine brain membranes was studied by precipitating proteins with polyethylene glycol and separating (/sup 3/H)forskolin bound to protein from free (/sup 3/H)forskolin by rapid filtration. The K/sub d/ for (/sup 3/H)forskolin binding to solubilized proteins was 14 nM which was similar to that for (/sup 3/H)forskolin binding sites in membranes from rat brain and human platelets. Forskolin analogs competed for (/sup 3/H)forskolin binding sites with the same rank potency in both brain membranes and in proteins solubilized from brain membranes. (/sup 3/H)forskolin bound to proteins solubilized from membranes with a Bmax of 38 fmolmg protein which increased to 94 fmolmg protein when GppNHp was included in the binding assay. In contrast, GppNHp had no effect on (/sup 3/H)forskolin binding to proteins solubilized from membranes preactivated with GppNHp. Solubilized adenylate cyclase from non-preactivated membranes had a basal activity of 130 pmolmgmin which was increased 7-fold by GppNHp. In contrast, adenylate cyclase from preactivated membranes had a basal activity of 850 pmolmgmin which was not stimulated by GppNHp or forskolin

  8. Characterization of Samples from the 3H Evaporator System Including Effects of Recycle

    SciTech Connect

    Wilmarth, W.R.

    2001-05-15

    Analysis of several series of samples from the 3H Evaporator System have been completed. The goal of this work was to determine the effects of 3H operation including recycle of concentrated supernate from Tank 30H into the sludge layer of Tank 32H.

  9. Morphine enhances the release of /sup 3/H-purines from rat brain cerebral cortical prisms

    SciTech Connect

    Wu, P.H.; Phillis, J.W.; Yuen, H.

    1982-10-01

    In vitro experiments have shown that /sup 3/H-purines can be released from /sup 3/H-adenosine preloaded rat brain cortical prisms by a KCl-evoked depolarization. The KCl-evoked release of /sup 3/H-purines is dependent on the concentration of KCl present in the superfusate. At concentrations of 10(-7) approximately 10(-5)M morphine did not influence the basal release of /sup 3/H-purines from the prisms, although it enhanced the KCl-evoked release of /sup 3/H-purines. The enhancement of KCl-evoked /sup 3/H-purine release by morphine was concentration-dependent and was antagonized by naloxone, suggesting the involvement of opiate receptors. Uptake studies with rat brain cerebral cortical synaptosomes show that morphine is a very weak inhibitor of adenosine uptake. Comparisons with dipyridamole, a potent inhibitor of adenosine uptake, suggest that this low level of inhibition of the uptake did not contribute significantly to the release of /sup 3/H-purine by morphine seen in our experiments. It is therefore suggested that morphine enhances KCl-evoked /sup 3/H-purine release by an interaction with opiate receptors and that the resultant increase in extracellular purine (adenosine) levels may account for some of the actions of morphine.

  10. Effect of Loss of Thymidine Kinase Activity on the Tumorigenicity of Clones of SV40-Transformed Hamster Cells*

    PubMed Central

    Rothschild, Henry; Black, Paul H.

    1970-01-01

    Cells deficient in the enzyme thymidine kinase were derived from transplantable SV40-transformed hamster cells. The resultant cell lines were less transplantable when inoculated into hamsters. Tumors which did arise from such cells had prolonged latent periods and were found to contain a mixture of enzyme-containing and enzyme-deficient cells. Revertant cell lines obtained either spontaneously or after mutagenesis in vitro contained intermediate levels of thymidine kinase activity and displayed an oncogenic potential which was intermediate between the wild type and enzyme-deficient cells. It is postulated that salvage pathway enzymes may play a rate-limiting role in tumorigenesis. PMID:4331716

  11. Behavior of substances labeled with /sup 3/H-proline and /sup 3/H-fucose in the cellular processes of odontoblasts and ameloblasts

    SciTech Connect

    Warshawsky, H.; Josephsen, K.

    1981-05-01

    Odontoblasts are cells with single cytoplasmic processes that grow longer as more dentin is elaborated. Ameloblasts also have single processes and it has been postulated that they too grow longer as more enamel is made. Support for this hypothesis was obtained using rat incisors to investigate the behavior of substances labeled with /sup 3/H-proline and /sup 3/H-fucose. A comparison was made between odontoblasts, which have processes known to grow and remain within the dentin, and the ameloblasts whose Tomes' processes are hypothesized to grow and leave remnants in the completed enamel. With /sup 3/H-proline, the odontoblast bodies are labeled at the early time intervals. With /sup 3/H-fucose, the cell bodies are labeled at the early intervals and the newly formed glycoproteins are deposited into the predentin. Almost immediately, these are progressively added to the dentin at the calcification front. With time a gradient of labeling extends from the unlabeled dentin toward the odontoblast bodies. Unlike the behavior of labeled proteins, by 1 and 2 days labeled glycoproteins appear along the entire length of the odontoblast processes. In the enamel, no Tomes' processes are present during maturation. With /sup 3/H-proline, reactions are adjacent to the cells and diffuse toward, but do not reach the dentino-enamel junction by 1 and 2 days. With /sup 3/H-fucose, reactions appear over the enamel near the cells. By 1 and 2 days no diffusive pattern is seen, but grains are concentrated near the dentino-enamel junction, in a region containing holes known to be the beginning of Tomes' processes. Since odontoblast glycoproteins migrate along odontoblast processes, it was postulated that cytoplasmic remnants were present in enamel along which ameloblast glycoproteins could also migrate to reach the holes at the dentino-enamel junction.

  12. Volumetric Properties of the Mixture Propenenitrile C3H3N + C3H6O2 Methyl ethanoate (VMSD1511, LB4267_V)

    NASA Astrophysics Data System (ADS)

    Cibulka, I.; Fontaine, J.-C.; Sosnkowska-Kehiaian, K.; Kehiaian, H. V.

    This document is part of Subvolume C 'Binary Liquid Systems of Nonelectrolytes III' of Volume 26 'Heats of Mixing, Vapor-Liquid Equilibrium, and Volumetric Properties of Mixtures and Solutions' of Landolt-Börnstein Group IV 'Physical Chemistry'. It contains the Chapter 'Volumetric Properties of the Mixture Propenenitrile C3H3N + C3H6O2 Methyl ethanoate (VMSD1511, LB4267_V)' providing data from direct measurement of low-pressure thermodynamic speed of sound at variable mole fraction and constant temperature, in the single-phase region(s).

  13. Volumetric Properties of the Mixture Propenenitrile C3H3N + C3H8O Propan-1-ol (VMSD1212, LB4918_V)

    NASA Astrophysics Data System (ADS)

    Cibulka, I.; Fontaine, J.-C.; Sosnkowska-Kehiaian, K.; Kehiaian, H. V.

    This document is part of Subvolume C 'Binary Liquid Systems of Nonelectrolytes III' of Volume 26 'Heats of Mixing, Vapor-Liquid Equilibrium, and Volumetric Properties of Mixtures and Solutions' of Landolt-Börnstein Group IV 'Physical Chemistry'. It contains the Chapter 'Volumetric Properties of the Mixture Propenenitrile C3H3N + C3H8O Propan-1-ol (VMSD1212, LB4918_V)' providing data by calculation of molar excess volume from low-pressure density measurements at variable mole fraction and constant temperature.

  14. Volumetric Properties of the Mixture Propenenitrile C3H3N + C3H7NO N,N-Dimethylmethanamide (VMSD1412, LB4271_V)

    NASA Astrophysics Data System (ADS)

    Cibulka, I.; Fontaine, J.-C.; Sosnkowska-Kehiaian, K.; Kehiaian, H. V.

    This document is part of Subvolume A 'Binary Liquid Systems of Nonelectrolytes I' of Volume 26 'Heats of Mixing, Vapor-Liquid Equilibrium, and Volumetric Properties of Mixtures and Solutions' of Landolt-Börnstein Group IV 'Physical Chemistry'. It contains the Chapter 'Volumetric Properties of the Mixture Propenenitrile C3H3N + C3H7NO N,N-Dimethylmethanamide (VMSD1412, LB4271_V)' providing data by calculation of isentropic compressibility from low-pressure density and thermodynamic speed of sound data at variable mole fraction and constant temperature, in the single-phase region(s).

  15. Volumetric Properties of the Mixture Propenenitrile C3H3N + C3H7NO N,N-Dimethylmethanamide (VMSD1511, LB4265_V)

    NASA Astrophysics Data System (ADS)

    Cibulka, I.; Fontaine, J.-C.; Sosnkowska-Kehiaian, K.; Kehiaian, H. V.

    This document is part of Subvolume A 'Binary Liquid Systems of Nonelectrolytes I' of Volume 26 'Heats of Mixing, Vapor-Liquid Equilibrium, and Volumetric Properties of Mixtures and Solutions' of Landolt-Börnstein Group IV 'Physical Chemistry'. It contains the Chapter 'Volumetric Properties of the Mixture Propenenitrile C3H3N + C3H7NO N,N-Dimethylmethanamide (VMSD1511, LB4265_V)' providing data from direct measurement of low-pressure thermodynamic speed of sound at variable mole fraction and constant temperature, in the single-phase region(s).

  16. Volumetric Properties of the Mixture Propenenitrile C3H3N + C3H8O Propan-1-ol (VMSD1511, LB4926_V)

    NASA Astrophysics Data System (ADS)

    Cibulka, I.; Fontaine, J.-C.; Sosnkowska-Kehiaian, K.; Kehiaian, H. V.

    This document is part of Subvolume C 'Binary Liquid Systems of Nonelectrolytes III' of Volume 26 'Heats of Mixing, Vapor-Liquid Equilibrium, and Volumetric Properties of Mixtures and Solutions' of Landolt-Börnstein Group IV 'Physical Chemistry'. It contains the Chapter 'Volumetric Properties of the Mixture Propenenitrile C3H3N + C3H8O Propan-1-ol (VMSD1511, LB4926_V)' providing data from direct measurement of low-pressure thermodynamic speed of sound at variable mole fraction and constant temperature, in the single-phase region(s).

  17. Volumetric Properties of the Mixture Propenenitrile C3H3N + C3H8O Propan-1-ol (VMSD1111, LB4910_V)

    NASA Astrophysics Data System (ADS)

    Cibulka, I.; Fontaine, J.-C.; Sosnkowska-Kehiaian, K.; Kehiaian, H. V.

    This document is part of Subvolume C 'Binary Liquid Systems of Nonelectrolytes III' of Volume 26 'Heats of Mixing, Vapor-Liquid Equilibrium, and Volumetric Properties of Mixtures and Solutions' of Landolt-Börnstein Group IV 'Physical Chemistry'. It contains the Chapter 'Volumetric Properties of the Mixture Propenenitrile C3H3N + C3H8O Propan-1-ol (VMSD1111, LB4910_V)' providing data from direct low-pressure measurement of mass density at variable mole fraction and constant temperature, in the single-phase region(s).

  18. Volumetric Properties of the Mixture Propenenitrile C3H3N + C3H6O2 Methyl ethanoate (VMSD1111, LB4251_V)

    NASA Astrophysics Data System (ADS)

    Cibulka, I.; Fontaine, J.-C.; Sosnkowska-Kehiaian, K.; Kehiaian, H. V.

    This document is part of Subvolume C 'Binary Liquid Systems of Nonelectrolytes III' of Volume 26 'Heats of Mixing, Vapor-Liquid Equilibrium, and Volumetric Properties of Mixtures and Solutions' of Landolt-Börnstein Group IV 'Physical Chemistry'. It contains the Chapter 'Volumetric Properties of the Mixture Propenenitrile C3H3N + C3H6O2 Methyl ethanoate (VMSD1111, LB4251_V)' providing data from direct low-pressure measurement of mass density at variable mole fraction and constant temperature, in the single-phase region(s).

  19. Volumetric Properties of the Mixture Propenenitrile C3H3N + C3H6O2 Methyl ethanoate (VMSD1212, LB4261_V)

    NASA Astrophysics Data System (ADS)

    Cibulka, I.; Fontaine, J.-C.; Sosnkowska-Kehiaian, K.; Kehiaian, H. V.

    This document is part of Subvolume C 'Binary Liquid Systems of Nonelectrolytes III' of Volume 26 'Heats of Mixing, Vapor-Liquid Equilibrium, and Volumetric Properties of Mixtures and Solutions' of Landolt-Börnstein Group IV 'Physical Chemistry'. It contains the Chapter 'Volumetric Properties of the Mixture Propenenitrile C3H3N + C3H6O2 Methyl ethanoate (VMSD1212, LB4261_V)' providing data by calculation of molar excess volume from low-pressure density measurements at variable mole fraction and constant temperature.

  20. Volumetric Properties of the Mixture Propenenitrile C3H3N + C3H7NO N,N-Dimethylmethanamide (VMSD1212, LB4259_V)

    NASA Astrophysics Data System (ADS)

    Cibulka, I.; Fontaine, J.-C.; Sosnkowska-Kehiaian, K.; Kehiaian, H. V.

    This document is part of Subvolume A 'Binary Liquid Systems of Nonelectrolytes I' of Volume 26 'Heats of Mixing, Vapor-Liquid Equilibrium, and Volumetric Properties of Mixtures and Solutions' of Landolt-Börnstein Group IV 'Physical Chemistry'. It contains the Chapter 'Volumetric Properties of the Mixture Propenenitrile C3H3N + C3H7NO N,N-Dimethylmethanamide (VMSD1212, LB4259_V)' providing data by calculation of molar excess volume from low-pressure density measurements at variable mole fraction and constant temperature.

  1. Volumetric Properties of the Mixture Propenenitrile C3H3N + C3H6O2 Methyl ethanoate (VMSD1412, LB4273_V)

    NASA Astrophysics Data System (ADS)

    Cibulka, I.; Fontaine, J.-C.; Sosnkowska-Kehiaian, K.; Kehiaian, H. V.

    This document is part of Subvolume C 'Binary Liquid Systems of Nonelectrolytes III' of Volume 26 'Heats of Mixing, Vapor-Liquid Equilibrium, and Volumetric Properties of Mixtures and Solutions' of Landolt-Börnstein Group IV 'Physical Chemistry'. It contains the Chapter 'Volumetric Properties of the Mixture Propenenitrile C3H3N + C3H6O2 Methyl ethanoate (VMSD1412, LB4273_V)' providing data by calculation of isentropic compressibility from low-pressure density and thermodynamic speed of sound data at variable mole fraction and constant temperature, in the single-phase region(s).

  2. Volumetric Properties of the Mixture Propenenitrile C3H3N + C3H7NO N,N-Dimethylmethanamide (VMSD1111, LB4254_V)

    NASA Astrophysics Data System (ADS)

    Cibulka, I.; Fontaine, J.-C.; Sosnkowska-Kehiaian, K.; Kehiaian, H. V.

    This document is part of Subvolume A 'Binary Liquid Systems of Nonelectrolytes I' of Volume 26 'Heats of Mixing, Vapor-Liquid Equilibrium, and Volumetric Properties of Mixtures and Solutions' of Landolt-Börnstein Group IV 'Physical Chemistry'. It contains the Chapter 'Volumetric Properties of the Mixture Propenenitrile C3H3N + C3H7NO N,N-Dimethylmethanamide (VMSD1111, LB4254_V)' providing data from direct low-pressure measurement of mass density at variable mole fraction and constant temperature, in the single-phase region(s).

  3. Pre- and post-dialysis quantitative dosage of thymidine in urine and plasma of a MNGIE patient by using HPLC-ESI-MS/MS.

    PubMed

    la Marca, G; Malvagia, S; Casetta, B; Pasquini, E; Pela, I; Hirano, M; Donati, M A; Zammarchi, E

    2006-05-01

    Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder characterized by severe gastrointestinal dysmotility, cachexia, ptosis, ophthalmoparesis, peripheral neuropathy and leukoencephalopathy. The disease is due to a thymidine phosphorylase defect. This enzyme catalyses the phosphorolysis of thymidine to thymine and deoxyribose 1-phosphate. For this reason, increased levels of thymidine in plasma and urine are found in MNGIE patients. Haemodialysis can reduce circulating plasma thymidine levels and can be beneficial in some MNGIE patients. We developed a fast analytical method based on HPLC-ESI-MS/MS capable of identifying pyrimidine nucleotides (thymine, cytosine, uracil) and nucleosides (thymidine, citidine, uridine) in plasma and urine after direct dilution of the samples without pre-treatment. In the patient studied, we observed a significant reduction of plasmatic and urinary thymidine levels during and after dialysis. However, we noted a progressive reduction of the initial thymidine level after some dialytic trials. This method will be useful not only for thymidine level follow-up during dialysis in MNGIE patients but also for the improvement of the diagnosis or diagnostic suspect in other pyrimidine defects such as dihydropyrimidine dehydrogenase deficiency, dihydropyrimidinase deficiency and ureidopropionase deficiency. PMID:16498612

  4. Localized binding of [3H]muscimol to synapses in chicken retina.

    PubMed Central

    Yazulla, S; Brecha, N

    1981-01-01

    Binding sites for [3H]muscimol, an analogue of gamma-aminobutyric acid (GABA) were localized in the synaptic layers of chicken retina by light microscopic and electron microscopic autoradiography. Light microscopic autoradiography of cryostat sections incubated in [3H]muscimol or [3H]GABA revealed identical binding patterns: a band over the inner plexiform layer (IPL) and a band over the outer plexiform layer (OPL). This binding pattern differed from the uptake pattern for [3H]GABA: labeling over horizontal, amacrine, and ganglion cell bodies as well as very intense labeling over lamina 5 in the proximal IPL. Statistical analysis of electron microscopic autoradiography data from the IPL indicated that only amacrine synapses bind [3H]muscimol (i.e., make GABAergic synapses). Processes of amacrine, bipolar, or ganglion cells can be postsynaptic to these amacrine synapses. The highest concentration of synapses binding [3H]muscimol occurred in laminae 2 and 4 of the IPL and not in lamina 5 as might be expected from the density of [3H]GABA uptake. In the OPL, [3H]muscimol binding occurred over specialized junctions proximal to photoreceptor terminals. In cone receptor terminals, [3H]muscimol binding was suspected near horizontal cell dendrite/receptor terminal membranes lateral to the synaptic ribbon, supporting the hypothesis that horizontal cells are involved in a GABAergic feedback loop with cone terminals. We conclude that the synaptic binding pattern provides a more accurate concept of GABAergic synaptic interaction than does the uptake pattern for [3H]GABA because the two patterns in the IPL are not related. Images PMID:6264454

  5. [3H]-lifarizine, a high affinity probe for inactivated sodium channels.

    PubMed Central

    MacKinnon, A. C.; Wyatt, K. M.; McGivern, J. G.; Sheridan, R. D.; Brown, C. M.

    1995-01-01

    1. [3H]-lifarizine bound saturably and reversibly to an apparently homogeneous class of high affinity sites in rat cerebrocortical membranes (Kd = 10.7 +/- 2.9 nM; Bmax = 5.10 +/- 1.43 pmol mg-1 protein). 2. The binding of [3H]-lifarizine was unaffected by sodium channel toxins binding to site 1 (tetrodotoxin), site 3 (alpha-scorpion venom) or site 5 (brevetoxin), Furthermore, lifarizine at concentrations up to 10 microM had no effect on [3H]-saxitoxin (STX) binding to toxin site 1. Lifarizine displaced [3H]-batrachotoxinin-A 20-alpha-benzoate (BTX) binding with moderate affinity (pIC50 7.31 +/- 0.24) indicating an interaction with toxin site 2. However, lifarizine accelerated the dissociation of [3H]-BTX and decreased both the affinity and density of sites labelled by [3H]-BTX, suggesting an allosteric interaction with toxin site 2. 3. The binding of [3H]-lifarizine was voltage-sensitive, binding to membranes with higher affinity than to synaptosomes (pIC50 for cold lifarizine = 7.99 +/- 0.09 in membranes and 6.68 +/- 0.14 in synaptosomes). Depolarization of synaptosomes with 130 mM KCl increased the affinity of lifarizine almost 10 fold (pIC50 = 7.86 +/- 0.25). This suggests that lifarizine binds selectively to inactivated sodium channels which predominate both in the membrane preparation and in the depolarized synaptosomal preparation. 4. There was negligible [3H]-lifarizine and [3H]-BTX binding to solubilized sodium channels, although [3H]-STX binding was retained under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7582509

  6. Tomato thymidine kinase-based suicide gene therapy for malignant glioma--an alternative for Herpes Simplex virus-1 thymidine kinase.

    PubMed

    Stedt, H; Samaranayake, H; Kurkipuro, J; Wirth, G; Christiansen, L S; Vuorio, T; Määttä, A-M; Piškur, J; Ylä-Herttuala, S

    2015-04-01

    Malignant gliomas (MGs) are the most common malignant primary brain tumors with a short life estimate accompanied by a marked reduction in the quality of life. Herpes Simplex virus-1 thymidine kinase ganciclovir (HSV-TK/GCV) system is the best characterized enzyme prodrug therapy in use. However, lipophobicity of GCV and low enzymatic activity of HSV-TK reduce the treatment efficacy. Tomato TK (ToTK) has shown high activity in combination with its specific substrate azidothymidine (AZT). The aim of this study was to evaluate whether ToTK/AZT could be used as an alternative to HSV-TK/GCV therapy. Both treatments demonstrated cytotoxicity in human MG cells in vitro. In vivo, both treatments decreased tumor growth and tumors were smaller in comparison with controls in mouse orthotopic MG model. Survival of ToTK/AZT-treated mice was significantly increased compared with control mice (*P<0.05) but not as compared with HSV-TK/GCV-treated mice. No significant differences were observed in clinical chemistry safety analyses. We conclude that both treatments showed a beneficial treatment response in comparison to controls on tumor growth and ToTK/AZT also on survival. There were no significant differences between these treatments. Therefore ToTK/AZT could be considered as an alternative treatment option for MG because of its favorable therapeutic characteristics. PMID:25613481

  7. Methodological issues in the preparation and assay of platelet 3H-imipramine binding.

    PubMed

    Severson, J A; Schneider, L S; Fredrickson, E R

    1990-07-01

    Several methodological factors in the preparation of platelets and the determination of platelet 3H-imipramine (3H-IMI) binding were examined. The ionic composition of the assay significantly affected platelet 3H-IMI binding. Approximately 25% of the specific binding of 3H-IMI to intact platelet preparations was retained in the absence of sodium and chloride ions. The addition of sodium ions enhanced the specific binding of 3H-IMI, but the addition of chloride in the presence of sodium had a more pronounced effect, enhancing binding approximately five-fold over that observed with the addition of sodium. Sodium was the only cation tested that enhanced binding. Only halides enhanced binding in the presence of sodium with the following order of potency: Cl- greater than Br- greater than I- = F-. Ions increased the density of binding sites (Bmax) and did not affect the affinity of the binding sites for 3H-IMI. In the presence of sodium and chloride, the use of serotonin (5HT) to define nonspecific binding in saturation experiments resulted in lower binding densities (Bmax) than when desipramine was used to define nonspecific binding. The component of binding that was insensitive to 5HT was roughly equal to the Bmax of 3H-IMI binding obtained in the absence of sodium and chloride using desipramine to define nonspecific binding. Overall, these data suggest that not all 3H-IMI binding that is displaced by desipramine is related to serotonergic mechanisms, and suggest that 5HT is a better choice than desipramine for the determination of the nonspecific binding of 3H-IMI. In addition, the binding of 3H-IMI to different platelet preparations was compared. The binding of 3H-IMI to intact platelets was less than that obtained using lysed platelet membranes when data were expressed per mg protein. The Coomassie Blue dye-binding method to determine platelet protein resulted in greater Bmax values than were obtained with the Folin phenol reagent method. The method of platelet

  8. A validation of the 3H/3He method for determining groundwater recharge

    NASA Astrophysics Data System (ADS)

    Solomon, D. K.; Schiff, S. L.; Poreda, R. J.; Clarke, W. B.

    1993-09-01

    Tritium and He isotopes have been measured at a site where groundwater flow is nearly vertical for a travel time of 100 years and where recharge rates are spatially variable. Because the mid-1960s 3H peak (arising from aboveground testing of thermonuclear devices) is well-defined, the vertical groundwater velocity is known with unusual accuracy at this site. Utilizing 3H and its stable daughter 3He to determine groundwater ages, we compute a recharge rate of 0.16 m/yr, which agrees to within about 5% of the value based on the depth of the 3H peak (measured both in 1986 and 1991) and two-dimensional modeling in an area of high recharge. Zero 3H/3He age occurs at a depth that is approximately equal to the average depth of the annual low water table, even though the capillary fringe extends to land surface during most of the year at the study site. In an area of low recharge (0.05 m/yr) where the 3H peak (and hence the vertical velocity) is also well-defined, the 3H/3He results could not be used to compute recharge because samples were not collected sufficiently far above the 3H peak; however, modeling indicates that the 3H/3He age gradient near the water table is an accurate measure of vertical velocities in the low-recharge area. Because 3H and 3He have different diffusion coefficients, and because the amount of mechanical mixing is different in the area of high recharge than in the low-recharge area, we have separated the dispersive effects of mechanical mixing from molecular diffusion. We estimate a longitudinal dispersivity of 0.07 m and effective diffusion coefficients for 3H (3HHO) and 3He of 2.4×10-5 and 1.3×10-4 m2/day, respectively. Although the 3H/3He age gradient is an excellent indicator of vertical groundwater velocities above the mid-1960s 3H peak, dispersive mixing and diffusive loss of 3He perturb the age gradient near and below the 3H peak.

