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Sample records for 3h thymidine incorporation

  1. LHRH inhibits (/sup 3/H)thymidine incorporation by pituitary cells cultured IN VITRO

    SciTech Connect

    Stepien, H.

    1981-11-01

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

  2. 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.

  3. Effect of 5-Fluoro-2′-Deoxyuridine on [3H]Thymidine Incorporation by Bacterioplankton in the Waters of Southwest Florida

    PubMed Central

    Jeffrey, Wade H.; Paul, John H.

    1988-01-01

    The effect of 5-fluoro-2′-deoxyuridine (FdUrd) on [methyl-3H] thymidine incorporation by bacterioplankton populations in subtropical freshwater, estuarine, and oceanic environments was examined. In estuarine waters, intracellular isotope dilution was inhibited by FdUrd, which enabled us to estimate both intracellular and extracellular isotope dilution. In 2 of 10 cases, extracellular isotope dilution was significant. At low concentrations of [methyl-3H]thymidine or [6-3H]thymidine, FdUrd completely inhibited incorporation of radioactivity into protein and RNA. At high concentrations of [3H]thymidine, however, FdUrd had little effect on labeling patterns. The dihydrofolate reductase inhibitors amethopterin and trimethoprim had no effect on macromolecular labeling patterns. These results suggest that thymidylate synthase is not involved in nonspecific labeling and that FdUrd inhibits nonspecific labeling by blocking some other enzyme involved in thymidine catabolism. In oligotrophic oceanic and freshwater samples, FdUrd did not inhibit intracellular isotope dilution or [3H]thymidine labeling of protein and RNA, but caused some inhibition of [3H]thymidine incorporation into DNA. The ability of FdUrd to inhibit nonspecific macromolecular labeling during [3H]thymidine incorporation was significantly correlated (r = 0.84) with total thymidine incorporation (in picomoles per liter per hour). The results are discussed in terms of applications of FdUrd to routine bacterial production measurements and the general assumptions of [3H]thymidine incorporation. PMID:16347546

  4. 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.

  5. 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.

  6. 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.

  7. Depressed 3H-thymidine incorporation by measles infected mononuclear cells can be corrected by treatment with isoprinosine or 5-fluoro-2-deoxyuridine.

    PubMed Central

    Lomnitzer, R; Rabson, A R

    1987-01-01

    Incorporation of thymidine by measles infected PHA-activated lymphocytes was found to be depressed although production of interleukin 2 (IL-2) and expression of IL-2 receptors on these cells was similar to that of non-infected cells. The decrease in incorporation of 3H-thymidine by infected cells was not due to cell death and could be restored by treating the cells with isoprinosine or 5-fluoro-2-deoxyuridine. These results suggest that the depressed incorporation of 3H-thymidine by measles-infected cells is not due to inhibition of early events required for lymphocyte proliferation, but is rather due to interference in the thymidine pathway required for the synthesis of DNA. PMID:2443285

  8. Effect of copper administration on the incorporation of (3H)-thymidine into the liver DNA of rats stimulated by dimethylnitrosamine and diethylnitrosamine

    SciTech Connect

    Sakai, K.; Murata, N.; Chiba, K.; Yamane, Y.

    1981-01-01

    The incorporation of (3H)thymidine into liver DNA of rats increased 6--8 times 48 h after a single injection of dimethylnitrosamine (DMN, 30 mg/kg) and diethylnitrosamine (DEN, 100 mg/kg). To test the suppressive effect of copper, the incorporation of (3H)thymidine into liver DNA in the DMN groups or DEN groups pretreated with copper was measured 48 h after the administration of DMN or DEN. The incorporation of (3H)thymidine into liver DNA of rats stimulated by the injection of DMN was strikingly suppressed by the injection of cupric acetate (20 mg Cu/kg), but that of rats stimulated by the injection of DEN was not suppressed by the injection of copper. Some other metal salts, silver nitrate (20 mg Ag/kg), nickel acetate (20 mg Ni/kg) and basic lead acetate (20 mg Pb/kg) did not significantly suppress the incorporation of (3H)thymidine stimulated by DMN or DEN. The accumulation of copper was much higher in the liver of copper-administered rats than that of nickel or lead in the liver of nickel-administered rats or lead-administered rats. The accumulation of silver was comparatively high in the liver of silver-administered rats.

  9. 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).

  10. Cell kinetic effects of incorporated /sup 3/H-thymidine on proliferating human lymphocytes: flow cytometric analysis using the DNA/nuclear protein method

    SciTech Connect

    Pollack, A.; Moulis, H.; Greenstein, D.B.; Block, N.L.; Irvin, G.L. 3d.