  9. Correlation between (/sup 3/H)dopamine specific uptake and (/sup 3/H)GBR 12783 specific binding during the maturation of rat striatum

    SciTech Connect

    Bonnet, J.J.; Costentin, J.

    1989-01-01

    The development of the specific uptake of dopamine in the rat striatum during the early postnatal period is compared with the ontogenetic changes of the specific binding of (/sup 3/H)GBR 12783 to the site of uptake inhibition. During maturation, the increase in the specific binding of (/sup 3/H)GBR 12783 parallels the increase in the specific uptake of dopamine. (/sup 3/H)GBR 12783 specific binding sites increase in number from day 1 postpartum until 40 days, when they reach the adult level. In 40 day-old rats, the weight of the striatum represents 80% of adult values. The affinity of (/sup 3/H)GBR 12783 for the inhibition site is similar in membrane preparations obtained from 6 day-old pups and adults; this results in a same ability of the inhibitor to block the specific uptake of dopamine into synaptosomes obtained from pups or adult rats. These data support the hypothesis of the existence of a single molecular entity including both the inhibition site and the carrier itself.

  10. Effects of selected muscarinic cholinergic antagonists on [3H]acetylcholine release from rat hippocampal slices.

    PubMed

    Pohorecki, R; Head, R; Domino, E F

    1988-01-01

    A number of cholinergic muscarinic (M) agonists and antagonists were studied for their ability to enhance tritiated acetylcholine ([3H]ACh) release from electrically field-stimulated rat hippocampal slices. A Ca++-free medium and carbachol, but not nicotine, inhibited [3H]ACh release. Atropine, methylatropine and dexetimide produced concentration-dependent increases in [3H]ACh release to a maximum of about 50% above control. Aprophen and benactyzine produced a maximal response 25 to 35% above control. The selective M1 antagonist pirenzepine had the least effect on [3H]ACh release. Of the nonspecific M1-M2 antagonists studied, benactyzine produced the least amount of [3H]ACh release. The order of potency of the M antagonists in promoting a 15% increase in [3H]ACh release was aprophen greater than benactyzine greater than methylatropine greater than dexetimide greater than pirenzepine greater than atropine. However, the order of promoting maximal release of [3H]ACh was atropine greater than dexetimide greater than methylatropine greater than aprophen greater than benactyzine greater than pirenzepine. PMID:3335998

  11. APOBEC3H polymorphisms and susceptibility to HIV-1 infection in an Indian population.

    PubMed

    Naruse, Taeko K; Sakurai, Daisuke; Ohtani, Hitoshi; Sharma, Gaurav; Sharma, Surendra K; Vajpayee, Madhu; Mehra, Narinder K; Kaur, Gurvinder; Kimura, Akinori

    2016-03-01

    Human APOBEC3H (A3H) is a member of APOBEC cytidine deaminase family intensively constraining the HIV-1 replication. A3H is known to be polymorphic with different protein stability and anti-HIV-1 activity in vitro. We recently reported that A3H haplotypes composed of two functional polymorphisms, rs139292 (N15del) and rs139297 (G105R), were associated with the susceptibility to HIV-1 infection in Japanese. To confirm the association of A3H and HIV-1 infection in another ethnic group, a total of 241 HIV-1-infected Indian individuals and ethnic-matched 286 healthy controls were analyzed for the A3H polymorphisms. The frequency of 15del allele was high in the HIV-1-infected subjects as compared with the controls (0.477 vs 0.402, odds ratio (OR)=1.36, P=0.014). Haplotype analysis showed that the frequencies of 15del-105R was high (0.475 vs 0.400, OR=1.36, permutation P=0.037) in the HIV-1-infected subjects, confirming the association of A3H polymorphisms with the susceptibility to HIV-1 infection. PMID:26559750

  12. Quantitative autoradiography of /sup 3/H-nomifensine binding sites in rat brain

    SciTech Connect

    Scatton, B.; Dubois, A.; Dubocovich, M.L.; Zahniser, N.R.; Fage, D.

    1985-03-04

    The distribution of /sup 3/H-nomifensine binding sites in the rat brain has been studied by quantitative autoradiography. The binding of /sup 3/H-nomifensine to caudate putamen sections was saturable, specific, of a highly affinity (Kd = 56 nM) and sodium-dependent. The dopamine uptake inhibitors benztropine, nomifensine, cocaine, bupropion and amfonelic acid were the most potent competitors of /sup 3/H-nomifensine binding to striatal sections. The highest levels of (benztropine-displaceable) /sup 3/H-nomifensine binding sites were found in the caudate-putamen, the olfactory tubercle and the nucleus accumbens. 6-Hydroxy-dopamine-induced lesion of the ascending dopaminergic bundle resulted in a marked decrease in the /sup 3/H-ligand binding in these areas. Moderately high concentrations of the /sup 3/H-ligand were observed in the bed nucleus of the stria terminalis, the anteroventral thalamic nucleus, the cingulate cortex, the lateral septum, the hippocampus, the amygdala, the zona incerta and some hypothalamic nuclei. There were low levels of binding sites in the habenula, the dorsolateral geniculate body, the substantia nigra, the ventral tegmental area and the periaqueductal gray matter. These autoradiographic data are consistent with the hypothesis that /sup 3/H-nomifensine binds primarily to the presynaptic uptake site for dopamine but also labels the norepinephrine uptake site. 33 references, 2 figures, 1 table.

  13. High affinity binding of (/sup 3/H)cocaine to rat liver microsomes

    SciTech Connect

    El-Maghrabi, E.A.; Calligaro, D.O.; Eldefrawi, M.E.

    1988-01-01

    )/sup 3/H)cocaine bound reversible, with high affinity and stereospecificity to rat liver microsomes. Little binding was detected in the lysosomal, mitochondrial and nuclear fractions. The binding kinetics were slow and the kinetically calculated K/sub D/ was 2 nM. Induction of mixed function oxidases by phenobarbital did not produce significant change in (/sup 3/H)cocaine binding. On the other hand, chronic administration of cocaine reduced (/sup 3/H)cocaine binding drastically. Neither treatment affected the affinity of the liver binding protein for cocaine. Microsomes from mouse and human livers had less cocaine-binding protein and lower affinity for cocaine than those from rat liver. Binding of (/sup 3/H)cocaine to rat liver microsomes was insensitive to monovalent cations and > 10 fold less sensitive to biogenic amines than the cocaine receptor in rat striatum. However, the liver protein had higher affinity for cocaine and metabolites except for norcocaine. Amine uptake inhibitors displaced (/sup 3/H)cocaine binding to liver with a different rank order of potency than their displacement of (/sup 3/H)cocaine binding to striatum. This high affinity (/sup 3/H)cocaine binding protein in liver is not likely to be monooxygenase, but may have a role in cocaine-induced hepatotoxicity

  14. Quantitative autoradiography of (/sup 3/H)CTOP binding to mu opioid receptors in rat brain

    SciTech Connect

    Hawkins, K.N.; Knapp, R.J.; Gehlert, D.R.; Lui, G.K.; Yamamura, M.S.; Roeske, L.C.; Hruby, V.J.; Yamamura, H.I.

    1988-01-01

    (/sup 3/H)H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 ((/sup 3/H)CTOP), a potent and highly selective mu opioid antagonist, was used to localize the mu receptors in rat brain by light microscopic autoradiography. Radioligand binding studies with (/sup 3/H)CTOP using slide-mounted tissue sections of rat brain produced a Kd value of 1.1 nM with a Bmax value of 79.1 fmol/mg protein. Mu opioid agonists and antagonists inhibited (/sup 3/H)CTOP binding with high affinity (IC50 values of 0.2-2.4nM), while the delta agonist DPDPE, delta antagonist ICI 174,864, and kappa agonist U 69,593 were very weak inhibitors of (/sup 3/H)CTOP binding. Light microscopic autoradiography of (/sup 3/H)CTOP binding sites revealed regions of high density and regions of moderate labeling. The cerebral cortex showed a low density of (/sup 3/H)CTOP binding.

  15. ( sup 3 H)cytisine binding to nicotinic cholinergic receptors in brain

    SciTech Connect

    Pabreza, L.A.; Dhawan, S.; Kellar, K.J. )

    1991-01-01

    Cytisine, a ganglionic agonist, competes with high affinity for brain nicotinic cholinergic receptors labeled by any of several nicotinic {sup 3}H-agonist ligands. Here we have examined the binding of ({sup 3}H)cytisine in rat brain homogenates. ({sup 3}H)Cytisine binds with high affinity (Kd less than 1 nM), and specific binding represented 60-90% of total binding at all concentrations examined up to 15 nM. The nicotinic cholinergic agonists nicotine, acetylcholine, and carbachol compete with high affinity for ({sup 3}H)cytisine binding sites, whereas among nicotinic receptor antagonists only dihydro-beta-erythroidine competes with high affinity (in the nanomolar range). Comparison of binding in several brain regions showed that ({sup 3}H)cytisine binding is higher in the thalamus, striatum, and cortex than in the hippocampus, cerebellum, or hypothalamus. The pharmacology and brain regional distribution of ({sup 3}H)cytisine binding sites are those predicted for neuronal nicotinic receptor agonist recognition sites. The high affinity and low nonspecific binding of ({sup 3}H)cytisine should make it a very useful ligand for studying neuronal nicotinic receptors.

  16. In vivo binding of /sup 3/H-N-methylspiperone to dopamine and serotonin receptors

    SciTech Connect

    Frost, J.J.; Smith, A.C.; Kuhar, M.J.; Dannals, R.F.; Wagner, H.N. Jr.

    1987-03-09

    /sup 3/H-N-methylspiperone (/sup 3/H-NMSP) was used to label dopamine-2 and serotonin-2 in vivo in the mouse. The striatum/cerebellum binding ratio reached a maximum of 80 eight hours after intravenous administration of /sup 3/H-NMSP. The frontal cortex/cerebellum ratio was 5 one hour after injection. The binding of /sup 3/H-NMSP was saturable in the frontal cortex and cerebellum between doses of 10 and 1000 ..mu..g/kg. Between 0.01 and 10 ..mu..g/kg the ratio total/nonspecific binding increased from 14 to 21. Inhibition of /sup 3/H-NMSP binding in the frontal cortex and striatum by ketanserin, a selective serotonin-2 antagonist, demonstrated that 20% of the total binding in the striatum was to serotonin-2 rectors and 91% of the total binding in the frontal cortex was to serotonin-2 receptors. Compared to /sup 3/H-spiperone, /sup 3/H-NMSP 1) results in a much higher specific/nonspecific binding ratio in the striatum and frontal cortex and 2) displays more than a two-fold higher brain uptake. 18 references, 4 figures.

  17. Studies with the high-affinity antiestrogen, (/sup 3/H)HI285

    SciTech Connect

    Keene, J.L.

    1985-01-01

    Antiestrogens are compounds that inhibit some of the actions of estrogens. Certain antiestrogens, notably the triphenylethylene, tamoxifen, are useful in the treatment of female breast cancer. The triphenylethylene antiestrogen, HI285, was labelled with radioactive hydrogen ((/sup 3/H)) for use as a probe of antiestrogen action. Radioactive HI285 ((/sup 3/H)HI285) bound to the cytosolic estrogen receptor from both rat and calf uterus and competed with estradiol for the estrogen specific binding site. In both animals (/sup 3/H)HI285 displayed a higher affinity for the estrogen receptor and a slower dissociation rate from the receptor than did estradiol. (/sup 3/H)HI285, as well as estradiol, appeared to trigger receptor activation. Studies using the activation blocker, sodium molybdate, indicated that (/sup 3/H)HI285 triggered activation in a manner different from estradiol. The non-activated estrogen receptor from calf uterus, when occupied by (/sup 3/H)estradiol, existed as two discrete forms that could be separated by ion-exchange chromatography. In contrast, the (/sup 3/H)HI285-occupied receptor existed as a single form.

  18. Ascorbic acid enables reversible dopamine receptor /sup 3/H-agonist binding

    SciTech Connect

    Leff, S.; Sibley, D.R.; Hamblin, M.; Creese, I.

    1981-11-16

    The effects of ascorbic acid on dopaminergic /sup 3/H-agonist receptor binding were studied in membrane homogenates of bovine anterior pituitary and caudate, and rat striatum. In all tissues virtually no stereospecific binding (defined using 1uM (+)butaclamol) of the /sup 3/H-agonists N-propylnorapomorphine (NPA), apomorphine, or dopamine could be demonstrated in the absence of ascorbic acid. Although levels of total /sup 3/H-agonist binding were three to five times greater in the absence than in the presence of 0.1% ascorbic acid, the increased binding was entirely non-stereospecific. Greater amounts of dopamine-inhibitable /sup 3/H-NPA binding could be demonstrated in the absence of 0.1% ascorbic acid, but this measure of ''specific binding'' was demonstrated not to represent dopamine receptor binding since several other catecholamines and catechol were equipotent with dopamine and more potent than the dopamine agonist (+/-)amino-6,7-dihydroxy-1,2,3,4-tetrahydronapthalene (ADTN) in inhibiting this binding. High levels of dopamine-displaceable /sup 3/H-agonist binding were detected in fresh and boiled homogenates of cerebellum, an area of brain which receives no dopaminergic innervation, further demonstrating the non-specific nature of /sup 3/H-agonist binding in the absence of ascorbic acid. These studies emphasize that under typical assay conditions ascorbic acid is required in order to demonstrate reversible and specific /sup 3/H-agonist binding to dopamine receptors.

  19. Anti-retroelement Activity of APOBEC3H was Lost Twice in Recent Human Evolution

    PubMed Central

    OhAinle, Molly; Kerns, Julie A.; Li, Melody M.H.; Malik, Harmit S.

    2008-01-01

    SUMMARY The primate APOBEC3 gene locus encodes a family of proteins (APOBEC3A-H) with various antiviral and anti-retroelement activities. Here, we trace the evolution of APOBEC3H activity in hominoids to identify a human-specific loss of APOBEC3H antiviral activity. Reconstruction of the predicted ancestral human APOBEC3H protein shows that human ancestors encoded a stable form of this protein with potent antiviral activity. Subsequently, the antiviral activity of APOBEC3H was lost via two polymorphisms that are each independently sufficient to destabilize the protein. Nonetheless, an APOBEC3H allele that encodes a stably expressed protein is still maintained at high frequency, primarily in African populations. This stable APOBEC3H protein has potent activity against retroviruses and retrotransposons, including HIV and LINE-1 elements. The surprising finding that APOBEC3H antiviral activity has been lost in the majority of humans may have important consequences for our susceptibility to retroviral infections as well as ongoing retroelement proliferation in the human genome. PMID:18779051

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

    NASA Astrophysics Data System (ADS)

    Panajotovic, Radmila

    2009-05-01

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

  1. Synthesis, biological evaluation, and radioiodination of halogenated closo-carboranyl thymidine analogues1

    PubMed Central

    Tiwari, Rohit; Toppino, Antonio; Agarwal, Hitesh K.; Huo, Tianyao; Byun, Youngjoo; Gallucci, Judith; Hasabelnaby, Sherifa; Khalil, Ahmed; Goudah, Ayman; Baiocchi, Robert A.; Darby, Michael V.; Barth, Rolf. F.; Tjarks, Werner

    2012-01-01

    The synthesis and initial biological evaluation of 3-carboranyl thymidine analogues (3CTAs) that are (radio)halogenated at the closo-carborane cluster is described. Radiohalogenated 3CTAs have the potential to be used in the radiotherapy and imaging of cancer, as they may be selectively entrapped in tumor cells through monophosphorylation by human thymidine kinase 1 (hTK1). Two strategies for the synthesis of a 127I-labeled form of a specific 3CTA, previously designated as N5, are described: 1) direct iodination of N5 with iodine monochloride (ICl) and aluminum chloride (AlCl3) to obtain N5-127I and 2) initial monoiodination of o-carborane to 9-iodo-o-carborane followed by its functionalization to N5-127I. The former strategy produced N5-127I in low yields along with di-, tri-, and tetra-iodinated N5 as well as decomposition products, whereas the latter method produced only N5-127I in high yields. N5-127I was subjected to nucleophilic halogen- and isotope exchange reactions using Na79/81Br and Na125I, respectively, in the presence of Herrmann's catalyst to obtain N5-79/81Br and N5-125I, respectively. Two intermediate products formed using the second strategy, 1-(tert-butyldimethylsilyl)-9-iodo-o-carborane and 1-(tert-butyldimethylsilyl)-12-iodo-o-carborane, were subjected to x-ray diffraction studies to confirm that substitution at a single carbon atom of 9-iodo-o-carborane resulted in the formation of two structural isomers. To the best of our knowledge, this is the first report of halogen and isotope exchange reactions of B-halocarboranes that have been conjugated to a complex biomolecule. Human TK1 phosphorylation rates of N5, N5-127I, and N5-79/81Br ranged from 38.0 % to 29.6% relative to that of thymidine, the endogenous hTK1 substrate. The in vitro uptake of N5, N5-127I, and N5-79/81Br in L929 TK1 (+) cells was 2.0 ×, 1.8 ×, and 1.4 × greater than that in L929 TK1 (−) cells. PMID:22175713

  2. Autoradiographic localization of /sup 3/H-paroxetine-labeled serotonin uptake sites in rat brain

    SciTech Connect

    De Souza, E.B.; Kuyatt, B.L.

    1987-01-01

    Paroxetine is a potent and selective inhibitor of serotonin uptake into neurons. Serotonin uptake sites have been identified, localized, and quantified in rat brain by autoradiography with 3H-paroxetine; 3H-paroxetine binding in slide-mounted sections of rat forebrain was of high affinity (KD = 10 pM) and the inhibition affinity constant (Ki) values of various drugs in competing 3H-paroxetine binding significantly correlated with their reported potencies in inhibiting synaptosomal serotonin uptake. Serotonin uptake sites labeled by 3H-paroxetine were highly concentrated in the dorsal and median raphe nuclei, central gray, superficial layer of the superior colliculus, lateral septal nucleus, paraventricular nucleus of the thalamus, and the islands of Calleja. High concentrations of 3H-paroxetine binding sites were found in brainstem areas containing dopamine (substantia nigra and ventral tegmental area) and norepinephrine (locus coeruleus) cell bodies. Moderate concentrations of 3H-paroxetine binding sites were present in laminae I and IV of the frontal parietal cortex, primary olfactory cortex, olfactory tubercle, regions of the basal ganglia, septum, amygdala, thalamus, hypothalamus, hippocampus, and some brainstem areas including the interpeduncular, trigeminal, and parabrachial nuclei. Lower densities of 3H-paroxetine binding sites were found in other regions of the neocortex and very low to nonsignificant levels of binding were present in white matter tracts and in the cerebellum. Lesioning of serotonin neurons with 3,4-methylenedioxyamphetamine caused large decreases in 3H-paroxetine binding. The autoradiographic distribution of 3H-paroxetine binding sites in rat brain corresponds extremely well to the distribution of serotonin terminals and cell bodies as well as with the pharmacological sites of action of serotonin.

  3. Felbamate increases [3H]glycine binding in rat brain and sections of human postmortem brain.

    PubMed

    McCabe, R T; Sofia, R D; Layer, R T; Leiner, K A; Faull, R L; Narang, N; Wamsley, J K

    1998-08-01

    The anticonvulsant compound felbamate (2-phenyl-1,3-propanediol dicarbamate; FBM) appears to inhibit the function of the N-methyl-D-aspartate (NMDA) receptor complex through an interaction with the strychnine-insensitive glycine recognition site. Since we have demonstrated previously that FBM inhibits the binding of [3H]5, 7-dichlorokynurenic acid (DCKA), a competitive antagonist at the glycine site, we assessed the ability of FBM to modulate the binding of an agonist, [3H]glycine, to rat forebrain membranes and human brain sections. In contrast to its ability to inhibit [3H]5,7-DCKA binding, FBM increased [3H]glycine binding (20 nM; EC50 = 485 microM; Emax = 211% of control; nH = 1.8). FBM, but not carbamazepine, phenytoin, valproic acid or phenobarbital, also increased [3H]glycine binding (50 nM; EC50 = 142 microM; Emax = 157% of control; nH = 1.6) in human cortex sections. Autoradiographic analysis of human brain slices demonstrated that FBM produced the largest increases in [3H]glycine binding in the cortex, hippocampus and the parahippocampal gyrus. Because various ions can influence the binding of glycine-site ligands, we assessed their effects on FBM-modulation of [3H]glycine binding. FBM-enhanced [3H]glycine binding was attenuated by Zn++ and not inhibited by Mg++ in human brain. These results suggest that FBM increases [3H]glycine binding in a manner sensitive to ions which modulate the NMDA receptor. These data support the hypothesis that FBM produces anticonvulsant and neuroprotective effects by inhibiting NMDA receptor function, likely through an allosteric modulation of the glycine site. PMID:9694960

  4. Platelet-derived growth factor stimulated mechanisms of glucosamine incorporation

    SciTech Connect

    Harrington, M.A.; Pledger, W.J. )

    1987-10-01

    Platelet-derived growth factor (PDGF) treatment of density-arrested BALB/c-3T3 cells results in increased ({sup 3}H)glucosamine (GlcN) incorporation into cellular material. The enhanced GlcN incorporation is not due to a preferential increase in proteoglycan synthesis as measured by ({sup 35}S)H{sub 2}SO{sub 4} incorporation. Approximately 50% of the GlcN incorporated in PDGF or platelet-poor plasma (PPP)-treated cultures enters N-linked glycoproteins. Addition of dolichol-phosphate (dolichol-P), a required intermediate in N-linked glycosylation, did not alter ({sup 3}H)GlcN incorporation in PDGF-treated cells but did increase incorporation in PPP-treated cultures to a level comparable to that observed for PDGF-treated cultures. PDGF-treated cultures contained twofold greater quantities of ({sup 3}H)GlcN dolichol intermediates and lipid-free glycoprotein. Over a 12-h time course 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase) activity was similar in cultures treated with PDGF or PPP. Results of these studies reveal that enhanced protein glycosylation in response to PDGF treatment is not the result of a direct effect on HMG CoA reductase.

  5. Ligand-Promoted Borylation of C(sp(3))-H Bonds with Palladium(II) Catalysts.

    PubMed

    He, Jian; Jiang, Heng; Takise, Ryosuke; Zhu, Ru-Yi; Chen, Gang; Dai, Hui-Xiong; Dhar, T G Murali; Shi, Jun; Zhang, Hao; Cheng, Peter T W; Yu, Jin-Quan

    2016-01-11

    A quinoline-based ligand effectively promotes the palladium-catalyzed borylation of C(sp(3))-H bonds. Primary β-C(sp(3))-H bonds in carboxylic acid derivatives as well as secondary C(sp(3))-H bonds in a variety of carbocyclic rings, including cyclopropanes, cyclobutanes, cyclopentanes, cyclohexanes, and cycloheptanes, can thus be borylated. This directed borylation method complements existing iridium(I)- and rhodium(I)-catalyzed C-H borylation reactions in terms of scope and operational conditions. PMID:26611496

  6. Modeling 3H-3He Gas-Liquid Phase Transport for Interpretation of Groundwater Age

    NASA Astrophysics Data System (ADS)

    Carle, S. F.; Esser, B.; Moran, J. E.

    2009-12-01

    California’s Groundwater Ambient Monitoring and Assessment (GAMA) Program has measured many hundreds of tritium (3H) and helium-3 (3He) concentrations in well water samples to derive estimates of groundwater age at production and monitoring wells in California basins. However, a 3H-3He age differs from an ideal groundwater age tracer in several respects: (1) the radioactive decay of 3H results in the accumulation of 3He being first-order with respect to 3H activity (versus a zero-order age-mass accumulation process for an ideal tracer), (2) surface concentrations of 3H as measured in precipitation over the last several decades have not been uniform, and (3) the 3H-3He “clock” begins at the water table and not at the ground surface where 3H source measurements are made. To better understand how these non-idealities affect interpretation of 3H-3He apparent groundwater age, we are modeling coupled gas-liquid phase flow and 3H-3He transport including processes of radiogenic decay, phase equilibrium, and molecular diffusion for water, air, 3H, and 3He components continuously through the vadose zone and saturated zone. Assessment of coupled liquid-gas phase processes enables consideration of 3H-3He residence time and dispersion within the vadose zone, including partitioning of tritiogenic 3He to the gas phase and subsequent diffusion into the atmosphere. The coupled gas-liquid phase modeling framework provides direct means to compare apparent 3H-3He age to ideal mean or advective groundwater ages for the same groundwater flow conditions. Examples are given for common groundwater flow systems involving areal recharge, discharge to streams or long-screened wells, and aquifer system heterogeneity. The Groundwater Ambient Monitoring and Assessment program is sponsored by the California State Water Resources Control Board and carried out in cooperation with the U.S. Geological Survey. This work was performed under the auspices of the U.S. Department of Energy by

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

    SciTech Connect

    Allen, Bryan G.; Johnson, Monika; Marsh, Anne E.; Dornfeld, Kenneth J. . E-mail: kenneth-dornfeld@uiowa.edu

    2006-08-01

    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.

  8. Ultrafast IR pump-probe and 2D-IR photon echo spectroscopy of adenosine-thymidine base pairs

    NASA Astrophysics Data System (ADS)

    Greve, Christian; Preketes, Nicholas K.; Costard, Rene.; Koeppe, Benjamin; Fidder, Henk; Nibbering, Erik T. J.; Temps, Friedrich; Mukamel, Shaul; Elsaesser, Thomas

    2013-03-01

    We characterize diagonal and off-diagonal anharmonicities of N-H stretching vibrations in adenosine and thymidine monomers and in A•T-base pairs in chloroform solution, showing the important role of coupling between vibrationally excited N-H stretching states.

  9. Dual roles of ERK1/2 in cellular senescence induced by excess thymidine in HeLa cells.

    PubMed

    Kudo, Ikuru; Nozawa, Megumi; Miki, Kensuke; Takauji, Yuki; En, Atsuki; Fujii, Michihiko; Ayusawa, Dai

    2016-08-15

    DNA damage response is crucially involved in cellular senescence. We have previously shown that excess thymidine, which stalls DNA replication forks, induces cellular senescence in human cells, and ERK1/2 play a key role in the induction of it. In this study, we found that Chk1 and ERK1/2 were activated to promote cell survival upon addition of excess thymidine. Knockdown of ERK1/2 activated Chk1, and conversely, knockdown of Chk1 activated ERK1/2, which observations suggested a mechanism for compensatory activation of Chk1 and ERK1/2 in the absence of ERK1/2 and Chk1, respectively. We also found that Chk1 functioned mainly at the onset of cellular senescence, and on the other hand, ERK1/2 functioned for a more extended period to induce cellular senescence. Our findings suggested that Chk1 and ERK1/2 were activated to promote cell survival upon addition of excess thymidine, but prolonged activation of ERK1/2 led to cellular senescence. This implies a pleiotropic effect of ERK1/2 in cellular senescence induced by excess thymidine. PMID:27443255

  10. 5-Bromo (or chloro)-6-azido-5,6-dihydro-2' -deoxyuridine and -thymidine derivatives with potent antiviral activity.

    PubMed

    Kumar, Rakesh

    2002-02-11

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

  11. In vitro induction of non-responsiveness in cloned normal inducer T cells by antigen and purified Ia incorporated into planar membranes

    SciTech Connect

    Quill, H.; Fox, B.; Carlson, L.; Pardoll, D.; Schwartz, R.H.

    1986-03-05

    Incubation of cytochrome c-specific E/sub ..beta..//sup k/E/sub ..cap alpha..//sup k/-containing planar membranes and an antigenic peptide analogue of moth cytochrome c resulted in a specific increase in cell volume of 40-50% as measured by Coulter Counter analysis. No change in cell volume was seen in the absence of antigen, or when A/sub ..beta..//sup k/A/sub ..cap alpha..//sup k/-planar membranes were used. T cell proliferation was never detected at any time from one to eight days after incubation with E/sub ..beta..//sup k/E/sub ..cap alpha..//sup k/-membranes at a wide range of antigen concentrations. Furthermore, only trace amounts of IL-2 were detected and no increase in IL-2 receptor expression was seen. IL-3 production, however, could be detected. T cells pre-incubated for one day with E/sub ..beta..//sup k/E/sub ..cap alpha..//sup k/-membranes plus antigen became non-responsive to subsequent normal stimulation with antigen and APC. Incorporation of /sup 3/H-thymidine was reduced by more than 90% and the production of both IL-2 and IL-3 was inhibited. Non-responsiveness persisted for at least eight days after exposure to E/sub ..beta..///sup k/E/sub ..cap alpha..//sup k/-membranes plus antigen. In contrast, T cells pre-incubated under control conditions remained fully responsive. These results demonstrate the specific induction of non-responsiveness in inducer T cells by antigen and purified E/sub ..beta..//sup k/E/sub ..cap alpha..//sup k/ in planar membranes.

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

    NASA Technical Reports Server (NTRS)

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

    1972-01-01

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

  13. Expression and characterization of the thymidine kinase gene of African swine fever virus.

    PubMed Central

    Martin Hernandez, A M; Tabares, E

    1991-01-01

    The thymidine kinase (TK) gene of African swine fever virus (ASFV) was located within the viral genome by using two degenerate oligonucleotide probes derived from sequences of the vaccinia virus and cellular TK genes. The TK gene was mapped within a 0.72-kbp BglII-XhoI fragment (0.242 to 0.246 map units) derived from a 23.9-kbp SalI-B fragment of the ASFV genome. Identification of this region as the ASFV TK gene was confirmed by expression of TK in Escherichia coli and by the synthesis of active TK in a cell-free system programmed with RNA synthesized in vitro. The sequenced gene for TK includes an open reading frame of 588 nucleotides encoding a protein of 196 amino acids. The deduced amino acid sequence shows 32.4% identity with the TK of vaccinia virus. Images PMID:1987368

  14. Prevalence of thymidine-dependent Staphylococcus aureus in patients with cystic fibrosis.

    PubMed Central

    Gilligan, P H; Gage, P A; Welch, D F; Muszynski, M J; Wait, K R

    1987-01-01

    During a 1-year period, the prevalence of thymidine-dependent (TD) Staphylococcus aureus in patients at two geographically distinct cystic fibrosis (CF) centers was determined. Of 200 CF patients who had their respiratory secretions cultured, 95 harbored S. aureus, and 20 (21%) had TD S. aureus as their predominant staphylococcal isolate. All 20 TD S. aureus-positive patients had received trimethoprim-sulfamethoxazole for an average of 30.9 months. It was also observed that TD S. aureus exhibited aberrant colony morphologies or did not grow on media commonly used in CF centers for S. aureus isolation, suggesting that this organism could be missed by routine culture methods. In contrast, all 20 isolates had typical staphylococcal morphology on mannitol salt agar after 48 h of incubation. Mannitol salt agar is recommended for primary isolation of TD S. aureus. PMID:3497170

  15. Liver as a source for thymidine phosphorylase replacement in mitochondrial neurogastrointestinal encephalomyopathy.

    PubMed

    Boschetti, Elisa; D'Alessandro, Roberto; Bianco, Francesca; Carelli, Valerio; Cenacchi, Giovanna; Pinna, Antonio D; Del Gaudio, Massimo; Rinaldi, Rita; Stanghellini, Vincenzo; Pironi, Loris; Rhoden, Kerry; Tugnoli, Vitaliano; Casali, Carlo; De Giorgio, Roberto

    2014-01-01

    Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal recessive mitochondrial disease associated with mutations in the nuclear TYMP gene. As a result, the thymidine phosphorylase (TP) enzyme activity is markedly reduced leading to toxic accumulation of thymidine and therefore altered mitochondrial DNA. MNGIE is characterized by severe gastrointestinal dysmotility, neurological impairment, reduced life expectancy and poor quality of life. There are limited therapeutic options for MNGIE. In the attempt to restore TP activity, allogenic hematopoietic stem cell transplantation has been used as cellular source of TP. The results of this approach on ∼ 20 MNGIE patients showed gastrointestinal and neurological improvement, although the 5-year mortality rate is about 70%. In this study we tested whether the liver may serve as an alternative source of TP. We investigated 11 patients (7M; 35-55 years) who underwent hepatic resection for focal disorders. Margins of normal liver tissue were processed to identify, quantify and localize the TP protein by Western Blot, ELISA, and immunohistochemistry, and to evaluate TYMP mRNA expression by qPCR. Western Blot identified TP in liver with a TP/GAPDH ratio of 0.9 ± 0.5. ELISA estimated TP content as 0.5 ± 0.07 ng/μg of total protein. TP was identified in both nuclei and cytoplasm of hepatocytes and sinusoidal lining cells. Finally, TYMP mRNA was expressed in the liver. Overall, our study demonstrates that the liver is an important source of TP. Orthotopic liver transplantation may be considered as a therapeutic alternative for MNGIE patients. PMID:24802030

  16. Liver as a Source for Thymidine Phosphorylase Replacement in Mitochondrial Neurogastrointestinal Encephalomyopathy

    PubMed Central

    Boschetti, Elisa; D’Alessandro, Roberto; Bianco, Francesca; Carelli, Valerio; Cenacchi, Giovanna; Pinna, Antonio D.; Gaudio, Massimo Del; Rinaldi, Rita; Stanghellini, Vincenzo; Pironi, Loris; Rhoden, Kerry; Tugnoli, Vitaliano; Casali, Carlo; De Giorgio, Roberto

    2014-01-01

    Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal recessive mitochondrial disease associated with mutations in the nuclear TYMP gene. As a result, the thymidine phosphorylase (TP) enzyme activity is markedly reduced leading to toxic accumulation of thymidine and therefore altered mitochondrial DNA. MNGIE is characterized by severe gastrointestinal dysmotility, neurological impairment, reduced life expectancy and poor quality of life. There are limited therapeutic options for MNGIE. In the attempt to restore TP activity, allogenic hematopoietic stem cell transplantation has been used as cellular source of TP. The results of this approach on ∼20 MNGIE patients showed gastrointestinal and neurological improvement, although the 5-year mortality rate is about 70%. In this study we tested whether the liver may serve as an alternative source of TP. We investigated 11 patients (7M; 35–55 years) who underwent hepatic resection for focal disorders. Margins of normal liver tissue were processed to identify, quantify and localize the TP protein by Western Blot, ELISA, and immunohistochemistry, and to evaluate TYMP mRNA expression by qPCR. Western Blot identified TP in liver with a TP/GAPDH ratio of 0.9±0.5. ELISA estimated TP content as 0.5±0.07 ng/μg of total protein. TP was identified in both nuclei and cytoplasm of hepatocytes and sinusoidal lining cells. Finally, TYMP mRNA was expressed in the liver. Overall, our study demonstrates that the liver is an important source of TP. Orthotopic liver transplantation may be considered as a therapeutic alternative for MNGIE patients. PMID:24802030

  17. Clinical significance of thymidine kinase in Egyptian children with acute lymphoblastic leukemia

    PubMed Central

    Hagag, Adel A.; Saad, Mohamed A.; Mohamed, Sohair A.

    2015-01-01

    Background: Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy, representing one-third of pediatric cancers. Thymidine kinase-1 (TK-1) is expressed in proliferating cells so elevated TK-1 indicates active tumor growth. Objective: To study the clinical significance of TK-1 in children with ALL. Patients and Methods: This study was carried out on 40 children with newly diagnosed ALL who were admitted to Oncology Unit, Pediatric department, Tanta University (26 males and 14 females) with their ages ranged from 4 to 10 years and 30 healthy children of matched age and sex as a control group. For all patients the following were done: Complete blood picture, bone marrow examination, immunophenotyping and TK-1 serum levels. Results: Mean TK-1 level was significantly higher in patients at diagnosis than controls and significantly higher in patients with unfavorable outcome than patients with favorable outcome. Mean TK-1 level was significantly higher in patients in relapse than patients in remission and controls. No significant differences in mean TK-1 level between patients in remission and controls. There were statistically significant differences in disease free survival and overall survival between patients with favorable and unfavorable outcome. Conclusion: From this study we concluded that TK is a helpful marker in diagnosis and follow-up of patients with ALL. Recommendations: Thymidine kinase-1 should be routinely assessed at diagnosis and during follow-up in ALL patients for better diagnostic and prognostic assessment and should be taken in consideration in designing future therapeutic strategies based on patients-specific risk factors. PMID:25992345

  18. Transcriptional and posttranscriptional mechanisms regulate murine thymidine kinase gene expression in serum-stimulated cells.

    PubMed Central

    Lieberman, H B; Lin, P F; Yeh, D B; Ruddle, F H

    1988-01-01

    We previously isolated and characterized the structure of murine thymidine kinase (tk) genomic and cDNA sequences to begin a study designed to identify regions of the tk gene important for regulated expression during the transition of cells from G0 to a proliferating state. In this report, we describe the stable transfection of the cloned gene into L-M(TK-) cells and show that both thymidine kinase (TK) enzyme activity and DNA synthesis increase in parallel when transfectants in G0 arrest are stimulated by serum. To define promoter and regulatory regions more precisely, we have constructed a series of tk minigenes and have examined their expression in stable transfectants after serum stimulation. We have identified a 291-base-pair DNA fragment at the 5' end of the tk gene that has promoter function, and we have determined its sequence. In addition, we have found that DNA sequences which mediate serum-induced expression of TK are transcribed, since expression of the murine tk cDNA, fused to a promoter from either the murine tk gene, the simian virus 40 early region, or the herpes simplex virus tk gene, is stimulated by serum. Our constructs also reveal that the murine tk polyadenylation signal is not required for regulation, nor is most of the 3' untranslated region. RNA dot blot analysis indicates that murine cytoplasmic tk mRNA levels always parallel TK enzyme activity. Nuclear runon transcription assays show less than a 2-fold increase in transcription from the cloned tk gene in serum-stimulated transfectants, but an 11-fold increase in mouse L929 cells, which are inherently TK+. These results taken together suggest that the murine tk gene is controlled in serum-stimulated cells by a transcriptional mechanism influenced by DNA sequences that flank tk and also by a posttranscriptional system linked to gene sequences that are transcribed. Images PMID:3244356

  19. Angiotensin AT1 and AT2 receptor antagonists modulate nicotine-evoked [3H]dopamine and [3H]norepinephrine release

    PubMed Central

    Narayanaswami, Vidya; Somkuwar, Sucharita S.; Horton, David B.; Cassis, Lisa A.; Dwoskin, Linda P.

    2014-01-01

    Tobacco smoking is the leading preventable cause of death in the United States. A major negative health consequence of chronic smoking is hypertension. Untoward addictive and cardiovascular sequelae associated with chronic smoking are mediated by nicotine-induced activation of nicotinic receptors (nAChRs) within striatal dopaminergic and hypothalamic noradrenergic systems. Hypertension involves both brain and peripheral angiotensin systems. Activation of angiotensin type-1 receptors (AT1) release dopamine and norepinephrine. The current study determined the role of AT1 and angiotensin type-2 (AT2) receptors in mediating nicotine-evoked dopamine and norepinephrine release from striatal and hypothalamic slices, respectively. The potential involvement of nAChRs in mediating effects of AT1 antagonist losartan and AT2 antagonist, 1-[[4-(dimethylamino)-3-methylphenyl]methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid (PD123319) was evaluated by determining their affinities for α4β2* and α7* nAChRs using [3H]nicotine and [3H]methyllycaconitine binding assays, respectively. Results show that losartan concentration-dependently inhibited nicotine-evoked [3H]dopamine and [3H]norepinephrine release (IC50: 3.9±1.2 and 2.2±0.7 μM; Imax: 82±3 and 89±6%, respectively). In contrast, PD123319 did not alter nicotine-evoked norepinephrine release, and potentiated nicotine-evoked dopamine release. These results indicate that AT1 receptors modulate nicotine-evoked striatal dopamine and hypothalamic norepinephrine release. Furthermore, AT1 receptor activation appears to be counteracted by AT2 receptor activation in striatum. Losartan and PD123319 did not inhibit [3H]nicotine or [3H]methyllycaconitine binding, indicating that these AT1 and AT2 antagonists do not interact with the agonist recognition sites on α4β2* and α7* nAChRs to mediate these effects of nicotine. Thus, angiotensin receptors contribute to the effects of nicotine on

  20. Lack of [3H]quinuclidinyl benzylate binding to biologically relevant binding sites on mononuclear cells.

    PubMed

    Adams, E M; Lubrano, T M; Gordon, J; Fields, J Z

    1992-09-01

    We analyzed the binding characteristics of [3H]quinuclidinyl benzylate ([3H]QNB), a muscarinic cholinergic ligand, to rat and human mononuclear cells (MNC). Under various assay conditions, atropine-sensitive, saturable binding occurred with an apparent Kd of 10 nM. Conditions which disrupted the MNC membrane reduced total binding and eliminated specific binding. Muscarinic agonists were unable to inhibit [3H]QNB binding to MNC at concentrations up to 10(-2) M. Stereoisomers dexetimide and levetimide were equipotent inhibitors of binding (IC50 2 x 10(-5) M). We conclude that, although atropine-sensitive binding of [3H]QNB to MNC occurs, the binding is not consistent with the presence of a biologically relevant muscarinic cholinergic receptor. PMID:1392105

  1. Alpha 2 adrenergic receptors in hyperplastic human prostate: identification and characterization using (/sup 3/H) rauwolscine

    SciTech Connect

    Shapiro, E.; Lepor, H.

    1986-05-01

    (/sup 3/H)Rauwolscine ((/sup 3/H)Ra), a selective ligand for the alpha 2 adrenergic receptor, was used to identify and characterize alpha 2 adrenergic receptors in prostate glands of men with benign prostatic hyperplasia. Specific binding of (/sup 3/H)Ra to prostatic tissue homogenates was rapid and readily reversible by addition of excess unlabelled phentolamine. Scatchard analysis of saturation experiments demonstrates a single, saturable class of high affinity binding sites (Bmax = 0.31 +/- 0.04 fmol./microgram. DNA, Kd = 0.9 +/- 0.11 nM.). The relative potency of alpha adrenergic drugs (clonidine, alpha-methylnorepinephrine and prazosin) in competing for (/sup 3/H)Ra binding sites was consistent with the order predicted for an alpha 2 subtype. The role of alpha 2 adrenergic receptors in normal prostatic function and in men with bladder outlet obstruction secondary to BPH requires further investigation.

  2. Deuterated C3H2 as a clue to deuterium chemistry

    NASA Technical Reports Server (NTRS)

    Gerin, M.; Combes, F.; Wootten, H. A.; Boulanger, F.; Peters, W. L., III; Kuiper, T. B. H.

    1987-01-01

    The deuterated cyclopropenylidene ring molecule, C3HD, has been detected toward several sources in four rotational lines, at 19, 79, 104, and 107 GHz. The relative integrated intensities of the 2-sub-12 - 1-sub-01 lines of C3HD and C3H2 are found in the ratio 1:5, indicating a high deuterium fractionation ratio for cyclopropenylidene. The detection of the C-13 isotope of C3H2 at the same position allows a determination of the optical thickness (about 3) of the line. The detection of such a large enhancement in the deuterated form of C3H2 very strongly suggests that a molecular ion is the chemical precursor of the molecules. Consideration of the amount of the enhancement relative to that in other molecules suggests that the precursor ion is C3H3+.

  3. Phylogenetic distribution of (/sup 3/H)cyclohexyladenosine binding sites in nervous tissue

    SciTech Connect

    Siebenaller, J.F.; Murray, T.F.

    1986-05-29

    The specific binding of the A/sub 1/ adenosine receptor ligand. (/sup 3/H)CHA, was investigated in membrane fractions prepared from brains of eleven vertebrate species and ganglia of four invertebrate species. Substantial amounts of specific (/sup 3/H)CHA binding sites were demonstrated in brain membranes of all vertebrate species examined; however, (/sup 3/H)CHA binding sites were not detectable in nervous sites in vertebrate brains increase in higher vertebrates. Moreover, the pharmacological characteristics of the site labeled by (/sup 3/H)CHA in two divergent classes of vertebrates were similar. The broad phylogenetic distribution of A/sub 1/ adenosine receptors in primitive as well as advanced vertebrate species suggests a fundamental role for adenosine in neuronal modulation.

  4. Photodisintegration of /sup 3/H and /sup 3/He. [Threshold to 25 MeV

    SciTech Connect

    Faul, D.D.

    1980-09-01

    The photoneutron cross sections for /sup 3/H and /sup 3/He have been measured from threshold to approx. 25 MeV with monoenergetic photons from the annihilation in flight of fast positrons at the LLL Electron-Positron Linear Accelerator facility. These reactions include the two-body breakup of /sup 3/H and the three-body breakup of both /sup 3/H and /sup 3/He; these measurements for /sup 3/H are the first to span the energy region across the peaks of the cross sections. An efficient BF/sub 3/-tube-and-paraffin neutron detector and high-pressure gaseous samples of several moles each (the activity of the /sup 3/H sample was approx. 200,000 Ci) were employed in these measurements. Measurements on /sup 16/O and /sup 2/H also were performed to verify the absolute cross-section scale. The results, when compared with each other and with results for the two-body breakup cross section for /sup 3/He from the literature, show that the two-body breakup cross sections for /sup 3/H and /sup 3/He have nearly the same shape, but the one for /sup 3/He lies lower in magnitude; the three-body breakup cross section for /sup 3/He lies higher in magnitude and is broader in the peak region and also rises less sharply from threshold than that for /sup 3/H; and these measured differences between the cross sections for the breakup modes largely compensate in their sum, so that the total photon absorption cross sections for /sup 3/H and /sup 3/He are nearly the same in both size and shape at energies near and above their peaks. Theoretical results from the literature disagree with the experimental results to a certain extent over the entire photon-energy region for which the photoneutron cross sections were measured. 50 figures, 7 tables.

  5. 3H/3He age data in assessing the susceptibility of wells to contamination

    USGS Publications Warehouse

    Manning, A.H.; Solomon, D.K.; Thiros, S.A.

    2005-01-01

    Regulatory agencies are becoming increasingly interested in using young-ground water dating techniques, such as the 3H/3He method, in assessing the susceptibility of public supply wells (PSWs) to contamination. However, recent studies emphasize that ground water samples of mixed age may be the norm, particularly from long-screened PSWs, and tracer-based "apparent" ages can differ substantially from actual mean ages for mixed-age samples. We present age and contaminant data from PSWs in Salt Lake Valley, Utah, that demonstrate the utility of 3H and 3He measurements in evaluating well susceptibility, despite potential age mixing. Initial 3H concentrations (measured 3H + measured tritiogenic 3He) are compared to those expected based on the apparent 3H/3He age and the local precipitation 3H record. This comparison is used to determine the amount of modern water (recharged after ???1950) vs. prebomb water (recharged before ???1950) samples might contain. Concentrations of common contaminants were also measured using detection limits generally lower than those used for regulatory purposes. A clear correlation exists between the potential magnitude of the modern water fraction and both the occurrence and concentration of contaminants. For samples containing dominantly modern water based on their initial 3H concentrations, potential discrepancies between apparent 3H/ 3He ages and mean ages are explored using synthetic samples that are random mixtures of different modern waters. Apparent ages can exceed mean ages by up to 13 years for these samples, with an exponential age distribution resulting in the greatest discrepancies.

  6. Synthetic studies towards putative yuremamine using an iterative C(sp(3))-H arylation strategy.

    PubMed

    Calvert, Matthew B; Sperry, Jonathan

    2016-06-28

    An overview of an iterative, 8-aminoquinoline (AQ)-directed C(sp(3))-H arylation strategy towards the pyrroloindole structure initially assigned to the alkaloid yuremamine is described. During initial efforts using a model indane system, it was discovered that the iodoresorcinol unit was not a viable C(sp(3))-H arylation partner when masked as its dimethyl ether but upon switching to a MOM group, the ether oxygen served to stabilise the high valent Pd intermediate during the reaction, thus promoting reductive elimination and leading to acceptable yields of the C(sp(3))-H arylation product. The second C(sp(3))-H arylation with an iodopyrogallol gave a 1,3-diarylated model yuremamine system possessing the desired 1,3-cis relationship. When the successful model studies were applied to a pyrroloindole system in pursuit of yuremamine, it became apparent that C9 underwent competing C(sp(2))-H arylation if left vacant, but installing a tryptamine side chain at this site prevented the desired C(sp(3))-H arylation from occurring altogether. However, a C9-methyl pyrroloindole underwent iterative C(sp(3))-H arylation at C1 with an iodoresorcinol followed by C3 with an iodopyrogallol to give a diarylated product with the aryl groups in the undesired 1,3-trans-relationship, arising from epimerisation at C1 during the second C(sp(3))-H arylation event. Although the synthesis of putative yuremamine was not accomplished, several findings are disclosed that will serve as useful additions to the burgeoning field of directed C(sp(3))-H arylations and related C-H functionalization reactions. PMID:26891188

  7. Chemical integrity of ( sup 3 H)GABA used in binding studies

    SciTech Connect

    Balcar, V.J. )

    1989-07-01

    A method which is claimed to be able to determine the proportion of true GABA within radiolabeled GABA used in binding studies was tested using (3H)GABA. The method was found to be unsuitable for {sup 3}H-labeled GABA and, furthermore, both theoretical considerations and the present experimental data indicated that it could also produce misleading results with ({sup 14}C)GABA.

  8. N-(/sup 3/H)acetyl-labeling, a convenient method for radiolabeling of glycosaminoglycans

    SciTech Connect

    Hook, M.; Riesenfeld, J.; Lindahl, U.