    1985-09-01

    Phytohemagglutinin-stimulated human peripheral blood lymphocytes incorporating high concentrations of /sup 3/H-thymidine accumulate in G2 and show a consequent reduction in the number of cells entering M (division delay). The simultaneous flow cytometric analysis of DNA content (propidium iodide fluorescence) and nuclear protein content (fluorescein isothiocyanate fluorescence) allows for the accurate quantitation of these events; G2 and M are separated in the bivariate distributions. A good correlation was observed between mitotic indices, quantitated by manually counting mitotic cells, and integration of the M area in DNA/nuclear protein histograms. Moreover, significant differences in G2 nuclear protein levels were found between untreated and /sup 3/H-thymidine-treated lymphocytes. In order to characterize this effect, G2 was empirically divided into low nuclear protein (G2A) and high nuclear protein (G2B) compartments. /sup 3/H-thymidine caused an initial accumulation of lymphocytes in G2A, followed within 3-6 h by a gradual movement of some cells into G2B, with a subsequent accumulation of cells in G2B. The results suggest that the distribution of cells in G2 (G2A and G2B), the average nuclear protein content of G2B cells, and the proportion of cells in M are parameters that when used in combination provide a unique description of radiobiological effects.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. Evaluation of [3H]thymidine uptake method for studying growth of spiroplasmas under various conditions.

    PubMed Central

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

    1988-01-01

    [3H]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. PMID:3182999

  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. 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.

  18. Cytoenzymology and 3H-thymidine uptake of retro-ocular connective tissue cultures in experimental endocrino-exophthalmos.

    PubMed

    Vaida, E; Petrescu, R; Ghinea, E; Stefaneanu, L

    1976-01-01

    The in vitro retro-ocular connective tissue cultures from guinea pigs with endocrine exophthalmos were studied before and after retro-ocular treatment with cortisol and hyaluronidase. Both cortisol and hyaluronidase inhibited the cell proliferation, the cytoenzymic activities of oxydoreductases, the 3H-thymidine uptake, the number of mitoses and the protein content of cultivated cells.

  19. Fate of 3H-thymidine labelled myogenic cells in regeneration of muscle isografts.

    PubMed

    Gutmann, E; Mares, V; Stichová, J

    1976-03-05

    Intact and denervated extensor digitorum longus (EDL) muscles of 20-day-old inbred Lewis-Wistar rats were labelled with 3H-thymidine. Ninety minutes after the injection of the isotope 4.0% of the nuclei were labelled in the intact (i.e. innervated) and 9.6% in the muscles, denervated 3 days before administration of the isotope. The labelled EDL muscles were grafted into the bed of the previously removed EDL muscles of inbred animals and these isografts were studied 30 days later. In the EDL muscles, regenerated from innervated isografts only occasionally labelled endothelial cells were found whereas in the muscles regenerated from denervated isografts also parenchymal muscle nuclei were regularly labelled. The incidence of labelled nuclei in the regenerated EDL muscles was, however, about 20 times lower than in the donor EDL muscles. The presen experiments provide a direct proof of utilization of donor satelite cell nuclei for regeneration in grafted muscle tissue. With respect to the low incidence of labelled nuclei in regenerated EDL muscles, other sources of cells apparently also contribute to the regeneration process.

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

    PubMed

    Dubravsky, N B; Maor, M H; Withers, H R

    1985-08-01

    Radiation damage can be measured by decreased incorporation of 3H-TdR. The early effect of total body irradiation of mice, with doses up to 300-400 rad, of gamma rays of neutrons, on thymidine-3H incorporation into the DNA of murine proliferating normal and tumor cells are described. Total body irradiation with single doses up to 300 rad resulted in a steep dose-dependent depression of 3H-TdR incorporation into the DNA of the jejunal crypt, testis, spleen, fibrosarcoma (FSa), and FSa metastasis cells. The dose required to depress 3H-TdR incorporation values to 37% of control level (D37/thymidine) after gamma-irradiation was calculated to be 405, 443, 72, 303, and 531 rad, for jejunal crypt, testis, spleen, FSa metastasis, and FSa tumor cells, respectively. The depression progressed during the first 3 hours after irradiation. After neutron irradiation, the D37/thymidine was calculated to be 81, 140, 35, and 155 rad for jejunal crypt, testis, spleen, and FSa metastasis cells, respectively. The RBEn/gamma derived from these results were 5.00, 3.16, 2.06, and 1.95 for jejunal crypt, testis, spleen, and FSa metastasis cells, respectively. These results of D37/thymidine after gamma-irradiation and the RBEn/gamma correlate well with the 1Do for the initial slope of the survival curve and RBEn/gamma published in the literature for C3H/Kam mice using the same gamma and neutron beams. These findings show that cell survival after small doses of irradiation correlate with the effect of irradiation on the actively proliferating cells at the time of irradiation.

  3. 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.

  4. 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.

  5. Clinical correlations with chemosensitivities measured in a rapid thymidine incorporation assay

    SciTech Connect

    Sondak, V.K.; Bertelsen, C.A.; Tanigawa, N.; Hildebrand-Zanki, S.U.; Morton, D.L.; Korn, E.L.; Kern, D.H.