    1982-01-15

    A method for the introduction of N-(/sup 3/H)acetyl groups into glycosaminoglycans is described. The procedure is based on (/sup 3/H)acetylation of N-unsubstituted hexosamine residues by treating the polysaccharides with (/sup 3/H)acetic anhydride. Preparations of heparin and heparin sulfate were found to contain significant numbers of N-unsubstituted hexosamine residues, as isolates. In contrast, such units could not be detected in chondroitin sulfate, dermatan sulfate, or hyaluronic acid. These polysaccharides were therefore subjected to partial N-deacetylation by reaction with hydrazine in the presence of hydrazine sulfate. After treatment with (/sup 3/H)acetic anhydride, the specific activities of the resulting labeled polysaccharide preparations ranged between 0.1 X 10/sup 6/ and 0.6 X 10/sup 6/ cpm /sup 3/H/..mu..g of uronic acid. The /sup 3/H-labeled polysaccharide preparations did not differ significantly from the corresponding unlabeled starting materials with regard to polyanion properties (chromatography on DEAE-cellulose) or polymer chain size (gel chromatography). Further, the radiolabeled polysaccharide derivatives were susceptible to specific enzymatic degradation (chondroitinase ABC and mammalian heparitinase) and retained their ability to interact specifically with certain proteins - for example, (/sup 3/H)heparin with antithrombin (/sup 3/H)hyaluronic acid oligosaccharides with chondroitin sulfate proteoglycan. These findings indicate that the labeling procedures did not induce any major structural derangement of the polysaccharide molecules. The method developed should be useful in providing labeled glycosaminoglycans for metabolic and enzymatic experiments as well as for studies on the interacion between glycosaminoglycans and other bilogical macromolecules.

  9. Purification of L-( sup 3 H) Nicotine eliminates low affinity binding

    SciTech Connect

    Romm, E.; Marks, M.J.; Collins, A.C. ); Lippiello, P.M. )

    1990-01-01

    Some studies of L-({sup 3}H) nicotine binding to rodent and human brain tissue have detected two binding sites as evidenced by nonlinear Scatchard plots. Evidence presented here indicated that the low affinity binding site is not stereospecific, is not inhibited by low concentrations of cholinergic agonists and is probably due to breakdown products of nicotine since purification of the L-({sup 3}H)nicotine eliminates the low affinity site.

  10. Ligand-Promoted C(sp(3) )-H Olefination en Route to Multi-functionalized Pyrazoles.

    PubMed

    Yang, Weibo; Ye, Shengqing; Schmidt, Yvonne; Stamos, Dean; Yu, Jin-Quan

    2016-05-17

    A Pd-catalyzed/N-heterocycle-directed C(sp(3) )-H olefination has been developed. The monoprotected amino acid ligand (MPAA) is found to significantly promote Pd-catalyzed C(sp(3) )-H olefination for the first time. Cu(OAc)2 instead of Ag(+) salts are used as the terminal oxidant. This reaction provides a useful method for the synthesis of alkylated pyrazoles. PMID:26991450

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

    SciTech Connect

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

    1988-07-22

    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.

  12. High-affinity binding of (/sup 3/H)acetylcholine to muscarinic cholinergic receptors

    SciTech Connect

    Kellar, K.J.; Martino, A.M.; Hall, D.P. Jr.; Schwartz, R.D.; Taylor, R.L.

    1985-06-01

    High-affinity binding of (/sup 3/H)acetylcholine to muscarinic cholinergic sites in rat CNS and peripheral tissues was measured in the presence of cytisin, which occupies nicotinic cholinergic receptors. The muscarinic sites were characterized with regard to binding kinetics, pharmacology, anatomical distribution, and regulation by guanyl nucleotides. These binding sites have characteristics of high-affinity muscarinic cholinergic receptors with a Kd of approximately 30 nM. Most of the muscarinic agonist and antagonist drugs tested have high affinity for the (/sup 3/H)acetylcholine binding site, but pirenzepine, an antagonist which is selective for M-1 receptors, has relatively low affinity. The ratio of high-affinity (/sup 3/H)acetylcholine binding sites to total muscarinic binding sites labeled by (/sup 3/H)quinuclidinyl benzilate varies from 9 to 90% in different tissues, with the highest ratios in the pons, medulla, and heart atrium. In the presence of guanyl nucleotides, (/sup 3/H) acetylcholine binding is decreased, but the extent of decrease varies from 40 to 90% in different tissues, with the largest decreases being found in the pons, medulla, cerebellum, and heart atrium. The results indicate that (/sup 3/H)acetylcholine binds to high-affinity M-1 and M-2 muscarinic receptors, and they suggest that most M-2 sites have high affinity for acetylcholine but that only a small fraction of M-1 sites have such high affinity.

  13. Autoradiographic localization of specific (/sup 3/H)dexamethasone binding in fetal lung

    SciTech Connect

    Beer, D.G.; Butley, M.S.; Cunha, G.R.; Malkinson, A.M.

    1984-10-01

    The cellular and subcellular localization of specific (/sup 3/H)dexamethasone binding was examined in fetal mouse lung at various stages of development and in human fetal lung at 8 weeks of gestation using a rapid in vitro steroid incubation technique followed by thaw-mount autoradiography. Competition studies with unlabeled steroids demonstrate the specificity of (/sup 3/H)dexamethasone labeling, and indicate that fetal lung mesenchyme is a primary glucocorticoid target during lung development. Autoradiographs of (/sup 3/H)dexamethasone binding in lung tissue at early stages of development demonstrate that the mesenchyme directly adjacent to the more proximal portions of the bronchiolar network is heavily labeled. In contrast, the epithelium which will later differentiate into bronchi and bronchioles, is relatively unlabeled. Distal portions of the growing epithelium, destined to become alveolar ducts and alveoli, do show nuclear localization of (/sup 3/H)dexamethasone. In addition, by utilizing a technique which allows the simultaneous examination of extracellular matrix components and (/sup 3/H)dexamethasone binding, a relationship is observed between extensive mesenchymal (/sup 3/H)dexamethasone binding and extensive extracellular matrix accumulation. Since glucocorticoids stimulate the synthesis of many extracellular matrix components, these results suggest a role for these hormones in affecting mesenchymal-epithelial interactions during lung morphogenesis.

  14. Transmission of /sup 3/H-compounds corresponding to the senescence signal in soybean

    SciTech Connect

    Nooden, L.D.; Finkelstein, D.; Wetzel, P.

    1987-04-01

    To detect compounds transmitted from the pods to the leaves, the pods of explants at various stages were injected with /sup 3/H-acetate and incubated for 24 hr. To avoid /sup 3/H contamination, the leaf blades, pods, and stem were each vented separately with air (pods, leaves) or water (stem). The leaf blades were extracted with MeOH/CHCl/sub 3//formic acid/H/sub 2/O (12:2:1:2 v/v), and after reduction to an aqueous phase, the /sup 3/H was partitioned. Most /sup 3/H entered the acid ether (50%) and aqueous (30%) phases with much less in the neutral and basic phases. The most /sup 3/H was transmitted during mid and late podfill when the pods induce senescence. When chromatographed on TLC (silica gel) with n-BuOH/HAc/H/sub 2/O) (450:112:188 v/v), the acid ether phase gave one sharp peak of /sup 3/H, while the aqueous phase produced a broad peak. Most (80%) of the former peak, which corresponded to IAA and ABA, could be resolved from these compounds by reverse phase HPLC on a C/sub 8/ column with a MeOH/gradient. Thus, some compounds are transmitted from the pods to the leaves during induction of monocarpic senescence, and at least the acid ether-soluble compounds are of limited heterogeneity.

  15. FIRST DETECTION OF c-C{sub 3}H{sub 2} IN A CIRCUMSTELLAR DISK

    SciTech Connect

    Qi Chunhua; Wilner, David J.; Rosenfeld, Katherine A.; Oeberg, Karin I.

    2013-03-01

    We report the first detection of c-C{sub 3}H{sub 2} in a circumstellar disk. The c-C{sub 3}H{sub 2} J = 6-5 line (217.882 GHz) is detected and imaged through Atacama Large Millimeter Array (ALMA) Science Verification observations toward the disk around the Herbig Ae star HD 163296 at 0.''8 resolution. The emission is consistent with that arising from a Keplerian rotating disk. Two additional c-C{sub 3}H{sub 2} transitions are also tentatively detected, bolstering the identification of this species, but with insufficient signal-to-noise ratio to constrain the spatial distribution. Using a previously developed model for the physical structure of this disk, we fit a radial power-law distribution model to the c-C{sub 3}H{sub 2} 6-5 emission and find that c-C{sub 3}H{sub 2} is present in a ring structure from an inner radius of about 30 AU to an outer radius of about 165 AU. The column density is estimated to be 10{sup 12}-10{sup 13} cm{sup -2}. The clear detection and intriguing ring structure suggest that c-C{sub 3}H{sub 2} has the potential to become a useful probe of radiation penetration in disks.

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

    PubMed

    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. PMID:24447935

  17. Imperfection works: Survival, transmission and persistence in the system of Heliothis virescens ascovirus 3h (HvAV-3h), Microplitis similis and Spodoptera exigua

    PubMed Central

    Li, Shun-Ji; Hopkins, Richard J.; Zhao, Yi-Pei; Zhang, Yun-Xuan; Hu, Jue; Chen, Xu-Yang; Xu, Zhi; Huang, Guo-Hua

    2016-01-01

    Ascoviruses are insect-specific large DNA viruses that mainly infect noctuid larvae, and are transmitted by parasitoids in the fields. Heliothis virescens ascovirus 3h (HvAV-3h) has been recently isolated from Spodoptera exigua, without parasitoid vector identified previously. Here we report that Microplitis similis, a solitary endoparasitoid wasp, could transmit HvAV-3h between S. exigua larvae in the laboratory. When the female parasitoid wasp acquired the virus and served as a vector, the period of virion viability on the ovipositor was 4.1 ± 1.4 days. Infected host larvae were still acceptable for egg laying by parasitoids, and the parasitoids thereafter transmitted virus to healthy hosts. Virus acquisition occurred only from donor hosts between 3 and 9 days post infection. The peak of virus acquisition (80.9 ± 6.3%) was found when M. similis wasps oviposited in larvae that had been inoculated with the virus 7 days previously. When virus infection of the host took place during the life cycle of the parasitoid wasp, it caused 1- to 4-day-old immature parasitoids death in the host, whilst a small proportion of 5- to 6-day-old and the majority of 7-day-old parasitoids larvae survived from the virus-infected hosts. Viral contamination did not reduce the life span or fecundity of female M. similis. PMID:26878829

  18. Imperfection works: Survival, transmission and persistence in the system of Heliothis virescens ascovirus 3h (HvAV-3h), Microplitis similis and Spodoptera exigua.

    PubMed

    Li, Shun-Ji; Hopkins, Richard J; Zhao, Yi-Pei; Zhang, Yun-Xuan; Hu, Jue; Chen, Xu-Yang; Xu, Zhi; Huang, Guo-Hua

    2016-01-01

    Ascoviruses are insect-specific large DNA viruses that mainly infect noctuid larvae, and are transmitted by parasitoids in the fields. Heliothis virescens ascovirus 3h (HvAV-3h) has been recently isolated from Spodoptera exigua, without parasitoid vector identified previously. Here we report that Microplitis similis, a solitary endoparasitoid wasp, could transmit HvAV-3h between S. exigua larvae in the laboratory. When the female parasitoid wasp acquired the virus and served as a vector, the period of virion viability on the ovipositor was 4.1 ± 1.4 days. Infected host larvae were still acceptable for egg laying by parasitoids, and the parasitoids thereafter transmitted virus to healthy hosts. Virus acquisition occurred only from donor hosts between 3 and 9 days post infection. The peak of virus acquisition (80.9 ± 6.3%) was found when M. similis wasps oviposited in larvae that had been inoculated with the virus 7 days previously. When virus infection of the host took place during the life cycle of the parasitoid wasp, it caused 1- to 4-day-old immature parasitoids death in the host, whilst a small proportion of 5- to 6-day-old and the majority of 7-day-old parasitoids larvae survived from the virus-infected hosts. Viral contamination did not reduce the life span or fecundity of female M. similis. PMID:26878829

  19. A validation of the [sup 3]H/[sup 3]He method for determining groundwater recharge

    SciTech Connect

    Solomon, D.K. ); Schiff, S.L. ); Poreda, R.J. ); Clarke, W.B. )

    1993-09-01

    Tritium and He isotopes have been measured at a site where groundwater flow is nearly vertical for a travel time of 100 years and where recharge rates are spatially variable. Because the mid-1960s [sup 3]H peak (arising from aboveground testing of thermonuclear devices) is well-defined, the vertical groundwater velocity is known with unusual accuracy at this site. Utilizing [sup 3]H and its stable daughter [sup 3]He to determine groundwater ages, we compute a recharge rate of 0.16 m/yr, which agrees to within about 5% of the value based on the depth of the [sup 3]H peak (measured both in 1986 and 1991) and two-dimensional modeling in an area of high recharge. Zero [sup 3]H/[sup 3]He age occurs at a depth that is approximately equal to the average depth of the annual low water table, even though the capillary fringe extends to land surface during most of the year at the study site. In an area of low recharge (0.05 m/yr) where the [sup 3]H peak (and hence the vertical velocity) is also well-defined, the [sup 3]H/[sup 3]He results could not be used to compute recharge because samples were not collected sufficiently far above the [sup 3]H peak; however, modeling indicates that the [sup 3]H/[sup 3]He age gradient near the water table is an accurate measure of vertical velocities in the low-recharge area. Because [sup 3]H and [sup 3]He have different diffusion coefficients, and because the amount of mechanical mixing is different in the area of high recharge than in the low-recharge area, we have separated the dispersive effects of mechanical mixing from molecular diffusion. We estimate a longitudinal dispersivity of 0.07 m and effective diffusion coefficients for [sup 3]H ([sup 3]HHO) and [sup 3]He of 2.4 x 10[sup [minus]5] and 1.3 x 10[sup [minus]4] m[sup 2]/day, respectively. 26 refs., 8 figs., 1 tab.

  20. Uptake of /sup 3/H prednisolone by fetal lung explants: role of intercellular contacts in epithelial maturation

    SciTech Connect

    Leung, C.K.; Adamson, I.Y.; Bowden, D.H.

    1980-06-01

    Epithelial maturation in the developing lung is accelerated by glucocorticoids and it has been suggested that a fibroblast-derived factor may be important in regulating this response. In the present study, uptake of /sup 3/H prednisolone by cultured explants of fetal rat lung is used to correlate cellular location of the steroid with changes in epithelial--mesenchymal cell relationships at different stages of development. At days 17 and 18 of gestation, there is substantial incorporation of steroid by epithelial and interstitial cells with no morphologic evidence of accelerated maturation. At days 19 and 20, uptake of prednisolone is maximal with preferential incorporation by Type 2 cells which now contain an increased number of lamellar bodies. In the bronchioles, steroid uptake is observed in Clara cells but not in ciliated cells. Lung development results in a progressively closer anatomic relationship between alveolar epithelial and interstitial cells and at the time of maximal steroid response, day 19-20, narrow ''gap junctions'' are observed between these cell types. The results suggest that, although steroid is bound to several cell types from an early stage of development, the effects on epithelial maturation occur relatively late in gestation when close epithelial-mesenchymal cell contacts are made.

  1. Whole-body autoradiographic localization of (/sup 3/H)phencyclidine and its metabolites in mice

    SciTech Connect

    Fand, I.; McNally, W.P.; Koul, O.; Yonekura, Y.; Som, P.; Brill, A.B.; Deutsch, D.G.

    1988-05-01

    When evaluated by whole-body autoradiography (WBAR) and quantitative densitometry, (3H)phencyclidine (PCP) equivalents were found to be removed rapidly from blood, after a single iv dose in mice, and avidly taken up as early as 1 min after dosage by glandular tissues including thyroid, salivary glands, pancreas, pituitary and, most prominently, by stomach mucosa. Stomach:blood (3H)PCP concentration ratios showed that rapid secretion of (3H)PCP from mucosa to the stomach contents occurred within 2 min after dosing. During early intervals, chromatographic analysis of tissue sections demonstrated that PCP was present in brain, liver, and gut primarily in its unaltered chemical form. Mice killed at 60 and 120 min showed persistently high levels of (3H)PCP equivalents within the stomach and intestines, these levels being the highest of all other tissues densitometrically measured. The early time course and magnitude of (3H)PCP uptake by stomach glandular mucosa strongly suggests that cycling of PCP occurs principally through gastroenteric recirculation. Very striking was the high concentration of (3H)PCP radioactivity observed within the adrenal as early as 5 min. The concentration of (3H)PCP equivalents in pituitary, choroid plexus, cortex, hippocampus, and thalamus was highest at 1-20 min following injection. Application of high-resolution quantitative WBAR was found to be a useful tool in the study of the biodistribution of labeled PCP, especially during very early post-treatment time points where alternative tissue counting techniques would not be feasible.

  2. Characterization and regulation of (/sup 3/H)-serotonin uptake and release in rodent spinal

    SciTech Connect

    Stauderman, K.A.

    1986-01-01

    The uptake and release of (/sup 3/H)-serotonin were investigated in rat spinal cord synaptosomes. In the uptake experiments, sodium-dependent and sodium-independent (/sup 3/H)-serotonin accumulation processes were found. Sodium-dependent (/sup 3/H)-serotonin accumulation was: linear with sodium concentrations up to 180 mM; decreased by disruption of membrane integrity or ionic gradients; associated with purified synaptosomal fractions; and reduced after description of descending serotonergic neurons in the spinal cord. Of the uptake inhibitors tested, the most potent was fluoxetine (IC/sub 50/ 75 nM), followed by desipramine (IC/sub 50/ 430 nM) and nomifensine (IC/sub 50/ 950 nM). The sodium-independent (/sup 3/H)-serotonin accumulation process was insensitive to most treatments and probably represents nonspecific membrane binding. Thus, only sodium-dependent (/sup 3/H)-serotonin uptake represents the uptake process of serotonergic nerve terminals in rat spinal cord homogenates. In the release experiments, K/sup +/-induced release of previously accumulated (/sup 3/H)-serotonin was Ca/sup 2 +/-dependent, and originated from serotonergic synaptosomes. Exogenous serotonin and 5-methyoxy-N,N-dimethyltryptamine inhibited (/sup 3/H)-serotonin release in a concentration-dependent way. Of the antagonists tested, only methiothepin effectively blocked the effect of serotonin. These data support the existence of presynaptic serotonin autoreceptors on serotonergic nerve terminals in the rat spinal cord that act to inhibit a voltage and Ca/sup 2 +/-sensitive process linked to serotonin release. Alteration of spinai cord serotonergic function may therefore be possible by drugs acting on presynaptic serotonin autoreceptors in the spinal cord.

  3. Tissue distribution of sup 3 H-nicotine in rats after bolus or constant injection

    SciTech Connect

    Chowdhury, P.; Pasley, J.N.; Rayford, P.L. )

    1989-01-01

    Two groups of rats, (N = 7), were fasted for 24 hrs prior to the study. On the day of the experiment, the animals were anesthetized and infused with either 5 ml nicotine solution (200 {mu}g/L) in saline containing 5 {mu}c {sup 3}H-nicotine, (sp. activity 50-80 mCi/mol) for 90 minutes or injected as a bolus with 0.5 ml of the same nicotine (200 {mu}g/L) solution. The animals were sacrificed 60 minutes after the injection or after the infusion was stopped. Blood and tissue samples were counted by liquid scintillation counting. Percent distribution of {sup 3}H-nicotine per gm of tissue was calculated from the total radioactivity recovered in individual tissues over the total activity injected into the rat and the values were compared using student's t test. Results: Distribution of {sup 3}H-nicotine was found highest in kidney (45-49%) among all tissues examined and was not different between routes of administration. Significantly higher retention of {sup 3}H-nicotine was found with continuous infusion in esophagus, fundus, antrum, spleen, cecum, pancreas, testes, heart and muscle when {sup 3}H-nicotine retentions were compared with bolus injection. In contrast, the distribution of {sup 3}H-nicotine in adrenal gland, was significantly lower in continuous infusion group. Distribution in blood was 6 fold higher in continuous infusion (7.26%) compared to bolus (1.11%) injection. The distribution {sup 3}H-nicotine in other tissues were not different by either routes of injection.

  4. Depletion of (/sup 3/H)methyltrienolone cytosol binding in glucocorticoid-induced muscle atrophy (42001)

    SciTech Connect

    Kurowski, T.T.; Capaccio, J.A.; Chatterton, R.T. Jr.; Hickson, R.C.

    1985-01-01

    The present study was undertaken to determine cytosol binding properties of (/sup 3/H)methyltrienolone, a synthetic androgen, in comparison with (/sup 3/)dexamethasone, a synthetic glucocorticoid, under conditions of glucocorticoid excess in skeletal muscle. Male hypophysectomized rats received either seven daily subcutaneous injections of cortisone acetate (CA) (100 mg x kg/sup -1/ body wt) or the vehicle, 1% carboxymethyl cellulose. Following treatment, both (/sup 3/H)dexamethansone and (/sup 3/H)methyltrienolone-receptor concentrations were decreased from those in vehicle-treated rats by more than 90 and 80%, respectively, in CA-treated animals. Scatchard analysis of (/sup 3/H)methyltrienolone binding in muscles of vehicle-treated animals became nonlinear at high concentrations of labeled ligand and were reanalyzed by a two-component binding model. The lower affinity, higher capacity component, which was attributed to binding of methyltrienolone to a dexamethasone component, which was attributed to binding of methyltrienolone to a dexamethasone component, disappeared in muscles of CA-treated rats and Scatchard plots were linear. Receptor concentrations of the higher affinity lower capacity methyltrienolone component were similar in muscles of vehicle-treated and CA-treated groups. From competition studies, the high relative specificities of glucocorticoids for (/sup 3/H)methyltrienolone binding in muscles of vehicle-treated animals were markedly reduced by CA treatment. In addition, the binding specificity data also showed strong competition by progesterone and methyltrienolone for (/sup 3/H)dexamethasone binding and estradiol-17..beta.. for (/sup 3/H)methyltrienolone binding.

  5. Characterizing a sewage plume using the 3H-3He dating technique

    USGS Publications Warehouse

    Shapiro, Stephanie Dunkle; LeBlanc, Denis; Schlosser, Peter; Ludin, Andrea

    1999-01-01

    An extensive 3H-3He study was performed to determine detailed characteristics of a regional flow system and a sewage plume over a distance of 4 km in a sand and gravel aquifer at Otis Air Base in Falmouth, Massachusetts. 3H-3He ages increase with depth in individual piezometer clusters and with distance along flowpaths. However, the age gradient with depth (Δt/Δz) is smaller in the plume than that in the regional waters, due to the intense recharge in the infiltration beds. The 1960s bomb peak of tritium in precipitation is archived longitudinally along a flowline through the main axis of the plume and vertically in individual piezometer clusters. On the eastern side of the sampling area, where water from Ashumet Pond forces plume water deeper into the flow system, 3H-3He ages are young at depth because the 3H-3He "clock" is reset due to outgassing of helium in the pond. A reconstruction of the tritium input functions for the regional and plume samples shows that there is no offset in the peak [3H]+[3Hetrit] concentrations for the plume and regional water, indicating that the water from supply wells for use on the base is young. The 3H-3He ages and detergent concentrations in individual wells are consistent with the beginning of use of detergents and the time period when their concentrations in sewage would have been greatest. Ages and hydraulic properties calculated using the 3H-3He data compare well with those from previous investigations and from particle-tracking simulations.

  6. Distributions of /sup 35/S-sulfate and /sup 3/H-glucosamine in the angular region of the hamster: light and electron microscopic autoradiography

    SciTech Connect

    Ohnishi, Y.; Taniguchi, Y.

    1983-06-01

    The distribution of /sup 35/S-sulfate and /sup 3/H-glucosamine in the angular region of the hamster was studied by light and electron microscopic autoradiography following intraperitoneal injection of these compounds to hamsters. Exposed silver grains of /sup 35/S-sulfate were concentrated in the trabecular meshwork, sclera, and cornea, and grains of /sup 3/H-glucosamine were localized in the trabecular region. The radioactivity of both isotopes was observed in the Golgi apparatuses of the endothelial cells of the angular aqueous plexus and the trabecular meshwork. The grains were noted over the entire cytoplasm, except for the nucleus, and then were incorporated into the amorphous substance and collagen fibers in the region adjacent to the angular aqueous sinus. These results suggest that endothelial cells in the angular region synthesize and secrete the sulfated glycosaminoglycans and hyaluronic acid.

  7. (/sup 3/H)-SK and F 101926, a novel radiolabeled vasopressin antagonist

    SciTech Connect

    Stassen, F.L.; Heckman, D.; Schmidt, D.; Landvatter, S.; Crooke, S.T.

    1986-05-01

    Vasopressin receptor binding studies have been carried out with radiolabeled agonists. They have labeled SK and F 101926 (desGlyd(CH2)5D-Tyr(Et)VAVP), a potent antagonist of vascular (V1) and renal (V2) vasopressin receptors, with (/sup 3/H)-Phe (37 Ci/mmol). They studied V1 receptors of cultured smooth muscle cells of rat aorta (A-10) and liver, and V2 receptors of pig kidney. (/sup 3/H)-SK and F 101926 binding to plasma membranes of A-10 cells was specific (non-specific binding with 10 ..mu..M AVP), saturable, and of high affinity. At 0.4nM, the specific binding was 50%. A linear Scatchard plot indicated one antagonist affinity (KD = 0.4nM; Bmax = 100-150 fmol/10/sup 6/ cells). In contrast, the Scatchard plot of (/sup 3/H)-AVP binding was curvilinear. Specific (/sup 3/H)-SK and F 101926 binding was inhibited by AVP and vasopressin antagonists d(CH2)5Tyr(Me)AVP > d(CH2)5DTyr(Et)VAVP > d(CH2)5Tyr(Et)VAVP > d(CH2)5D-IleVAVP. The rank orders of the antagonists for vasopressin receptors of rat liver and A-10 cells determined with (/sup 3/H)-SK and F 101926 and (/sup 3/H)-AVP were the same. GppNHp did not affect (/sup 3/H)-SK and F 101926 binding. In competition experiments with cell and liver membranes, GppNHp decreased the affinity of AVP but not of the antagonist d(CH2)5Tyr(Me)AVP. (/sup 3/H)-SK and F 101926 also appeared to bind specifically to crude membranes of pig kidney medulla. In conclusion, the antagonist (/sup 3/H)-SK and F 101926 binds specifically and with high affinity to vasopressin receptors and is, thus, a powerful new tool to study vasopressin receptors.

  8. An analysis of [3H]gamma-aminobutyric acid (GABA) binding in the human brain.

    PubMed

    Lloyd, K G; Dreksler, S

    1979-03-01

    The binding of [3H]GABA to membranes prepared from human brains obtained post morten was examined. This binding was independent of patient sex, age (16--80 years), postmortem interval (4--33 h) or storage time when frozen (0-64 months). In preparations from cerebellar cortex various compounds displaced [3H]GABA binding with the following order of potency: muscimol greater than 3-aminopropanesulfonic acid greater than GABA greater than imidazoleacet acid greater than delta-amino-n-valeric acid greater than 3-hydroxyGABA greater than bicuculline. Other compounds active 'in vitro' included strychnine, homocarnosine and some (e.g. clozapine, thioridazine, pimozide) but not all (chlorpromazine, haloperiodol) neuroleptics. Compounds inactive 'in vitro' included aminooxyacetic acid, baclofen, picrotoxin, anticholinergics, metrazole, anticonvulsants and naloxone. Triton X-100 augmented the [3H]GABA binding (25 nM) by about 10--20-fold in most brain regions. [3H]GABA binding (IC50) was altered in Huntington's chorea and Reye's syndrome, but not in schizophrenics (4-neuroleptic-treated patients) or sudden infant death syndrome. The data presented strongly support the proposal that the measurement of [3H]GABA binding in postmortem human brain offers a reflection of the state of the physiologically relevant GABA receptor. PMID:218679

  9. (4B-3H) NADH-H2O exchange reaction of the mitochondrial NADH dehydrogenase

    SciTech Connect

    Chen, S.; Guillory, R.J.

    1985-06-14

    The purified mitochondrial NADH dehydrogenase enzyme has been shown to catalyze a rapid (4B-/sup 3/H) NADH-H/sub 2/O exchange reaction. When the enzyme is subjected to a single freeze-thaw cycle there is a complete loss of NADH dehydrogenation without a measurable decrease in the (4B-/sup 3/H) NADH-H/sub 2/O exchange. Complete loss of the (4B-/sup 3/H) NADH-H/sub 2/O exchange follows brief exposure to ultraviolet photoirradiation. The differential sensitivity of the water exchange reaction and the dehydrogenase activity suggests a direct involvement of the enzymes flavin cofactor in the catalysis of the (4B-/sup 3/H) NADH-H/sub 2/O exchange. Arylazido-beta-alanyl NAD+ (A3'-0-(3-(N-4-azido-2-nitrophenyl)amino) propionyl)NAD+) is shown to be a potent photodependent inhibitor of the (4B-3H) NADH-H/sub 2/O exchange activity following photoirradiation with visible light. This is consistent with the observed photodependent inhibition of the dehydrogenase activity by this photoprobe.

  10. Characterization of ( sup 3 H)alprazolam binding to central benzodiazepine receptors

    SciTech Connect

    McCabe, R.T.; Mahan, D.R.; Smith, R.B.; Wamsley, J.K. )

    1990-10-01

    The binding of the triazolobenzodiazepine ({sup 3}H)alprazolam was studied to characterize the in vitro interactions with benzodiazepine receptors in membrane preparations of rat brain. Studies using nonequilibrium and equilibrium binding conditions for ({sup 3}H)alprazolam resulted in high specific to nonspecific (signal to noise) binding ratios. The binding of ({sup 3}H)alprazolam was saturable and specific with a low nanomolar affinity for benzodiazepine receptors in the rat brain. The Kd was 4.6 nM and the Bmax was 2.6 pmol/mg protein. GABA enhanced ({sup 3}H)alprazolam binding while several benzodiazepine receptor ligands were competitive inhibitors of this drug. Compounds that bind to other receptor sites had a very weak or negligible effect on ({sup 3}H)alprazolam binding. Alprazolam, an agent used as an anxiolytic and in the treatment of depression, acts in vitro as a selective and specific ligand for benzodiazepine receptors in the rat brain. The biochemical binding profile does not appear to account for the unique therapeutic properties which distinguish this compound from the other benzodiazepines in its class.

  11. Lack of specific (/sup 3/H) prazosin binding sites in dog and rabbit cerebral arteries

    SciTech Connect

    Ferron, P.M.; Banner, W. Jr.; Duckles, S.P.

    1984-11-19

    In order to explore the characteristics of alpha adrenergic receptors on cerebrovascular smooth muscle, specific binding sites for the alpha/sub 1/ adrenergic ligand, (/sup 3/H) prazosin, were studied in blood vessel homogenates. No specific (/sup 3/H) prazosin binding was found in either rabbit or dog cerebral arteries, but specific binding was demonstrated in the rabbit saphenous and ear arteries. In the ear artery /sup 3/H-prazosin binding was saturable with a K/sub d/ of 0.51 +/- 0.20 nM and a Bmax of 89 +/- 29 fmoles/mg protein. To confirm the adequacy of our membrane preparation, homogenates of both dog and rabbit cerebral arteries showed saturable specific binding with two different ligands: one for muscarinic receptors, (/sup 3/H)(-) quinuclidinyl benzilate (QNB) and one for alpha/sub 2/ adrenergic receptors, (/sup 3/H) yohimbine. The results of these studies demonstrate a lack of alpha/sub 1/ adrenergic receptors on cerebral blood vessels, confirming functional studies showing only a weak contractile response to norepinephrine. 29 references, 3 figures, 2 tables.

  12. Enkephalin convertase: Characterization and localization with ( sup 3 H)-guanidinoethylmercap-tosuccinic acid

    SciTech Connect

    Lynch, D.R.

    1988-01-01

    Enkephalin convertase (EC) has been characterized by the binding of its selective inhibitor ({sup 3}H)-guanidinoethylmercaptosuccinic acid (GEMSA). The pharmacology and affinity of ({sup 3}H)-GEMSA binding match the pharmacology of EC activity and the inhibition of EC activity by GEMSA. EC activity and ({sup 3}H)-GEMSA binding activity copurify to homogeneity demonstrating that ({sup 3}H)-GEMSA binds selectively to EC. The selective association of ({sup 3}H)-GEMSA for EC allows localization of membrane bound EC by in vitro autoradiography. EC is heterogeneously distributed in the rat brain with the highest levels in the outer zone of the median eminence and in the hypothalamic magnocellular nuclei. In the pituitary gland, autoradiography localizes EC to all three lobes with the highest levels in the intermediate lobe. In the adrenal EC is found exclusively in the medulla. In the gastrointestinal tract, EC is localized to epithelial surfaces where its function cannot be that of propeptide processing. In the heart EC is localized to atrium where it likely processes precursors of atrial natriuretic factor.

  13. Characteristics of central binding sites for ( sup 3 H) DAMGO in spontaneously hypertensive rats

    SciTech Connect

    Gulati, A.; Bhargava, H.N. )

    1990-01-01

    The binding of ({sup 3}H) DAMGO, a highly selective ligand for {mu}-opiate receptors, to membranes of discrete brain regions and spinal cord of 10 week old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats were determined. The brain regions examined were hypothalamus, amygdala, hippocampus, corpus striatum, pons and medulla, midbrain and cortex. ({sup 3}H) DAMGO bound to membranes of brain regions and spinal cord at a single high affinity site. The receptor density (B{sub max} value) and apparent dissociation constant (K{sub d} value) of ({sup 3}H) DAMGO to bind to membranes of hippocampus, corpus striatum, pons and medulla, cortex and spinal cord of WKY and SHR rats did not differ. The B{sub max} value of ({sup 3}H) DAMGO in membranes of hypothalamus and midbrain of SHR rats was significantly higher than in WKY rats but the K{sub d} values in the two strains did not differ. On the other hand, the B{sub max} value of ({sup 3}H) DAMGO in membranes of amygdala of SHR rats was lower than that of WKY rats but the K{sub d} values in the two strains were similar.

  14. Platelet 3H-imipramine binding sites in obsessive-compulsive behavior.

    PubMed

    Kim, S W; Dysken, M W; Pandey, G N; Davis, J M

    1991-09-01

    Several studies indicate a serotonergic dysfunction in patients with obsessive-compulsive disorder (OCD). We examined serotonergic function in OCD by determining platelet 3H-impiramine binding sites in patients with OCD during a drug-free baseline period as well as normal control volunteers. The maximum number of binding sites (Bmax) and apparent dissociation constant (Kd) was determined using 3H-imipramine (IMI) as the binding ligand. We observed that the mean 3H-IMI binding Bmax (fmol/mg protein) determined in 24 patients with OCD was not significantly different from that in 23 normal control subjects. There were no significant differences in the Kd between patients with OCD and normal control subjects. Our results are thus similar to those reported by Insel et al (1985) and Black et al (1990), who observed no significant differences in platelet 3H-IMI binding between OCD patients and controls; but different from those reported by Weizmann et al (1986), who observed decreased 3H-IMI Bmax in OCD patients. The discrepancy in the results is not clear, but may be related to several factors. Our results thus indicate that any abnormality in serotonergic function present in patients with OCD is not related to imipramine binding sites in the platelets. However, the possibility that there may be an abnormal platelet serotonin uptake or other serotonergic function in OCD cannot be ruled out. PMID:1657222

  15. Potassium activation of [3H]-choline accumulation by isolated sympathetic ganglia of the rat.

    PubMed Central

    Higgins, A. J.; Neal, M. J.

    1982-01-01

    1 The effect of K-depolarization on the uptake of low and high concentrations of [3H]-choline by isolated superior sympathetic ganglia of the rat has been studied. 2 In unstimulated ganglia, the uptake of [3H]-choline (0.1 microM) ('high affinity uptake') was unaffected by denervation or by hemicholinium-3 (HC-3), suggesting uptake by structures other than cholinergic nerve terminals. 3 K-depolarization of the ganglia increased [3H]-choline accumulation by the high affinity uptake process but in contrast the 'low affinity' accumulation of [3H]-choline (100 microM) was decreased. 4 The K-activated, 'high affinity' component of choline uptake was highly sodium-dependent, inhibited by HC-3, and was abolished by denervation. 5 In incubation conditions designed to prevent transmitter release (Ca-free medium and high-Mg medium), the K-activated uptake of [3H]-choline was abolished. 6 It is concluded that in unstimulated ganglia, there is little choline uptake by nerve terminals. However, when the terminals are depolarized, choline uptake is increased by the activation of a sodium-dependent, HC-3-sensitive transport process. The activation of this uptake process is apparently associated with the release of acetylcholine from the terminals, or by changes in ionic fluxes, and not by the depolarization per se. PMID:7150866

  16. Nuclear magnetic resonance studies of cis-syn, trans-syn, and 6-4 photodimers of thymidylyl(3'-5')thymidine monophosphate and cis-syn photodimers of thymidylyl(3'-5')thymidine cyanoethyl phosphotriester

    SciTech Connect

    Kan, L.; Voituriez, L.; Cadet, J.

    1988-07-26

    Three out of four possible photodimers of thymidylyl(3'-5')thymidine monophosphates (i.e., cis-syn, 6-4, and one of the trans-syn) and two structural isomers (i.e., R and S forms) of cis-syn-thymidylyl(3'-5')thymidine cyanoethyl phosphotriester have been isolated and purified from the reaction mixtures after UV irradiation and studied by multinuclear magnetic resonance spectroscopy. All five inter thymine base linked photodimers have grossly similar structures which are quite different from those of the parent thymidylyl(3'-5')thymidine. The base of Tp- is in the syn conformation, and that of -pT is in the anti conformation. The sugar puckering of Tp- is dominated by the /sup 2/E conformer, but in -pT it is in /sup 4/E; except for the conformer around the C/sub 5/'-O/sub 5/' bond, the 6-4 isomer is very similar to those of cis-syn and trans-syn conformation. As expected, there are sugar-phosphate backbone distortions in the phosphotriesters, due to the neutralization of the negative charge of the phosphate. In general the structures of all five photodimers are very close to those of the cis-syn photodimer of thymidylyl(3'-5')thymidine monophosphate cyanoethyl ester as studied by X-ray diffraction. While the trans-syn photodimer has two structural isomers, only one (C/sub 6/(of Tp-)-R) was produced by the UV irradiation and studied.

  17. Effect of urokinase on the proliferation of primary cultures of human prostatic cells

    SciTech Connect

    Kirchheimer, J.C.; Wojta, J.; Hienert, G.; Christ, G.; Heger, M.E.; Pflueger, H.B.; Binder, B.R.

    1987-11-01

    The effects of exogenously added urokinase type plasminogen activator, tissue type plasminogen activator, plasmin and thrombin on the proliferation of primary cultures of cells derived from prostatic hyperplasia or prostatic carcinomas were investigated by measuring the incorporation of /sup 3/H-thymidine into the cultures. Addition of urokinase type plasminogen activator (1.35 x 10(-9) M) or thrombin (10(-7) M) to the culture medium caused a two-fold increase of /sup 3/H-thymidine incorporation, regardless of the origin of the prostatic cells. Tissue type plasminogen activator did not alter the rate of /sup 3/H-thymidine incorporation, whereas plasmin caused a 25% decrease of /sup 3/H-thymidine incorporation in all cultures.

  18. Incorporated of tritiated water in fish

    SciTech Connect

    Sprous, D.G.; Fox, J.E.; Jackson, B.A.

    1989-01-01

    Tritiated water {sup 3}H{sub 2}O is routinely discharged into the environment near nuclear power plants and reactors. The radioactive water is rapidly equilibriated with cell water in the aquatic life forms. The purpose of this study was to determine the uptake of the radioactive hydrogen into the various lipid classes. Oak Ridge National Laboratory has been discharging tritiated water into White Oak Lake. Blue gill and mosquito fish from White Oak Lake were analyzed. An experimental fish tank with water having a specific activity of 1.2 {times} 10{sup 6} dpm of {sup 3}H/mL was set up. Mosquito fish were exposed to this level of radioactivity for thirty days. After this time the fish were lypolyzed an the lipids were extracted. The phospholipid fraction incorporated the greatest percentage of the radioactivity. Significant incorporation of activity was also seen in the triglyceride and cholesterol fractions. Phospholipids and cholesterol are important structural components of the cell, insuring persistence of the radioactivity in the organism. The long term effects are not known.

  19. Penetration of ( sup 3 H)T-2 mycotoxin through abraded and intact skin and methods to decontaminate ( sup 3 H)T-2 mycotoxin from abrasions

    SciTech Connect

    Solberg, V.B.; Broski, F.H.; Dinterman, R.E.; George, D.T.

    1990-01-01

    T-2 mycotoxin is a toxic metabolite of various fungi of the Fusarium species. T-2 is found naturally in moldy grain and concentrations as high as 2 ppm have been found in moldy corn. T-2 purportedly has been used as a biological warfare agent in Southeast Asia and Iran, causing human deaths and has been implicated in dermal diseases in grain handling workers. Radiolabeled T-2 has been shown to penetrate excised animal and human skin in liquid vehicles and while adsorbed onto corn dust. In experimental animals studies, (3H)T-2 penetration through skin caused symptoms ranging from erythema and skin lesions to death.

  20. An enzyme-linked immunosorbent assay for bromodeoxyuridine incorporation using fixed microcultures

    SciTech Connect

    Muir, D.; Varon, S.; Manthorpe, M. )

    1990-03-01

    We report a quantitative method by which a single microculture can be examined for cell morphology; cell number; DNA synthesis; and expression of cell antigens. This method first involves measuring by enzyme-linked immunosorbent assay (ELISA) the total bromodeoxyuridine (BrdU) incorporation into DNA by monolayer microcultures. The BrdU-ELISA measurement was followed by simultaneous immunostaining for BrdU-positive nuclei and for a cytoplasmic antigen. The method was applied to the measurement of mitogen-induced proliferation of rat sciatic nerve Schwann cell and cerebral astroglia microcultures. The ELISA measurement of BrdU incorporation compares favorably with measurements of tritiated thymidine incorporation and offers the additional advantages that the same microculture can subsequently be examined for cell number, for cell morphology, and for the percentage of cells having BrdU-labeled nuclei and other antigens.

  1. (/sup 3/H)muscimol binding sites increased in autopsied brains of chronic schizophrenics

    SciTech Connect

    Hanada, S.; Mita, T.; Nishino, N.; Tanaka, C.

    1987-01-19

    (/sup 3/H)muscimol binding and glutamic acid decarboxylase (GAD) activity in the prefrontal cortex and caudate nucleus of autopsied brains from 19 chronic schizophrenics and 17 control subjects were investigated. In the schizophrenics, saturation analysis with varying concentrations of (/sup 3/H)muscimol revealed an increase in the number GABA/sub A/ receptors, but there was no significant difference in the affinity. In addition, the enhancement of (/sup 3/H)muscimol binding by diazepam was significantly greater in schizophrenics than in controls. GAD activity did not differ between controls and schizophrenics. The possibility that GABAergic mechanisms might play a role in case of chronic schizophrenia should be given further attention.

  2. Characterization of central alpha-adrenoceptors using /sup 3/H-clonidine and its derivatives

    SciTech Connect

    Jarrott, B.; Louis, W.J.; Summers, R.J.

    1983-02-01

    alpha-Adrenoceptors in brain can be studied readily by radioligand binding techniques. This provides valuable information not only on the distribution of receptors in brain regions, but also on the regulation of receptors. The usefulness of this technique is dependent in part on a radioligand with high specificity for the receptor under study. Researchers' studies have shown that /sup 3/H-clonidine does not bind exclusively to alpha 2-adrenoceptor subtypes, but also interacts with alpha 1-adrenoceptors. In contrast, /sup 3/H-guanfacine labels a high affinity alpha 2 subtype with good selectivity, but /sup 3/H-lofexidine probably labels with both alpha 2 and alpha 1-adrenoceptor binding sites.

  3. Synthesis and characterization of [N-methyl-3H]loperamide.

    PubMed

    Filer, Crist N; Egan, Judith A; Nugent, Richard P

    2014-05-30

    Loperamide is a piperidine butyramide mu-opiate receptor agonist and currently employed to treat diarrhea. Because a single past report of tritiating loperamide was limited to only a very low specific activity product without technical details or extensive analysis, the synthesis of [N-methyl-(3)H]loperamide at high specific activity is now described in detail. An imine precursor was alkylated with [(3)H]methyl iodide to obtain a quaternary intermediate, which was then reacted with 4-(4-chlorophenyl)-4-hydroxypiperidine to afford the desired product [N-methyl-(3)H]loperamide, characterized by thin layer chromatography (TLC), HPLC, MS, UV, and proton-decoupled tritium NMR. PMID:24753311

  4. Direct Acylation of C(sp(3))-H Bonds Enabled by Nickel and Photoredox Catalysis.

    PubMed

    Joe, Candice L; Doyle, Abigail G

    2016-03-14

    Using nickel and photoredox catalysis, the direct functionalization of C(sp(3))-H bonds of N-aryl amines by acyl electrophiles is described. The method affords a diverse range of α-amino ketones at room temperature and is amenable to late-stage coupling of complex and biologically relevant groups. C(sp(3))-H activation occurs by photoredox-mediated oxidation to generate α-amino radicals which are intercepted by nickel in catalytic C(sp(3))-C coupling. The merger of these two modes of catalysis leverages nickel's unique properties in alkyl cross-coupling while avoiding limitations commonly associated with transition-metal-mediated C(sp(3))-H activation, including requirements for chelating directing groups and high reaction temperatures. PMID:26890705

  5. Autoradiographic localization of adenosine uptake sites in rat brain using (/sup 3/H)nitrobenzylthioinosine

    SciTech Connect

    Bisserbe, J.C.; Patel, J.; Marangos, P.J.

    1985-02-01

    The adenosine uptake site has been localized in rat brain by an in vitro light microscopic autoradiographic method, using (/sup 3/H)nitrobenzylthioinosine ((/sup 3/H)NBI) as the probe. The binding characteristics of (/sup 3/H)NBI on slide-mounted sections are comparable to those seen in studies performed on brain homogenates. A very high density of uptake sites occurs in the nucleus tractus solitarius, in the superficial layer of the superior colliculus, in several thalamic nuclei, and also in geniculate body nuclei. A high density of sites are also observed in the nucleus accumbens, the caudate putamen, the dorsal tegmentum area, the substantia nigra, and the central gray. The localization of the adenosine uptake site in brain may provide information on the functional activity of the site and suggests the involvement of the adenosine system in the central regulation of cardiovascular function.

  6. Synthesis of 3,4-cis-[3H]leucocyanidin and enzymatic reduction to catechin.

    PubMed

    Tanner, G J; Kristiansen, K N

    1993-03-01

    A novel method is presented for the synthesis and purification of (+)-2,3-trans-3,4-cis-[4-3H]leucocyanidin. Soluble enzyme extracts from developing barley grains and leaves of the forage legume Onobrychis viciifolia (sainfoin) catalyzed the NAD(P)H-dependent reduction of (+)-2,3-trans-3,4-cis-[4-3H]leucocyanidin to (+)-[4-3H]catechin. NADPH was the preferred substrate. With extracts of barley the rate of reaction with 1 mM NADH was 20% of the rate found with NADPH. With extracts from both tissues there was a broad pH optimum around pH 6.6. PMID:8470799

  7. Amino­silanes derived from 1H-benzimidazole-2(3H)-thione

    PubMed Central

    Palomo-Molina, Juliana; García-Báez, Efrén V.; Contreras, Rosalinda; Pineda-Urbina, Kayim; Ramos-Organillo, Angel

    2015-01-01

    Two new mol­ecular structures, namely 1,3-bis­(tri­methyl­silyl)-1H-benzimidazole-2(3H)-thione, C13H22N2SSi2, (2), and 1-tri­methyl­silyl-1H-benzimidazole-2(3H)-thione, C10H14N2SSi, (3), are reported. Both systems were derived from 1H-benzimidazole-2(3H)-thione. Noncovalent C—H⋯π inter­actions between the centroid of the benzmidazole system and the SiMe3 groups form helicoidal arrangements in (2). Dimerization of (3) results in the formation of R 2 2(8) rings via N—H⋯S inter­actions, along with parallel π–π inter­actions between imidazole and benzene rings. PMID:26322611

  8. Blood epididymal barrier to (/sup 3/H)-inulin in intact and vasectomized hamsters

    SciTech Connect

    Turner, T.T.; D'Addario, D.A.; Howards, S.S.

    1981-09-01

    The net transport of (/sup 3/H)-inulin into the fluids of the hamster seminiferous and caput, corpus, and cauda epididymal tubules was examined in both intact animals and those vasectomized 10 months previously. Mean isotope concentrations in reproductive tract tubule fluids did not exceeded 10 per cent of blood plasma isotope concentrations during the experiment. There were no significant differences in net transport of (/sup 3/H)-inulin into any of the tubule fluids sampled. Ten months after vasectomy, the seminiferous tubule, and all regions of the epididymal tubule retain the capacity to exclude (/sup 3/H)-insulin. Thus in the hamster 10 months after vasectomy, the blood testis and blood epididymal barriers to inulin are intact.

  9. Effect of membrane protein concentration on binding of /sup 3/H-imipramine in human platelets

    SciTech Connect

    Barkai, A.I.; Kowalik, S.; Baron, M.

    1985-02-01

    Binding of /sup 3/H-imipramine to platelet membranes has been implicated as a marker for depression. Comparing /sup 3/H-IMI binding between depressed patients and normal subjects we observed an increase in the dissociation constant Kd with increasing membrane protein. This phenomenon was studied more rigorously in five normal subjects. Platelet membranes were prepared and adjusted to four concentrations of protein ranging from 100 to 800 micrograms/ml. The /sup 3/H-IMI binding parameters of maximum binding sites number (Bmax) and Kd were obtained by Scatchard analysis at each membrane concentration. A positive linear relationship was found between K/sub d/ values and the concentration of membrane protein in the assay, but no change was observed in Bmax. The variability in Kd values reported in the literature may be accounted for in part by the different concentrations of membrane protein used in various studies.

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

    SciTech Connect

    Holzer, Georg W. . E-mail: falknef@baxter.com

    2005-07-05

    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. Production of inositol trisphosphates upon. cap alpha. -adrenergic stimulation in BC3H-1 muscle cells

    SciTech Connect

    Ambler, S.K.; Thompson, B.; Brown, J.H.; Taylor, P.

    1986-05-01

    Activation of ..cap alpha../sub 1/-adrenergic receptors in BC3H-1 muscle cells rapidly mobilizes intracellular and results in a paradoxically slower accumulation of inositol trisphosphate. A possible explanation for this discrepancy may be provided by the recent findings of Irvine et al. of additional Ins P3 isomers besides the Ca/sup + +/-mobilizing isomer, Ins 1,4,5-P3. They have eluted and separated the inositol phosphates of BC3H-1 cells with an NH/sub 4//sup +/ x HCO/sub 2//sup -//H/sub 3/PO/sub 4/ gradient on a Whatman Partisil 10SAX column using Hewlett-Packard HPLC. Commercial (/sup 3/H)Ins 1,4,5-P3 and (/sup 3/H)inositol phosphates from carbachol-stimulated parotid glands were used as standards. Little or no Ins 1,3,4-P3 could be detected in control or phenylephrine-treated BC3H-1 cells. Ins 1,4,5-P3 followed the pattern of agonist stimulation observed previously. As a positive control, Ins P3 isomers were also measured in 1321N1 astrocytoma cells. Muscarinic stimulation of 1321N1 cells results in both the rapid accumulation of Ins P3 and Ca/sup + +/ mobilization. There is no detectable basal Ins 1,3,4-P3, but carbachol stimulates a rapid production of this compound in 1321N1 cells. Agonist activation also results in a rapid increase in Ins 1,4,5-P3 above basal values. These studies indicate that Ins 1,3,4-P3 does not contribute to the InsP3 signal in BC3H-1 cells and multiple mechanisms may exist for the coupling of receptors to PI turnover.

  12. Distribution of 3H-GABA uptake sites in the nematode Ascaris

    SciTech Connect

    Guastella, J.; Stretton, A.O. )

    1991-05-22

    The distribution of uptake sites for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in the nematode Ascaris suum was examined by autoradiography of 3H-GABA uptake. Single neural processes in both the ventral and dorsal nerve cords were labeled with 3H-GABA. Serial section analysis identified the cells of origin of these processes as the RMEV-like and RMED-like neurons. These cells belong to a set of four neurons in the nerve ring, all of which are labeled by 3H-GABA. 3H-GABA labeling of at least two other sets of cephalic neurons was seen. One of these pairs consists of medium-sized lateral ganglia neurons, located at the level of the amphid commissure bundle. A second pair is located in the lateral ganglia at the level of the deirid commissure bundle. The position and size of these lateral ganglia cells suggest that they are the GABA-immunoreactive lateral ganglia cells frequently seen in whole-mount immunocytochemical preparations. Four neuronal cell bodies located in the retrovesicular ganglion were also labeled with 3H-GABA. These cells, which are probably cholinergic excitatory motor neurons, do not contain detectable GABA-like immunoreactivity. Heavy labeling of muscle cells was also observed. The ventral and dorsal nerve cord inhibitory motor neurons, which are known to contain GABA-like immunoreactivity, were not labeled above background with 3H-GABA. Together with the experiments reported previously, these results define three classes of GABA-associated neurons in Ascaris: (1) neurons that contain endogenous GABA and possess a GABA uptake system; (2) neurons that contain endogenous GABA, but that either lack a GABA uptake system or possess a GABA uptake system of low activity; (3) neurons that possess a GABA uptake system, but that lack endogenous GABA.

  13. Interactions of ( sup 3 H)amphetamine with rat brain synaptosomes. II. Active transport

    SciTech Connect

    Zaczek, R.; Culp, S.; De Souza, E.B. )

    1991-05-01

    The accumulation of 5 nM d-({sup 3}H)amphetamine (d-({sup 3}H)AMPH) into rat brain synaptosomes was examined using physiological buffer conditions. The accumulation of d-({sup 3}H)AMPH into striatal synaptosomes was saturable, of high affinity, ouabain-sensitive and temperature-dependent, suggesting an active transport phenomenon. Eadee-Hofstee analysis of striatal d-({sup 3}H)AMPH transport (AMT) saturation isotherms indicated an apparent Km of 97 nM and a Vmax of 3.0 fmol/mg tissue/min. Lesion of the striatal dopaminergic innervation led to equivalent decreases of ({sup 3}H) dopamine (DA) transport and AMT, indicating that AMT occurs in DA terminals. Furthermore, AMT was not evident in cerebral cortex, a brain region with a paucity of DA terminals. In competition studies, AMT was stereospecific; d-AMPH (IC50 = 60 nM) was an 8-fold more potent inhibitor of the transport than its I-isomer (IC50 = 466 nM). DA(IC50 = 257 nM), DA uptake blockers and substrates were found to be potent inhibitors of AMT: GBR12909 IC50 = 5 nM; methamphetamine IC50 = 48 nM; methylphenidate IC50 = 53 nM; and cocaine IC50 = 172 nM. In contrast, serotonin was relatively weak in inhibiting AMT (IC50 = 7.9 microM). There was a highly significant (P less than .001; slope = 1.2) linear correlation between the AMT-inhibiting potencies of AMPH analogs and their potencies in stimulating locomotor activity in rodents. AMT may be important in the low dose effects of AMPH such as increased locomotor activity in rodents and stimulant activity in man. Differences between AMT and d-({sup 3}H)AMPH sequestration described earlier, as well as their possible relevance to behavioral and neurochemical sequelae of AMPH administration are also discussed.

  14. MR-guided focused ultrasound: enhancement of intratumoral uptake of [3H]-docetaxel in vivo

    NASA Astrophysics Data System (ADS)

    Chen, Lili; Mu, Zhaomei; Hachem, Paul; Ma, C.-M.; Wallentine, Annie; Pollack, Alan

    2010-12-01

    The purpose of this study is to quantify the enhancement of [3H]-docetaxel in implanted prostate tumors treated with MR-guided pulsed focused ultrasound (MRgFUS). Human prostate cancer, LNCaP cells in 25 µl, were implanted into the prostates of male nude mice. The tumor growth was directly monitored on MRI. When the tumor reached a designated size, MRgFUS treatment was performed using a focused ultrasound treatment system (InSightec ExAblate 2000) with a 1.5 T GE MR scanner. The tumor-bearing animals were randomly divided into three groups: group 1, MRgFUS treatment + [3H]-docetaxel; group 2, [3H]-docetaxel only and group 3, as a control. Animals in group 1 were treated with MRgFUS non-invasively. Immediately after the treatment, the animals received a single dose of tail vein injection of docetaxel at 15 mg kg-1 mixed with [3H]-docetaxel at 50 uCi kg-1 in a total volume of 150 µl. Animals in group 2 were treated the same as in group one, however without MRgFUS treatment. Animals in group 3 were treated as a control. Animals were sacrificed 30 min after i.v. injections regardless of whether or not they received focused ultrasound. Tumors were removed and processed. The radioactivity of [3H]-docetaxel in the tumor tissue was quantitatively measured by a liquid scintillation counter. Our study showed that all animals tolerated the MRgFUS treatment well. Our data showed increased 3H-docetaxel concentration in the tumor in the MRgFUS-treated group (1079 ± 132 cmp/75 mg) versus those without MRgFUS treatment (524 ± 201 cmp/75 mg) with P = 0.037.

  15. Autoradiographic localization of /sup 3/H-digoxin binding by neural cells in the medulla

    SciTech Connect

    Traurig, H.H.; Bhagat, A.; Bass, N.H.

    1985-01-01

    The purpose of this investigation was to localize binding sites for the cardiac glycoside digoxin in the medulla of the rat in vivo. Adult male Sprague-Dawley rats were injected (IV) with /sup 3/H-digoxin and killed 30 minutes later. Autoradiographs of medullas showed evidence of /sup 3/H-digoxin binding to small- and medium-sized neural cells in the regions of the nucleus solitarius, dorsal motor nucleus of the vagus, area postrema, and in the zone between the area postrema and the underlying neuropil. However, the parasympathetic preganglionic neurons of the dorsal motor nucleus were not labeled. The /sup 3/H-digoxin-labeled cells in the medulla were located mainly in the commissural and medial portions of nucleus solitarius at the level of the area postrema. Animals injected with unlabeled digoxin followed by /sup 3/H-digoxin showed reduced binding of radioactivity. The small- and medium-sized neurons of the caudal portions of the nucleus solitarius are internuncial in position with respect to cardiovascular afferents of the glossopharyngeal and vagus nerves and sympathetic and parasympathetic cardiovascular efferent neurons of the medulla. The results of this study suggest that these /sup 3/H-digoxin-labeled cells, presumably neurons of nucleus solitarius, may possess high affinity binding sites for digoxin. Further, the area postrema, which lacks a blood-brain barrier, may provide a portal of entry for /sup 3/H-digoxin into regions of the medulla known to contain neurons that play a role in the regulation of cardiac rhythm.

  16. Palladium-Catalyzed Intramolecular Carbene Insertion into C(sp(3) )-H Bonds.

    PubMed

    Solé, Daniel; Mariani, Francesco; Bennasar, M-Lluïsa; Fernández, Israel

    2016-05-23

    A palladium-catalyzed carbene insertion into C(sp(3) )-H bonds leading to pyrrolidines was developed. The coupling reaction can be catalyzed by both Pd(0) and Pd(II) , is regioselective, and shows a broad functional group tolerance. This reaction is the first example of palladium-catalyzed C(sp(3) )-C(sp(3) ) bond assembly starting from diazocarbonyl compounds. DFT calculations revealed that this direct C(sp(3) )-H bond functionalization reaction involves an unprecedented concerted metalation-deprotonation step. PMID:27079473

  17. Radiometric assay of ghrelin hydrolase activity and 3H-ghrelin distribution into mouse tissues.

    PubMed

    Chen, Vicky Ping; Gao, Yang; Geng, Liyi; Brimijoin, Stephen

    2015-12-15

    A high-throughput radiometric assay was developed to characterize enzymatic hydrolysis of ghrelin and to track the peptide's fate in vivo. The assay is based on solvent partitioning of [(3)H]-octanoic acid liberated from [(3)H]-octanoyl ghrelin during enzymatic hydrolysis. This simple and cost-effective method facilitates kinetic analysis of ghrelin hydrolase activity of native and mutated butyrylcholinesterases or carboxylesterases from multiple species. In addition, the assay's high sensitivity facilitates ready evaluation of ghrelin's pharmacokinetics and tissue distribution in mice after i.v. bolus administration of radiolabeled peptide. PMID:26514871

  18. Specific binding of /sup 3/H-naloxone with isolated rat enterocytes

    SciTech Connect

    Yarygin, K.N.; Shitin, A.G.; Suiridov, D.D.; Titov, M.I.; Vinogradov, V.A.

    1985-12-01

    This paper presents data on the specific binding of naloxone with isolated rat enterocytes. Naloxone was bound with the cells in medium 199 containing 1 mg/ml of BSA. The incubation mixture contained 5 x 100 mM /sup 3/H-naloxone and, if indicated, other substances also. Dose dependence of binding of naloxone with rat enterocytes is shown. The kinetics of specific binding of naloxone with enterocytes at different temperatures is also shown, as is the irreversibility of binding of /sup 3/H-naloxone with isolated rat enterocytes. It was found that different ligands of opioid receptors can inhibit binding of naloxone competitively.

  19. Erythrocyte /sup 3/H-ouabain binding and digitalis treatment in ethanol addicted patients

    SciTech Connect

    Battaini, F.; Govoni, S.; Mauri, A.; Civelli, L.; Trabucchi, M.

    1987-06-29

    The binding of /sup 3/H-ouabain to human erythrocytes was analyzed in a population of hospitalized male ethanol addicted patients under long term digitalis treatment. In the non-alcoholic patient group the long term digitalis treatment induced an increase in Bmax and Kd values; such modification was not observed in the alcoholic patients. Chronic alcohol intake itself induced an increase in /sup 3/H-ouabain kinetic parameters. These observations confirm that ouabain binding to human erythrocytes is subject to pharmacological and toxicological regulation and that adaptive changes in peripheral tissues can be useful in predicting possible parallel modifications in other less accessible tissues. 22 references, 1 table.

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

    PubMed Central

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

    2014-01-01

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

  1. The phtC-phtD locus equips Legionella pneumophila for thymidine salvage and replication in macrophages.

    PubMed

    Fonseca, Maris V; Sauer, John-Demian; Crepin, Sebastien; Byrne, Brenda; Swanson, Michele S

    2014-02-01

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

  2. Tritritionikkomycin Z, (uracil-5- sup 3 H,pyridyl-2,4- sup 3 H sub 2 ): Radiolabeling of a potent inhibitor of fungal and insect chitin synthetase

    SciTech Connect

    Ando, Tetsu; Tecle, B.; Toia, R.F.; Casida, J.E. )

    1990-08-01

    Nikkomycin Z (NZ) (a potent fungicide, insecticide, miticide, and inhibitor of fungal and insect chitin synthetase) was converted to a mixture of specific mono-, di-, and tribromo derivatives (BrNZ, Br{sub 2}NZ, and Br{sub 3}NZ, respectively) on reaction with N-bromosuccinimide in N,N-dimethylformamide. Substitution by bromine occurred first at the 2-position of the 3-hydroxypyridyl moiety, second at the 5-position of the uracil moiety, and finally at the 4-position of the 3-hydroxypyridyl moiety as observed both for NZ and for mixtures of uridine and 3-hydroxy-6-methylpyridine as model compounds representative of the moieties of NZ. Following fractionation of the various bromonikkomycin derivatives by HPLC, their structures were assigned by NMR, MS, and UV analyses. Catalytic reductive debromination of Br{sub 3}NZ with tritium gas over palladium on carbon gave (uracil-5-{sup 3}H,pyridyl-2,4-{sup 3}H{sub 2})NZ. This material has sufficiently high specific activity ({approximately}60 Ci/mmol) and suitable positions of labeling to study its uptake, distribution, metabolism, and possible target site interactions in fungal and insect systems.

  3. Reduced permeation of /sup 14/C-sucrose, /sup 3/H-mannitol and /sup 3/H-inulin across blood-brain barrier in nephrectomized rats

    SciTech Connect

    Preston, E.; Haas, N.; Allen, M.

    1984-01-01

    Experiments were carried out to determine if changes in the concentration-time profile of a blood-borne radiotracer such as /sup 14/C-sucrose would spuriously alter measurements of its permeation across the blood-brain barrier (permeability-area product, PA) based on a 2-compartment (plasma/brain) simple diffusion model. Anesthetized rats which were bilaterally nephrectomized and given a standard intravenous bolus injection of /sup 14/C-sucrose, /sup 3/H-mannitol or /sup 3/H-inulin exhibited an elevated plasma tracer concentration compared to control animals. However, tracer concentration measured in brain parenchyma after 30 min was not proportionally elevated, and PA calculated from the ratio, parenchymal tracer concentration: plasma concentration-time integral, was significantly reduced below control values. In control rats, distortion and elevation of the plasma /sup 14/C-sucrose profile by continuous intravenous infusion did not result in lowered PA values. This suggested that the lowering of PA by nephrectomy reflected reduced cerebrovascular permeability or area or other cerebral influence rather than a deficiency in the 2-compartment model for PA measurement.

  4. Crystal structures of [NEt3H]5[XCoIIW11O39]·3H2O (X = P or As)

    USGS Publications Warehouse

    Evans, H.T., Jr.; Weakley, T.J.R.; Jameson, G.B.

    1996-01-01

    The orthorhombic crystal structures of [NEt3H]5[XCoIIW11O39]·3H2O for X = P and As have been determined with data collected at room temperature, and for X = P at –100 °C, using Mo-Kα radiation. For the latter the space group is Pna21, a= 21.670(11), b= 14.805(4), c= 20.393(5)Å and Z= 4. The structure consists of chains of α-Keggin-type molecules joined by W–O–links aligned in the a-axis direction. The Co/W occupancy at the link is disordered, with 61% Co on one side and 39% on the other. Further probable disorder, by lamellar merohedral twinning on (001) and by misorientation of the triethylammonium ions, has obscured the ethyl groups and the water molecules. In polarized light the crystals are deep wine-red normal to the chains (in the b direction), but nearly colourless in the a and c directions. The structure of the arsenate is similar to that of the phosphate.

  5. Pentamidine analogs as inhibitors of [3H]MK-801 and [3H]ifenprodil binding to rat brain NMDA receptors

    PubMed Central

    Berger, Michael L.; Maciejewska, Dorota; Vanden Eynde, Jean Jacques; Mottamal, Madhusoodanan; Żabiński, Jerzy; Kaźmierczak, Paweł; Rezler, Mateusz; Jarak, Ivana; Piantanida, Ivo; Karminski-Zamola, Grace; Mayence, Annie; Rebernik, Patrick; Kumar, Arvind; Ismail, Mohamed A.; Boykin, David W.; Huang, Tien L.

    2016-01-01

    The anti-protozoal drug pentamidine is active against opportunistic Pneumocystis pneumonia, but in addition has several other biological targets, including the NMDA receptor (NR). Here we describe the inhibitory potencies of 76 pentamidine analogs at 2 binding sites of the NR, the channel binding site labeled with [3H]MK-801 and the [3H]ifenprodil binding site. Most analogs acted weaker at the ifenprodil than at the channel site. The spermine-sensitivity of NR inhibition by the majority of the compounds was reminiscent of other long-chain dicationic NR blockers. The potency of the parent compound as NR blocker was increased by modifying the heteroatoms in the bridge connecting the 2 benzamidine moieties and also by integrating the bridge into a seven-membered ring. Docking of the 45 most spermine-sensitive bisbenzamidines to a recently described acidic interface between the N-terminal domains of GluN1 and GluN2B mediating polyamine stimulation of the NR revealed the domain contributed by GluN1 as the most relevant target. PMID:26117647

  6. Dithymine photodimers and photodecomposition products of thymidylyl-thymidine induced by ultraviolet radiation from 150 to 300 nm

    SciTech Connect

    Saitou, M.; Hieda, K.

    1994-11-01

    Solid thymidylyl-(3{prime} {yields} 5{prime})-thymidine (dTpdT) was irradiated in a vacuum with monochromatic photons from 150 to 300 nm; the photoproducts were analyzed quantitatively by high-performance liquid chromatography. The results of the experiment were as follows: (1) above 210 nm the major photoproducts were three dithymine photodimers [the cis-syn and trans-syn cyclobutane thymine dimers and the thymine (6-4) photoproduct]; below 210 nm, they were three photodecomposition products (thymine, thymidine 5{prime}-monophosphate and thymidine 3{prime}-monophosphate). This shows that 210 nm is the wavelength at which the major photoproducts change from dithymine photodimers (far-UV type) to photodecomposition products (X-ray type). (2) The yields of the three dithymine photodimers had a similar wavelength dependence with each other: the yields had a peak at 260 nm and gradually decreased toward shorter wavelengths to 150 nm. (3) The yields of the three photodecomposition products also had a very similar wavelength dependence with each other; the yields increased exponentially with a decrease in the wavelength. (4) The average ratios of the yield of the (6-4) photoproduct to that of the cis-syn dimer were 0.30 between 170 and 220 nm, but 0.16 between 240 and 290 nm. 18 refs., 4 figs.

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

    PubMed

    Gendaszewska-Darmach, Edyta; Szustak, Marcin

    2016-06-01

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

  8. Validation of a HPLC method for the measurement of erythrocyte encapsulated thymidine phosphorylase (EE-TP) activity.

    PubMed

    Fairbanks, Lynette D; Levene, Michelle; Bax, Bridget E

    2013-03-25

    A sensitive and simple reverse-phase high performance liquid chromatographic (HPLC) assay has been validated for the determination of thymine as a measure of thymidine phosphorylase activity encapsulated in erythrocytes (EE-TP), a formulation which is under clinical development as an enzyme replacement therapy for the treatment of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). Diluted erythrocyte lysates were incubated in 100mM sodium phosphate buffer and 10mM thymidine at 37°C for 10min and the reaction stopped with 40% trichloroacetic acid. Following centrifugation, the supernatant was washed with water saturated diethyl ether, and injected onto a Spherisorb C(18) column (125mm×4.6mm, 5μm), with a mobile phase (40mM ammonium acetate, 5mM tetrabutyl ammonium hydrogen sulphate, pH 2.70) delivered at a flow rate of 1.0ml/min and run time of 8min. Ultraviolet detection (UV) was employed at 254nm. The method was linear in the range of 5-500nmol/ml (r(2)=0.992), specific with intra- and inter-day precisions of <9.6 and accuracies within ±20%. Limits of detection and quantification were 1.2nmol/ml and 10nmol/ml, respectively. The method was applied to quantify thymidine phosphorylase activity in samples of in-process controls and batches of EE-TP manufactured for clinical use. PMID:23291437

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

    PubMed

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

    2009-04-01

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

  10. Octa(thymidine methanephosphonates) of partially defined stereochemistry: synthesis and effect of chirality at phosphorus on binding to pentadecadeoxyriboadenylic acid.

    PubMed Central

    Lesnikowski, Z J; Jaworska, M; Stec, W J

    1990-01-01

    Block condensation of MePOCI2 or MeP(NEt2)2 with appropriately protected tetra(thymidine methanephosphonates) of predetermined sense of chirality at asymmetric phosphonate centres gave two pairs of diastereomeric mixtures, namely (SpSpSpSpSpSpSp + SpSpSpRpSpSpSp) 5a and (RpRpRpRpRpRpRp + RpRpRpSpRpRpRp) 5b. A comparison of the CD spectra of 5a and 5b with those of octathymidylic acid (7) and a random mixture of diastereomers of octa(thymidine methanephosphonate) (6), and also a comparison of the Tm of complexes formed between 5a, 5b, 6 or 7, and pentadecadeoxyriboadenylic acid (8), indicates that octamer 5b and its complex with its complementary oligonucleotide has a well-ordered structure due to the 'outward' or 'pseudoequatorial' orientation of the methyl group of each internucleotide methanephosphonate function of Rp configuration. Results presented in this report clearly indicate that the stability of hybrids formed between octa(thymidine methanephosphonate) and pentadecadeoxyriboadenylic acid depends on the stereochemistry of each internucleotide methanephosphonate function and strongly suggests that stereoselective synthesis of P-chiral oligonucleotide analogues is an important goal. PMID:2336391

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

    PubMed Central

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

    2012-01-01

    Thymidylate kinase (TMK) is a potential chemotherapeutic target because it is directly involved in the synthesis of an essential component, thymidine triphosphate, in DNA replication. All reported TMK inhibitors are thymidine analogs, which might retard their development as potent therapeutics due to cell permeability and off-target activity against human TMK. A small molecule hit (1, IC50 = 58 μM), which has reasonable inhibition potency against Pseudomonas aeruginosa TMK (PaTMK), was identified by the analysis of the binding mode of thymidine or TP5A in a PaTMK homology model. This hit (1) was co-crystallized with PaTMK, and several potent PaTMK inhibitors (leads, 46, 47, 48, and 56, IC50 = 100–200 nM) were synthesized using computer aided design approaches including virtual synthesis/screening, which was used to guide the design of inhibitors. The binding mode of the optimized leads in PaTMK overlaps with that of other bacterial TMKs, but not with human TMK which shares few common features with the bacterial enzymes. Therefore, the optimized TMK inhibitors described here should be useful for the development of antibacterial agents targeting TMK without undesired off-target effects. In addition, an inhibition mechanism associated with the LID loop, which mimics the process of phosphate transfer from ATP to dTMP, was proposed based on X-ray co-crystal structures, homology models, and SAR results. PMID:22243413

  12. Carotenoid incorporation into microsomes: yields, stability and membrane dynamics

    NASA Astrophysics Data System (ADS)

    Socaciu, Carmen; Jessel, Robert; Diehl, Horst A.

    2000-12-01

    The carotenoids β-carotene (BC), lycopene (LYC), lutein (LUT), zeaxanthin (ZEA), canthaxanthin (CTX) and astaxanthin (ASTA) have been incorporated into pig liver microsomes. Effective incorporation concentrations in the range of about 1-6 nmol/mg microsomal protein were obtained. A stability test at room temperature revealed that after 3 h BC and LYC had decayed totally whereas, gradually, CTX (46%), LUT (21%), ASTA (17%) and ZEA (5%) decayed. Biophysical parameters of the microsomal membrane were changed hardly by the incorporation of carotenoids. A small rigidification may occur. Membrane anisotropy seems to offer only a small tolerance for incorporation of carotenoids and seems to limit the achievable incorporation concentrations of the carotenoids into microsomes. Microsomes instead of liposomes should be preferred as a membrane model to study mutual effects of carotenoids and membrane dynamics.

  13. Time-resolved infrared spectroscopy of the lowest triplet state of thymine and thymidine

    NASA Astrophysics Data System (ADS)

    Hare, Patrick M.; Middleton, Chris T.; Mertel, Kristin I.; Herbert, John M.; Kohler, Bern

    2008-05-01

    Vibrational spectra of the lowest energy triplet states of thymine and its 2'-deoxyribonucleoside, thymidine, are reported for the first time. Time-resolved infrared (TRIR) difference spectra were recorded over seven decades of time from 300 fs to 3 μs using femtosecond and nanosecond pump-probe techniques. The carbonyl stretch bands in the triplet state are seen at 1603 and ˜1700 cm -1 in room-temperature acetonitrile- d3 solution. These bands and additional ones observed between 1300 and 1450 cm -1 are quenched by dissolved oxygen on a nanosecond time scale. Density-functional calculations accurately predict the difference spectrum between triplet and singlet IR absorption cross sections, confirming the peak assignments and elucidating the nature of the vibrational modes. In the triplet state, the C4 dbnd O carbonyl exhibits substantial single-bond character, explaining the large (˜70 cm -1) red shift in this vibration, relative to the singlet ground state. Femtosecond TRIR measurements unambiguously demonstrate that the triplet state is fully formed within the first 10 ps after excitation, ruling out a relaxed 1nπ ∗ state as the triplet precursor.

  14. Kinetics and mechanistic study of competitive inhibition of thymidine phosphorylase by 5-fluoruracil derivatives.

    PubMed

    Petaccia, Manuela; Gentili, Patrizia; Bešker, Neva; D'Abramo, Marco; Giansanti, Luisa; Leonelli, Francesca; La Bella, Angela; Gradella Villalva, Denise; Mancini, Giovanna

    2016-04-01

    In a previous investigation, cationic liposomes formulated with new 5-FU derivatives, differing for the length of the polyoxyethylenic spacer that links the N(3) position of 5-FU to an alkyl chain of 12 carbon atoms, showed a higher cytotoxicity compared to free 5-FU, the cytotoxic effect being directly related to the length of the spacer. To better understand the correlation of the spacer length with toxicity, we carried out initial rate studies to determine inhibition, equilibrium and kinetic constants (KI, KM, kcat), and get inside inhibition activity of the 5-FU derivatives and their mechanism of action, a crucial information to design structural variations for improving the anticancer activity. The experimental investigation was supported by docking simulations based on the X-ray structure of thymidine phosphorylase (TP) from Escherichia coli complexed with 3'-azido-2'-fluoro-dideoxyuridin. Theoretical and experimental results showed that all the derivatives exert the same inhibition activity of 5-FU either as monomer and when embedded in lipid bilayer. PMID:26752208

  15. Clinicopathological significance of vascular endothelial growth factor, thymidine phosphorylase and microvessel density in colorectal cancer.

    PubMed

    Kimura, Yutaka; Morohashi, Satoko; Yoshizawa, Tadashi; Suzuki, Takahiro; Morohashi, Hajime; Sakamoto, Yoshiyuki; Koyama, Motoi; Murata, Akihiko; Kijima, Hiroshi; Hakamada, Kenichi

    2016-02-01

    Colorectal cancer is a common malignant disease, the incidence of which is increasing worldwide, therefore, identifying novel prognostic factors to improve adjuvant therapeutic strategies or postoperative monitoring is required. Angiogenesis, which is assessed by microvessel density (MVD), is significant in tumor growth and metastasis. However, the association between angiogenesis and clinical outcome remains controversial. In the present study, 84 surgically resected cases of colorectal cancer were examined to clarify the clinicopathological significance of vascular endothelial growth factor (VEGF), thymidine phosphorylase (TP) and cluster of differentiation (CD)34 expression levels. VEGF expression was identified to be significantly correlated with TP expression (r=0.45; P<0.0001) and MVD in the high VEGF expression group was observed to be significantly greater than that in the low VEGF expression group (P=0.0194). In the Dukes' stage D group, the MVD in the high TP expression group was significantly greater than that in the low TP expression group (P=0.0149). High VEGF expression was subsequently correlated with a short overall survival rate for patients exhibiting lymph node metastasis (P=0.0128); however, there was no significant difference in overall survival rate regarding the expression levels of TP and CD34. The results of the present study indicate that VEGF expression may serve as a prognostic factor for colorectal cancer patients exhibiting lymph node metastasis. Furthermore, angiogenesis, as assessed by MVD, is an important prognostic factor for tumor growth at the primary site. PMID:26676225

  16. Thymidine phosphorylase gene variant, platelet counts and survival in gastrointestinal cancer patients treated by fluoropyrimidines.

    PubMed

    Huang, Liu; Chen, Fengju; Chen, Yangyang; Yang, Xiaomei; Xu, Sanpeng; Ge, Shuwang; Fu, Shengling; Chao, Tengfei; Yu, Qianqian; Liao, Xin; Hu, Guangyuan; Zhang, Peng; Yuan, Xianglin

    2014-01-01

    The predictive value of thymidine phosphorylase gene variants (TP, also called platelet-derived endothelial cell growth factor) and thrombocytosis were controversial and worthy of further study in gastrointestinal cancer (GIC) patients. We screened all of the common missense single nucleotide polymorphisms (MAF ≥ 0.1) in fluoropyrimidines (FU) pathway genes (including TP, TS, ENOSF1 and DPD). Three of them were selected and genotyped using Sequenom MassARRAY in 141 GIC patients. TP expression was assessed by immunohistochemistry. Our aim was to evaluate the prognostic significance of studied genes and platelet counts in GIC patients. Multivariate analyses indicated in rs11479-T allele carriers, platelet counts negatively correlated to overall survival. In addition, T allele of TP: rs11479 was associated with higher TP expression in cancer tissues. We suggest TP: rs11479 variant combined with platelet counts may be useful prognostic makers in GIC patients receiving first-line FU chemotherapy and thrombopoietin factor should be used with caution in the rs11479 T allele bearing patients. PMID:25027354

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

    PubMed Central

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

    2010-01-01

    AIM: To evaluate the role of thymidine phosphorylase (TP) in cholangiocarcinoma using small interfering RNA (siRNA). METHODS: A human cholangiocarcinoma-derived cell line KKU-M139, which has a naturally high level of endogenous TP, had TP expression transiently knocked down using siRNA. Cell growth, migration, in vitro angiogenesis, apoptosis, and cytotoxicity were assayed in TP knockdown and wild-type cell lines. RESULTS: TP mRNA and protein expression were decreased by 87.1% ± 0.49% and 72.5% ± 3.2%, respectively, compared with control cells. Inhibition of TP significantly decreased migration of KKU-M139, and suppressed migration and tube formation of human umbilical vein endothelial cells. siRNA also reduced the ability of TP to resist hypoxia-induced apoptosis, while suppression of TP reduced the sensitivity of KKU-M139 to 5-fluorouracil. CONCLUSION: Inhibition of TP may be beneficial in decreasing angiogenesis-dependent growth and migration of cholangiocarcinoma but may diminish the response to 5-fluorouracil chemotherapy. PMID:20355241

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

    SciTech Connect

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

    1985-01-01

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

  19. Elevated levels of thymidine kinase 1 peptide in serum from patients with breast cancer

    PubMed Central

    Larsson, Anders; Lindman, Henrik

    2009-01-01

    Objectives Thymidine kinase (TK) has an important role in DNA synthesis and is thus related to cell proliferation and turn-over. Traditionally, TK has been measured by enzymatic activity or radioimmunoassays. These assays are difficult to adapt to random access instruments. The aim of this study was to evaluate a new immunological sandwich assay for detection of TK peptides in serum from breast cancer patients. Material and methods Serum samples were collected from patients with breast cancer and stored frozen at −70°C. The samples were collected after surgery, after metastatic tumor recurrence and after chemotherapy due to tumour recurrence. Patients’ serum samples were analysed by the TK enzyme-linked immunosorbent assay (ELISA). Results In receiver operating characteristics (ROC) analyses of TK1 for diagnosis of breast cancer, the area under the curve (AUC) collected four weeks after surgery was 0.56 (95% CI 0.47–0.65), for samples collected postsurgically after tumour recurrence 0.73 (95% CI 0.65–0.80), and after chemotherapy 0.64 (95% CI 0.56–0.72). Conclusions This study indicates that the tumour proliferation marker TK has a potential as a serum marker in breast cancer. Further studies are warranted to verify this observation. PMID:19396699

  20. A phase I clinical trial of thymidine kinase-based gene therapy in advanced hepatocellular carcinoma.

    PubMed

    Sangro, B; Mazzolini, G; Ruiz, M; Ruiz, J; Quiroga, J; Herrero, I; Qian, C; Benito, A; Larrache, J; Olagüe, C; Boan, J; Peñuelas, I; Sádaba, B; Prieto, J

    2010-12-01

    The aim of this phase I clinical trial was to assess the feasibility and safety of intratumoral administration of a first-generation adenoviral vector encoding herpes simplex virus thymidine kinase (HSV-TK) gene (Ad.TK) followed by systemic ganciclovir to patients with advanced hepatocellular carcinoma (HCC). Secondarily, we have analyzed its antitumor effect. Ten patients were enrolled in five dose-level cohorts that received from 10¹⁰ to 2 × 10¹² viral particles (vp). Ad.TK was injected intratumorally and patients received up to three doses at 30-day intervals. Positron emission tomography was used to monitor TK gene expression. Ad.TK injection was feasible in 100% of cases. Treatment was well tolerated and dose-limiting toxicity was not achieved. Cumulative toxicity was not observed. Hepatic toxicity was absent even in cirrhotic patients. Fever, flu-like syndrome, pain at the injection site and pancytopenia were the most common side effects. No partial responses were observed and 60% of patients showed tumor stabilization of the injected lesion. Importantly, two patients who received the highest dose showed signs of intratumoral necrosis by imaging procedures. One of them achieved a sustained stabilization and survived for 26 months. In conclusion, Ad.TK can be safely administered by intratumoral injection to patients with HCC up to 2 × 10¹² vp per patient. PMID:20689572

  1. Thymidine Kinase Suicide Gene-mediated Ganciclovir Ablation of Autologous Gene-modified Rhesus Hematopoiesis

    PubMed Central

    Barese, Cecilia N; Krouse, Allen E; Metzger, Mark E; King, Connor A; Traversari, Catia; Marini, Frank C; Donahue, Robert E; Dunbar, Cynthia E

    2012-01-01

    Despite the genotoxic complications encountered in clinical gene therapy trials for primary immunodeficiency diseases targeting hematopoietic cells with integrating vectors; this strategy holds promise for the cure of several monogenic blood, metabolic and neurodegenerative diseases. In this study, we asked whether the inclusion of a suicide gene in a standard retrovirus vector would allow elimination of vector-containing stem and progenitor cells and their progeny in vivo following transplantation, using our rhesus macaque transplantation model. Following stable engraftment with autologous CD34+ cells transduced with a retrovirus vector encoding a highly sensitive modified Herpes simplex virus thymidine kinase SR39, the administration of the antiviral prodrug ganciclovir (GCV) was effective in completely eliminating vector-containing cells in all hematopoietic lineages in vivo. The sustained absence of vector-containing cells over time, without additional GCV administration, suggests that the ablation of TkSR39 GCV-sensitive cells occurred in the most primitive hematopoietic long-term repopulating stem or progenitor cell compartment. These results are a proof-of-concept that the inclusion of a suicide gene in integrating vectors, in addition to a therapeutic gene, can provide a mechanism for later elimination of vector-containing cells, thereby increasing the safety of gene transfer. PMID:22910293

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

    PubMed

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

    2016-07-01

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

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

    PubMed

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

    2005-09-01

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

  4. The dual role of thymidine phosphorylase in cancer development and chemotherapy.

    PubMed

    Bronckaers, Annelies; Gago, Federico; Balzarini, Jan; Liekens, Sandra

    2009-11-01

    Thymidine phosphorylase (TP), also known as "platelet-derived endothelial cell growth factor" (PD-ECGF), is an enzyme, which is upregulated in a wide variety of solid tumors including breast and colorectal cancers. TP promotes tumor growth and metastasis by preventing apoptosis and inducing angiogenesis. Elevated levels of TP are associated with tumor aggressiveness and poor prognosis. Therefore, TP inhibitors are synthesized in an attempt to prevent tumor angiogenesis and metastasis. TP is also indispensable for the activation of the extensively used 5-fluorouracil prodrug capecitabine, which is clinically used for the treatment of colon and breast cancer. Clinical trials that combine capecitabine with TP-inducing therapies (such as taxanes or radiotherapy) suggest that increasing TP expression is an adequate strategy to enhance the antitumoral efficacy of capecitabine. Thus, TP plays a dual role in cancer development and therapy: on the one hand, TP inhibitors can abrogate the tumorigenic and metastatic properties of TP; on the other, TP activity is necessary for the activation of several chemotherapeutic drugs. This duality illustrates the complexity of the role of TP in tumor progression and in the clinical response to fluoropyrimidine-based chemotherapy. PMID:19434693

  5. Free water 3H concentrations in serum samples collected during 1969-1992 in Akita, Japan.

    PubMed

    Hisamatsu, Shun'ichi; Inoue, Yoshikazu; Hachiya, Noriyuki; Katoh, Kiyoshi; Nakagomi, Toyoko; Nakagomi, Osamu; Motohashi, Yutaka; Takizawa, Yukio

    2003-08-01

    The measurements for human and environmental samples from the 1960's and 1970's are important to understand the long-term transfer of 3H from the environment to the human body. The authors have previously reported 3H concentrations in diet samples collected in Akita Prefecture during 1969-1988. Serum samples from persons living in Akita Prefecture during 1969-1992 were recently obtained. The samples were originally gathered for medical examinations and stored in freezers at -20 degrees C. Composite samples from 100 persons on average were made for analysis. The free water 3H (FWT) concentrations in those samples were determined and compared with 3H concentrations in diet samples and precipitation. The long-term variation pattern of the FWT concentrations in the serum samples was similar to patterns in the diet samples and precipitation, but the FWT concentrations in the serum samples were slightly higher than those in the latter two. A single compartment model calculation showed that the apparent mean residence time of serum FWT was 1.4 y using precipitation as an input to the compartment. PMID:12938967

  6. IMPROVED SCORING OF CHEMICAL TRANSFORMATION OF C3H/10T1/2 CELLS

    EPA Science Inventory

    This research program was undertaken to improve the scoring of the transformation by chemical carcinogens of C3H/10T1/2 mouse embryo fibroblasts. (1) A probabilistic view of transformed focus formation in these cells induced by methylcholanthrene (MCA) treatment has been formulat...

  7. In Situ g-PHA Measurements of the 285-3H Cooling Tower Components

    SciTech Connect

    Salaymeh, S.R.

    2001-05-23

    The Analytical Development Section of Savannah River Technology Center was requested by the Facility Disposition Division to conduct in-situ gamma-ray pulse height analysis measurements to provide input toward the decision to unconditionally release the 285-3H cooling tower.

  8. Release of (/sup 3/H)-monoamines from superfused rat striatal slices by methylenedioxymethamphetamine (MDMA)

    SciTech Connect

    Levin, J.A.; Schmidt, C.J.; Lovenberg, W.

    1986-03-05

    MDMA is a phenylisopropylamine which is reported to have unique behavioral effects in man. Because of its structural similarities to the amphetamines the authors have compared the effects of MDMA and two related amphetamines on the spontaneous release of tritiated dopamine (DA) and serotonin (5HT) from superfused rat striatal slices. At concentrations of 10/sup -7/ - 10/sup -5/M MDMA and the serotonergic neurotoxin, p-chloroamphetamine, were equipotent releasers of (/sup 3/H)5HT being approximately 10x more potent than methamphetamine. However, methamphetamine was the more potent releaser of (/sup 3/H)DA by a factor of approximately 10x. MDMA-induced release of both (/sup 5/H)5HT and (/sup 3/H)DA was Ca/sup 2 +/-independent and inhibited by selective monoamine uptake blockers suggesting a carrier-dependent release mechanism. Synaptosomal uptake experiments with (+)(/sup 3/H)MDMA indicated no specific uptake of the drug further suggesting the effect of uptake blockers may be to inhibit the carrier-mediated export of amines displaced by MDMA.

  9. Biliary excretion of radioactivity after intravenous administration of (3H)25-hydroxyvitamin D3 in man

    SciTech Connect

    Ledger, J.E.; Watson, G.J.; Compston, J.E.

    1986-04-01

    The biliary excretion of radioactivity after intravenous (3H)25-hydroxyvitamin D3 was studied in nine patients with T-tube bile drainage. The mean +/- SD 24-hr radioactivity excretion in T-tube bile expressed as a percentage of the administered dose was 6.7 +/- 2.9%; after correction for incomplete bile collection, the value obtained was 16.0 +/- 11.1%. Chloroform solubility of biliary radioactivity increased from 27.4 +/- 8.9% to 72.9 +/- 10.1% following incubation with beta-glucuronidase. High-performance liquid chromatographic analysis of chloroform extracts of bile revealed that most of the eluted radioactivity was more polar than (3H)25-hydroxyvitamin D3. No free (3H)25-hydroxyvitamin D3 was demonstrated. Thus in man, most of the biliary radioactivity excreted following (3H)25-hydroxyvitamin D3 is in the form of water-soluble compounds, mainly glucuronides. However, our results suggest that glucuronides of metabolites other than 25-OHD3 are predominantly formed.

  10. Aminosilanes derived from 1H-benzimidazole-2(3H)-thione

    SciTech Connect

    Palomo-Molina, Juliana; García-Báez, Efrén V.; Pineda-Urbina, Kayim; Ramos-Organillo, Angel

    2015-08-12

    In two trimethylsilyl-substituted 1H-benzimidazole-2(3H)-thiones, noncovalent C—H⋯π interactions between the centroid of the benzmidazole system and the SiMe{sub 3} groups form helicoidal arrangements in one, and dimerization results in the formation of R{sub s} {sup 2}(8) rings via N—H⋯S interactions, along with parallel π–π interactions between imidazole and benzene rings, in the second compound. Two new molecular structures, namely 1,3-bis(trimethylsilyl)-1H-benzimidazole-2(3H)-thione, C{sub 13}H{sub 22}N{sub 2}SSi{sub 2}, (2), and 1-trimethylsilyl-1H-benzimidazole-2(3H)-thione, C{sub 10}H{sub 14}N{sub 2}SSi, (3), are reported. Both systems were derived from 1H-benzimidazole-2(3H)-thione. Noncovalent C—H⋯π interactions between the centroid of the benzmidazole system and the SiMe{sub 3} groups form helicoidal arrangements in (2). Dimerization of (3) results in the formation of R{sub 2}{sup 2}(8) rings via N—H⋯S interactions, along with parallel π–π interactions between imidazole and benzene rings.

  11. Anionic ordering and thermal properties of FeF3·3H2O.

    PubMed

    Burbano, Mario; Duttine, Mathieu; Borkiewicz, Olaf; Wattiaux, Alain; Demourgues, Alain; Salanne, Mathieu; Groult, Henri; Dambournet, Damien

    2015-10-01

    Iron fluoride trihydrate can be used to prepare iron hydroxyfluoride with the hexagonal-tungsten-bronze (HTB) type structure, a potential cathode material for batteries. To understand this phase transformation, a structural description of β-FeF3·3H2O is first performed by means of DFT calculations and Mössbauer spectroscopy. The structure of this compound consists of infinite chains of [FeF6]n and [FeF2(H2O)4]n. The decomposition of FeF3·3H2O induces a collapse and condensation of these chains, which lead to the stabilization, under specific conditions, of a hydroxyfluoride network FeF3-x(OH)x with the HTB structure. The release of H2O and HF was monitored by thermal analysis and physical characterizations during the decomposition of FeF3·3H2O. An average distribution of FeF4(OH)2 distorted octahedra in HTB-FeF3-x(OH)x was obtained subsequent to the thermal hydrolysis/olation of equatorial anionic positions involving F(-) and H2O. This study provides a clear understanding of the structure and thermal properties of FeF3·3H2O, a material that can potentially bridge the recycling of pickling sludge from the steel industry by preparing battery electrodes. PMID:26378743

  12. Anionic ordering and thermal properties of FeF3·3H2O

    DOE PAGESBeta

    Burbano, Mario; Duttine, Mathieu; Borkiewicz, Olaf; Wattiaux, Alain; Demourgues, Alain; Salanne, Mathieu; Groult, Henri; Dambournet, Damien

    2015-09-17

    In this study, iron fluoride tri-hydrate can be used to prepare iron hydroxyfluoride with the Hexagonal-Tungsten-Bronze (HTB) type structure, a potential cathode material for batteries. To understand this phase transformation, a structural description of β-FeF3·3H2O is first performed by means of DFT calculations and Mössbauer spectroscopy. The structure of this compound consists of infinite chains of [FeF6]n and [FeF2(H2O)4]n. The decomposition of FeF3·3H2O induces a collapse and condensation of these chains, which lead to the stabilization, under specific conditions, of a hydroxyfluoride network FeF3-x(OH)x with the HTB structure. The release of H2O and HF was monitored by thermal analysis andmore » physical characterizations during the decomposition of FeF3·3H2O. An average distribution of FeF4(OH)2 distorted octahedra in HTB-FeF3-x(OH)x was obtained subsequent to the thermal hydrolysis/olation of equatorial anionic positions involving F- and H2O. This study provides a clear understanding of the structure and thermal properties of FeF3·3H2O, a material that can potentially bridge the recycling of pickling sludge from the steel industry by preparing battery electrodes.« less

  13. MELATONIN ENHANCES JUNCTIONAL TRANSFER IN NORMAL C3H/1OT1/2 CELLS

    EPA Science Inventory

    There is strong evidence that pineal melatonin is involved in controlling neoplastic processes. e have reported that physiological, but not pharmacological or subphysiological, concentrations of melatonin enhance intercellular communication in normal C3H/1OT1/2 fibroblasts. ap ju...

  14. Comparison of sludge treatment by O3 and O3/H2O2.

    PubMed

    Yuxin, Zhao; Liang, Wang; Helong, Yu; Baojun, Jiang; Jinming, Jiang

    2014-01-01

    This work focuses on the comparison of sludge decomposition caused by ozone (O3) alone and by ozone/hydrogen peroxide (O3/H2O2). The content of carbonaceous organic materials, nitrogenous compounds and phosphoric substances in sludge supernatant were measured. The release of soluble chemical oxygen demand, total nitrogen (TN) and total phosphorus (TP) caused by O3/H2O2 treatment were more than by O3 alone. As a result, it can be concluded that the efficiency of sludge breakup in O3/H2O2 was better than that in O3 alone. However, a peak appeared in both systems for the biodegradable substances such as carbohydrate. Carbohydrate could be used as the carbon source for denitrification, and the releasing of TN and TP may become an additional burden for a subsequent biological system. So, it was of benefit for the enhancement of cryptic growth and cost reduction by raising and maintaining the content of biodegradable substance and reducing the concentrations of the nitrogenous and phosphoric substances as far as possible. Therefore, sludge treated by O3/H2O2 with lower O3 dose would be more suitable than O3 alone. PMID:25026588

  15. Synthesis of (+/-)-3H-epivincamine via a Rh(II)-triggered cyclization/cycloaddition cascade.

    PubMed

    England, Dylan B; Padwa, Albert

    2007-08-16

    A synthesis of (+/-)-3H-epivincamine is reported. Important steps include (1) a Rh(II)-catalyzed intramolecular [3+2]-cycloaddition of an alpha-diazo indolo amide, (2) a reductive ring opening of the cycloadduct, (3) a decarboethoxylation reaction, and (4) a base-induced keto-amide ring contraction. PMID:17658832

  16. DIBENZODIOXOCIN STRUCTURES INVOLVING P-HYDROXYPHENYL UNITS IN C3H DOWN-REGULATED LIGNINS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We previously reported that downregulation of the gene encoding 4-coumarate 3-hydroxylase (C3H) in alfalfa markedly increased the proportion of p-hydroxyphenyl (H) units relative to the normally dominant guaiacyl (G) and syringyl (S) units, as determined by 2D 13C-1H correlative NMR methods, thioaci...

  17. Cholecystokinin-8 suppressed /sup 3/H-etorphine binding to rat brain opiate receptors

    SciTech Connect

    Wang, X.J.; Fan, S.G.; Ren, M.F.; Han, J.S.

    1989-01-01

    Radioreceptor assay (RRA) was adopted to analyze the influence of CCK-8 on /sup 3/H-etorphine binding to opiate receptors in rat brain synaptosomal membranes (P2). In the competition experiment CCK-8 suppressed the binding of /sup 3/H-etorphine. This effect was completely reversed by proglumide at 1/mu/M. Rosenthal analysis for saturation revealed two populations of /sup 3/H-etorphine binding sites. CCK-8 inhibited /sup 3/H-etorphine binding to the high affinity sites by an increase in Kd and decrease in Bmax without significant changes in the Kd and Bmax of the low affinity sites. This effect of CCK-8 was also completely reversed by proglumide at 1/mu/M. Unsulfated CCK-8 produced only a slight increase in Kd of the high affinity sites without affecting Bmax. The results suggest that CCK-8 might be capable of suppressing the high affinity opioid binding sites via the activation of CCK receptor.

  18. TOOLS FOR LIGNIN STRUCTURAL ANALYSIS; APPLICATIONS TO C3H-DOWNREGULATION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effects on lignification of downregulating most of the genes for enzymes on the monolignol biosynthetic pathway have been reasonably well studied. The exception to this is the crucial hydroxylase, cinnamate 3-hydroxylase (C3H), taking p-coumarate to caffeate. The Noble Foundation group has been ...

  19. Pharmacokinetics of buspirone as determined by ex vivo (/sup 3/H)-DPAT binding

    SciTech Connect

    Sethy, V.H.; Francis, J.W.

    1988-01-01

    Ex vivo (/sup 3/H)-8-hydroxy-2-(di-n-propylamino)-tetraline ((/sup 3/H)-DPAT) binding to the hippocampus has been utilized to determine the pharmacokinetic parameters of buspirone after i.v. and oral administration of this drug to rats. Intravenous buspirone rapidly penetrated the brain as demonstrated by a maximum inhibition of (/sup 3/H)-DPAT binding at 1 min. Elimination of drug from the brain was biphasic, with a first component half-life of 24.8 min and a second component half-life of 96 min. Oral buspirone at 3 times the i.v. dose produced less than one-third the maximum inhibition of (/sup 3/H)-DPAT binding compared to that observed with i.v. buspirone. The pharmacokinetic parameters of buspirone observed in the present study are in agreement with those reported previously. Thus, the ex vivo binding assay could be utilized to determine the bioavailability of the drug to the brain, and its duration of action. 20 references, 2 figures, 5 tables.

  20. Binding of /sup 3/H-acetylcholine to cholinergic receptors in bovine cerebral arteries

    SciTech Connect

    Shimohama, S.; Tsukahara, T.; Taniguchi, T.; Fujiwara, M.

    1985-11-18

    Cholinergic receptor sites in bovine cerebral arteries were analyzed using radioligand binding techniques with the cholinergic agonist, /sup 3/H-acetylcholine (ACh), as the ligand. Specific binding of /sup 3/H-ACh to membrane preparations of bovine cerebral arteries was saturable, of two binding sites, with dissociation constant (K/sub D/) values of 0.32 and 23.7 nM, and maximum binding capacity (Bmax) values of 67 and 252 fmol/mg protein, respectively. Specific binding of /sup 3/H-ACh was displaced effectively by muscarinic cholinergic agents and less effectively by nicotinic cholinergic agents. IC/sub 50/ values of cholinergic drugs for /sup 3/H-ACh binding were as follows: atropine, 38.5 nM; ACh, 59.8 nM; oxotremorine, 293 nM; scopolamine 474 nM; carbamylcholine, 990 nM. IC/sub 50/ values of nicotinic cholinergic agents such as nicotine, cytisine and ..cap alpha..-bungarotoxin exceeded 50 ..mu..M. Choline acetyltransferase activity was 1.09 nmol/mg protein/hour in the cerebral arteries. These findings suggest that the cholinergic nerves innervate the bovine cerebral arteries and that there are at least two classes of ACh binding sites of different affinities on muscarinic reporters in these arteries. 18 references, 2 figures, 2 tables.

  1. The discovery of CCR3/H1 dual antagonists with reduced hERG risk.

    PubMed

    Bahl, Ash; Barton, Patrick; Bowers, Keith; Brough, Steven; Evans, Richard; Luckhurst, Christopher A; Mochel, Tobias; Perry, Matthew W D; Rigby, Aaron; Riley, Robert J; Sanganee, Hitesh; Sisson, Adam; Springthorpe, Brian

    2012-11-01

    A series of dual CCR3/H(1) antagonists based on a bispiperidine scaffold were discovered. Introduction of an acidic group overcame hERG liability. Bioavailability was optimised by modulation of physico-chemical properties and physical form to deliver a compound suitable for clinical evaluation. PMID:23031591

  2. Excretion of (3H)prednisolone in clinically normal and experimentally infected bovine udders

    SciTech Connect

    Geleta, J.N.; Shimoda, W.; Mercer, H.D.

    1984-08-01

    The excretion rate of (3H)prednisolone from clinically normal and experimentally infected udders of 10 lactating cows was studied. Each quarter of 6 cows was injected with a single dose of (3H)prednisolone mixed with non-radioactive prednisolone equivalent to 10 mg in 10 ml of peanut oil base. Each of the remaining 4 cows was given 40 mg of nonradioactive prednisolone and (3H)prednisolone in 60% ethanol IV. Control and postadministration samples of blood, milk, and urine were examined for radioactivity. The effects of (3H)prednisolone were evaluated in the same cows, first in clinically normal udders, then 2 weeks later in udders experimentally infected with Streptococcus agalactiae. Absorption and elimination of prednisolone were the same before and after induced infection. Within 3 hours after intramammary injection, 95% of the labeled prednisolone was absorbed systemically, less than 5% of this dose was recovered in milk, and 29% was excreted in urine. After IV injection of (3H)prednisolone, less than 0.2% of the total radioactivity was recovered in milk and less than 46% was excreted in urine. Clinical mastitis induced by S agalactiae was moderate. Circulating blood leukocytes and somatic cells in the milk of normal cows remained essentially unchanged. The leukocyte response to induced infection was rapid in blood and milk. Large numbers of leukocytes were noticed in the milk and a severe leukopenia occurred. Prednisolone treatment did not alter the number of somatic cells in milk or reduce the inflammatory response of experimentally infected cows.

  3. A study of the C3H2 isomers and isotopologues: first interstellar detection of HDCCC

    NASA Astrophysics Data System (ADS)

    Spezzano, S.; Gupta, H.; Brünken, S.; Gottlieb, C. A.; Caselli, P.; Menten, K. M.; Müller, H. S. P.; Bizzocchi, L.; Schilke, P.; McCarthy, M. C.; Schlemmer, S.

    2016-02-01

    The partially deuterated linear isomer HDCCC of the ubiquitous cyclic carbene (c-C3H2) was observed in the starless cores TMC-1C and L1544 at 96.9 GHz, and a confirming line was observed in TMC-1 at 19.38 GHz. To aid the identification in these narrow line sources, four centimetre-wave rotational transitions (two in the previously reported Ka = 0 ladder and two new ones in the Ka = 1 ladder) and 23 transitions in the millimetre band between 96 and 272 GHz were measured in high-resolution laboratory spectra. Ten spectroscopic constants in a standard asymmetric top Hamiltonian allow the main transitions of astronomical interest in the Ka ≤ 3 rotational ladders to be calculated to within 0.1 km s-1 in radial velocity up to 400 GHz. Conclusive identification of the two astronomical lines of HDCCC was provided by the VLSR, which is the same as for the normal isotopic species (H2CCC) in the three narrow line sources. In these sources, deuterium fractionation in singly substituted H2CCC (HDCCC/H2CCC ~4-19%) is comparable to that in c-C3H2 (c-C3H2/c-C3HD ~5-17%) and similarly in doubly deuterated c-C3H2 (c-C3D2/c-C3HD ~3-17%), implying that the efficiency of the deuteration processes in the H2CCC and c-C3H2 isomers are comparable in dark clouds. Based on observations carried out with the IRAM 30 m Telescope. IRAM is supported by INSU/CNRS (France), MPG (Germany) and IGN (Spain).

  4. Cycloheximide resistance of Physarum polycephalum

    SciTech Connect

    Evans, T.E.; Evans, H.H.

    1980-08-01

    In the presence of cycloheximide, wild-type plasmodia of Physarum polycephalum exhibit an immediate decrease in deoxyribonucleic acid synthesis, a reduction in the incorporation of (/sup 3/H)thymidine into thymidine triphosphate, and an increase in the level of thymidine triphosphate, as well as a decrease in protein synthesis. In this study, we have utilized a cycloheximide-resistant (Cyc/sup r/) amoebic strain selected from a population of cells mutagenized with nitrosoguanidine.

  5. Diffusion of intracerebrally injected (1-/sup 14/C)arachidonic acid and (2-/sup 3/H)glycerol in the mouse brain. Effects of ischemia and electroconvulsive shock

    SciTech Connect

    Pediconi, M.F.; Rodriguez de Turco, E.B.; Bazan, N.G.

    1982-12-01

    (2-/sup 3/H)Glycerol and (1-/sup 14/C)arachidonic acid were injected into the region of the frontal horn of the left ventricle of mice and were distributed rapidly throughout the brain. After 10 sec, most of the radioactive fatty acid was found in the hemisphere near the injection site; after 10 min, it was recovered in similar proportions in the cerebellum and brain stem. (2-/sup 3/H)Glycerol showed a heterogeneous distribution, with most of the label remaining in the left hemisphere even after 10 min. On a fresh weight basis, cerebrum, cerebellum, and brain stem were found to contain similar amounts of labeled glycerol. However, the amount of (1-/sup 14/C)arachidonate in cerebrum was only 50% of that recovered from cerebellum or brain stem. Brain ischemia or a single electroconvulsive shock reduced the spread of the label, producing an accumulation of radioactivity in the injected hemisphere, except for an increase in (2-/sup 3/H)glycerol in the brain stem during ischemia. Despite the significant decrease in available precursor in the cerebellum and brain stem after electroshock, the amount of label incorporated into lipids was not altered in these areas and only slightly diminished in the cerebrum.

  6. The role of native defects in the transport of charge and mass and the decomposition of Li4BN3H10.

    PubMed

    Hoang, Khang; Janotti, Anderson; Van de Walle, Chris G

    2014-12-14

    Li4BN3H10 is of great interest for hydrogen storage and for lithium-ion battery solid electrolytes because of its high hydrogen content and high lithium-ion conductivity, respectively. The practical hydrogen storage application of this complex hydride is, however, limited due to irreversibility and cogeneration of ammonia (NH3) during the decomposition. We report a first-principles density-functional theory study of native point defects and defect complexes in Li4BN3H10, and propose an atomistic mechanism for the material's decomposition that involves mass transport mediated by native defects. In light of this specific mechanism, we argue that the release of NH3 is associated with the formation and migration of negatively charged hydrogen vacancies inside the material, and it can be manipulated by the incorporation of suitable electrically active impurities. We also find that Li4BN3H10 is prone to Frenkel disorder on the Li sublattice; lithium vacancies and interstitials are highly mobile and play an important role in mass transport and ionic conduction. PMID:25335576

  7. Differential effects of pertussis toxin on insulin-stimulated phosphatidylcholine hydrolysis and glycerolipid synthesis de novo. Studies in BC3H-1 myocytes and rat adipocytes

    SciTech Connect

    Hoffman, J.M.; Standaert, M.L.; Nair, G.P.; Farese, R.V. )

    1991-04-02

    Insulin-induced increases in diacylglycerol (DAG) have been suggested to result from stimulation of de novo phosphatidic acid (PA) synthesis and phosphatidylcholine (PC) hydrolysis. Presently, the authors found that insulin decreased PC levels of BC3H-1 myocytes and rat adipocytes by approximately 10-25% within 30 s. These decreases were rapidly reversed in both cell types, apparently because of increased PC synthesis de novo. In BC3H-1 myocytes, pertussis toxin inhibited PC resynthesis and insulin effects on the pathway of de novo PA-DAG-PC synthesis, as evidenced by changes in ({sup 3}H)glycerol incorporation, but did not inhibit insulin-stimulated PC hydrolysis. Pertussis toxin also blocked the later, but not the initial, increase in DAG production in the myocytes. Phorbol esters activated PC hydrolysis in both myocytes and adipocytes, but insulin-induced stimulation of PC hydrolysis was not dependent upon activation of PKC, since this hydrolysis was not inhibited by 500 {mu}M sangivamycin, an effective PKC inhibitor. The results indicate that insulin increases DAG by pertussis toxin sensitive and insensitive (PC hydrolysis) mechanisms, which are mechanistically separate, but functionally interdependent and integrated. PC hydrolysis may contribute importantly to initial increases in DAG, but later sustained increases are apparently largely dependent on insulin-induced stimulation of the pathway of de novo phospholipid synthesis.

  8. Synthesis and migration of /sup 3/H-fucose-labeled glycoproteins in the retinal pigment epithelium of albino rats, as visualized by radioautography

    SciTech Connect

    Haddad, A.; Bennett, G.

    1987-03-01

    /sup 3/H-fucose was injected into the vitreous body of the eye(s) of 250-gm rats, which were then killed by means of an intracardiac perfusion with glutaraldehyde after intervals of 10 min, 1 and 4 hr, and 1 and 7 days. The eyes were removed and further fixed, and pieces of retina were processed for light and electron microscope radioautography. Light microscope radioautography showed that the pigment epithelial cells actively incorporated /sup 3/H-fucose label. The intensity of reaction peaked at 4 hr after injection of the label and then slowly declined. Quantitative electron microscope radioautography revealed that, at 10 min after /sup 3/H-fucose injection, over 70% of the label was localized to the Golgi apparatus, indicating that fucose residues are added to newly synthesized glycoproteins principally at this site. With time the proportion of label associated with the Golgi apparatus decreased, but that assigned to the infolded basal plasma membrane, the apical microvilli, and various apical lysosomes increased. These results indicate that in retinal pigment epithelial cells newly synthesized glycoproteins continuously migrate from the Golgi apparatus to lysosomes and to various regions of the plasma membrane. In this case, the membrane glycoproteins may play specific roles in receptor functions of the basal plasma membrane or phagocytic activities at the apical surface. Very little label migrated to Bruch's membrane, indicating either a very slow turnover or a paucity of fucose-containing glycoproteins at this site.

  9. Comparative potencies of 3,4-methylenedioxymethamphetamine (MDMA) analogues as inhibitors of [3H]noradrenaline and [3H]5-HT transport in mammalian cell lines

    PubMed Central

    Montgomery, T; Buon, C; Eibauer, S; Guiry, P J; Keenan, A K; McBean, G J

    2007-01-01

    Background and purpose: Illegal ‘ecstasy' tablets frequently contain 3,4-methylenedioxymethamphetamine (MDMA)-like compounds of unknown pharmacological activity. Since monoamine transporters are one of the primary targets of MDMA action in the brain, a number of MDMA analogues have been tested for their ability to inhibit [3H]noradrenaline uptake into rat PC12 cells expressing the noradrenaline transporter (NET) and [3H]5-HT uptake into HEK293 cells stably transfected with the 5-HT transporter (SERT). Experimental approach: Concentration–response curves for the following compounds at both NET and SERT were determined under saturating substrate conditions: 4-hydroxy-3-methoxyamphetamine (HMA), 4-hydroxy-3-methoxymethamphetamine (HMMA), 3,4-methylenedioxy-N-hydroxyamphetamine (MDOH), 2,5-dimethoxy-4-bromophenylethylamine (2CB), 3,4-dimethoxymethamphetamine (DMMA), 3,4-methylenedioxyphenyl-2-butanamine (BDB), 3,4-methylenedioxyphenyl-N-methyl-2-butanamine (MBDB) and 2,3-methylenedioxymethamphetamine (2,3-MDMA). Key results: 2,3-MDMA was significantly less potent than MDMA at SERT, but equipotent with MDMA at NET. 2CB and BDB were both significantly less potent than MDMA at NET, but equipotent with MDMA at SERT. MBDB, DMMA, MDOH and the MDMA metabolites HMA and HMMA, were all significantly less potent than MDMA at both NET and SERT. Conclusions and implications: This study provides an important insight into the structural requirements of MDMA analogue affinity at both NET and SERT. It is anticipated that these results will facilitate understanding of the likely pharmacological actions of structural analogues of MDMA. PMID:17891159

  10. Assays for in vitro monitoring of proliferation of human airway smooth muscle (ASM) and human pulmonary arterial vascular smooth muscle (VSM) cells.

    PubMed

    Goncharova, Elena A; Lim, Poay; Goncharov, Dmitry A; Eszterhas, Andrew; Panettieri, Reynold A; Krymskaya, Vera P

    2006-01-01

    Vascular and airway remodeling, which are characterized by airway smooth muscle (ASM) and pulmonary arterial vascular smooth muscle (VSM) proliferation, contribute to the pathology of asthma, pulmonary hypertension, restenosis and atherosclerosis. To evaluate the proliferation of VSM and ASM cells in response to mitogens, we perform a [3H]thymidine incorporation assay. The proliferation protocol takes approximately 48 h and includes stimulating cells synchronized in G0/G1 phase of the cell cycle with agonists, labeling cells with [3H]thymidine and examining levels of [3H]thymidine incorporation by scintillation counting. Although using radiolabeled [3H]thymidine incorporation is a limitation, the greatest benefit of the assay is providing reliable and statistically significant data. PMID:17406550

  11. Thymidine kinase-mediated shut down of bone morphogenetic protein-4 expression allows regulated bone production.

    PubMed

    Lombardo, Barbara; Rocco, Teresa; Esposito, Maria T; Cantilena, Bruno; Gargiulo, Sara; Greco, Adelaide; Montanaro, Donatella; Brunetti, Arturo; Pastore, Lucio

    2013-06-01

    Bone morphogenetic Proteins (BMPs) are growth factors also involved in ossification and chondrogenesis that have generated interest for their efficiency in inducing bone neo-synthesis. BMPs expression in engineered cells has been successful in stimulating osteoblastic differentiation and ectopic and orthotopic bone formation in vivo. We have previously shown that an adenoviral vector expressing bone morphogenetic protein type-4 (BMP-4) is able to efficiently drive bone formation in a rabbit model of discontinuous bone lesions. However, unregulated secretion of BMPs has also been implicated in bone overproduction and exostosis. We have constructed a replication-defective first generation adenoviral (FG-Ad) vector containing a cassette for the expression of BMP-4 associated with the Herpes Simplex virus thymidine kinase (TK) gene (FG-B4TK) in order to shut down BMP-4 expression and, therefore, regulate bone production. TK expression does not interfere with BMP-4 ability to induce ectopic bone formation in athymic nude mice. Administration of ganciclovir blocks ectopic bone production in quadriceps muscle transduced with the FG-B4TK with no effect on the contralateral muscle transduced with a vector expressing only BMP-4. Histological findings confirmed the pro-apoptotic activity of TK and the reduction of mineralized areas in the quadriceps transduced with FG-B4TK in mice treated with ganciclovir. We have generated a system to block BMP-4 secretion by inducing apoptosis in transduced cells therefore blocking unwanted bone formation. This system is an additional tool to generate regulated amount of bone in discontinuous bone lesions and can be easily coupled with biomaterials capable of recruiting cells and generating a local bioreactor. PMID:23317056

  12. Bacterial Thymidine Kinase as a Non-Invasive Imaging Reporter for Mycobacterium tuberculosis in Live Animals

    PubMed Central

    Davis, Stephanie L.; Be, Nicholas A.; Lamichhane, Gyanu; Nimmagadda, Sridhar; Pomper, Martin G.; Bishai, William R.; Jain, Sanjay K.

    2009-01-01

    Background Bacteria can be selectively imaged in experimentally-infected animals using exogenously administered 1-(2′deoxy-2′-fluoro-β-D-arabinofuranosyl)-5-[125I]-iodouracil ([125I]-FIAU), a nucleoside analog substrate for bacterial thymidine kinase (TK). Our goal was to use this reporter and develop non-invasive methods to detect and localize Mycobacterium tuberculosis. Methodology/Principal Findings We engineered a M. tuberculosis strain with chromosomally integrated bacterial TK under the control of hsp60 - a strong constitutive mycobacterial promoter. [125I]FIAU uptake, antimicrobial susceptibilities and in vivo growth characteristics were evaluated for this strain. Using single photon emission computed tomography (SPECT), M. tuberculosis Phsp60 TK strain was evaluated in experimentally-infected BALB/c and C3HeB/FeJ mice using the thigh inoculation or low-dose aerosol infection models. M. tuberculosis Phsp60 TK strain actively accumulated [125I]FIAU in vitro. Growth characteristics of the TK strain and susceptibility to common anti-tuberculous drugs were similar to the wild-type parent strain. M. tuberculosis Phsp60 TK strain was stable in vivo and SPECT imaging could detect and localize this strain in both animal models tested. Conclusion We have developed a novel tool for non-invasive assessment of M. tuberculosis in live experimentally-infected animals. This tool will allow real-time pathogenesis studies in animal models of TB and has the potential to simplify preclinical studies and accelerate TB research. PMID:19606217

  13. Treatment of experimental human mesothelioma using adenovirus transfer of the herpes simplex thymidine kinase gene.

    PubMed Central

    Smythe, W R; Hwang, H C; Elshami, A A; Amin, K M; Eck, S L; Davidson, B L; Wilson, J M; Kaiser, L R; Albelda, S M

    1995-01-01

    OBJECTIVE: The authors demonstrate the ability of an adenovirus vector expressing the herpes simplex thymidine kinase (HSVtk) gene to treat human malignant mesothelioma growing within the peritoneal cavity of severe combined immunodeficient (SCID) mice. BACKGROUND DATA: Introduction of the HSVtk gene into tumor cells renders them sensitive to the antiviral drug ganciclovir (GCV). This approach has been used previously to treat experimental brain tumors. Although malignant mesothelioma is refractory to current therapies, its localized nature and the accessibility of the pleural space make it a potential target for a similar type of in vivo gene therapy using adenovirus. METHODS: An adenovirus containing the HSVtk gene (Ad.RSVtk) was used to transduce mesothelioma cells in vitro. These cells were then injected into the flanks of SCID mice. Ad.RSVtk was also injected directly into the peritoneal cavity of SCID mice with established human mesothelioma tumors. Mice were subsequently treated for 7 days with GCV at a dose of 5 mg/kg. RESULTS: Mesothelioma cells transduced in vitro with Ad.RSVtk formed nodules when injected in the subcutaneous tissue. These tumors could be eliminated by the administration of GCV, even when as few as 10% of cells were transduced to express HSVtk (bystander effect). Administration of Ad.RSVtk into the peritoneal space of animals with established multifocal human mesothelioma followed by GCV therapy resulted in the eradication of macroscopic tumor in 90% of animals and microscopic tumor in 80% of animals when evaluated after 30 days. The median survival of animals treated with Ad.RSVtk/GCV was significantly longer than that of control animals treated with similar protocols. CONCLUSION: These results indicate that an adenoviral vector containing the HSVtk gene is effective in treating established malignant mesothelioma in an in vivo setting and raise the possibility of using adenovirus transfer of HSVtk for clinical trials in mesothelioma and

  14. Cytosolic thymidine kinase is a specific histopathologic tumour marker for breast carcinomas.

    PubMed

    He, Qimin; Mao, Yongrong; Wu, Jainping; Decker, Catrine; Merza, Malik; Wang, Naining; Eriksson, Staffan; Castro, Juan; Skog, Sven

    2004-10-01

    Thymidine kinase 1 (TK1), an enzyme involved in the synthesis of precursors for DNA, and thus proliferation dependent, has been suggested as a good tumour marker. We have recently developed poly/monoclonal antibodies against TK1, which proved useful for diagnostics in both serum and immunohistochemistry of cancer patients. The anti-TK1 monoclonal antibodies (mAbs) 1D11 and 1E3 were characterized by Western blot, immunoprecipitation and flow cytometry. TK1 mAbs and Ki-67 mAb were then used for immunohistochemistry staining of tumour sections from 54 patients with ductal infiltrated breast carcinoma. Results showed the relative number of patients with positively stained tumours for TK1 (mAb 1D11) and for Ki-67 (mAb MIB-1) were 47 and 41%, respectively, significantly related (p=0.007). Combination of TK1 mAbs 1D11 and 1E3 increased this number to 56%, due to detection of a significantly higher number of patients with grade 2 tumours. Patients with stage II and grade 2 tumours showed significantly higher TK1 staining when compared to stage I and grade 1. Ki-67 staining was significantly higher in stage III and grade 3. The tumours only stained for TK1 represented higher stages and grades, while tumours staining only for Ki-67 were of lower stages and grades. Combining TK1 and Ki-67 increased the number of patients with positively stained tumours to 69%. In conclusion, TK1 is a reliable marker for identification of patients with grade 2 tumours. The highest number of patients with positively stained tumours were obtained when both TK1 and Ki-67 markers were used. PMID:15375544

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

    PubMed

    Khan, Zahidul; Knecht, Wolfgang; Willer, Mette; Rozpedowska, Elzbieta; Kristoffersen, Peter; Clausen, Anders Ranegaard; Munch-Petersen, B