    1984-04-01

    A rapid assay for in vitro chemosensitivity testing measuring (3H)thymidine incorporation has been developed. Results of this assay correlate highly with chemosensitivities determined by the soft-agar clonogenic assay. A correlative study was carried out on 219 solid tumor specimens to assess the ability of the rapid assay to predict clinical response to antineoplastic therapy. One hundred forty-two of 219 tumors (65%) yielded chemosensitivity data. Of these, 33 were evaluable for in vitro-in vivo correlations. In vitro sensitivity (greater than or equal to 80% inhibition of thymidine uptake) was associated with clinical response in 6 of 13 patients. In vitro resistance was associated with progressive disease in 20 of 20 patients. The rapid assay offers several advantages over the soft-agar clonogenic assay, including higher success rate, avoidance of clumping artifact, shorter time course (5 days), and very low false-negative rate. Further refinement may be necessary, but the rapid assay appears to have potential for individualizing solid tumor chemotherapy.

  6. 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.

  7. Inhibitory Effect of Solar Radiation on Thymidine and Leucine Incorporation by Freshwater and Marine Bacterioplankton

    PubMed Central

    Sommaruga, R.; Obernosterer, I.; Herndl, G. J.; Psenner, R.

    1997-01-01

    We studied the effect of solar radiation on the incorporation of [(sup3)H]thymidine ([(sup3)H]TdR) and [(sup14)C]leucine ([(sup14)C]Leu) by bacterioplankton in a high mountain lake and the northern Adriatic Sea. After short-term exposure (3 to 4 h) of natural bacterial assemblages to sunlight just beneath the surface, the rates of incorporation of [(sup3)H]TdR and [(sup14)C]Leu were reduced at both sites by up to (symbl)70% compared to those for the dark control. Within the solar UV radiation (290 to 400 nm), the inhibition was caused exclusively by UV-A radiation (320 to 400 nm). However, photosynthetically active radiation (PAR) (400 to 700 nm) contributed almost equally to this effect. Experiments with samples from the high mountain lake showed that at a depth of 2.5 m, the inhibition was caused almost exclusively by UV-A radiation. At a depth of 8.5 m, where chlorophyll a concentrations were higher than those in the upper water column, the rates of incorporation of [(sup3)H]TdR were higher in those samples exposed to full sunlight or to UV-A plus PAR than in the dark control. In laboratory experiments with artificial UV light, the incorporation of [(sup3)H]TdR and [(sup14)C]Leu by mixed bacterial lake cultures was also inhibited mainly by UV-A. In contrast, in the presence of the green alga Chlamydomonas geitleri at a chlorophyll a concentration of 2.5 (mu)g liter(sup-1), inhibition by UV radiation was significantly reduced. These results suggest that there may be complex interactions among UV radiation, heterotrophic bacteria, and phytoplankton and their release of extracellular organic carbon. Our findings indicate that the wavelengths which caused the strongest inhibition of TdR and Leu incorporation by bacterioplankton in the water column were in the UV-A range. However, it may be premature to extrapolate this effect to estimates of bacterial production before more precise information on how solar radiation affects the transport of TdR and Leu into the cell

  8. 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.

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

    PubMed

    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. 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-H]adenine incorporation into adenylate residues in both RNA and DNA in parallel with the [H]thymidine experiments and compared the degree of nonspecific labeling by [H]adenine with that derived from [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 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.

  10. Thymidine Incorporation by Free-Living and Particle-Bound Bacteria in a Eutrophic Dimictic Lake

    PubMed Central

    Lovell, Charles R.; Konopka, Allan

    1985-01-01

    The percentage of [methyl-3H]thymidine incorporated into samples from a dimictic eutrophic lake and retained on polycarbonate membranes of 3.0-, 1.0-, and 0.2-μm pore size was studied in a lake with filamentous cyanobacteria as the dominant phytoplankton type throughout the period of thermal stratification. Water samples were also examined by epifluorescence microscopy for evidence of algal senescence and bacterial colonization of intact and damaged cyanobacterial filaments. A small percentage (2 to 20%) of bacterial activity was retained by filters with pore sizes ≥ 1 μm in epilimnetic samples. Epilimnetic samples also had a small percentage of cyanobacterial filaments, either intact or damaged, which were visibly colonized by bacteria in summer and fall samples. A significant proportion (20 to 35%) of bacterial activity was retained by filters with pore sizes ≥ 1 μm in samples collected from the metalimnion and hypolimnion during late summer and fall. The proportion of damaged cyanobacterial filaments was higher in these samples than in those from the epilimnion or from those obtained early in the summer. Furthermore, the filaments in these samples were more heavily colonized by bacteria. Overall, particle-bound production accounted for only 2 to 19% of total bacterial production from April to August in all water layers. It appears that the supply of colonizable particles (damaged cyanobacterial filaments) is an important factor affecting the level of particle-bound bacterial activity in this lake. PMID:16346744

  11. 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.

  12. 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.

  13. A microassay for colony-stimulating factor based on thymidine incorporation.

    PubMed Central

    Prystowsky, M. B.; Naujokas, M. F.; Ihle, J. N.; Goldwasser, E.; Fitch, F. W.

    1984-01-01

    A variety of growth factors and lectins were tested; only colony-stimulating factors CSF-1, Interleukin 3, and a T-lymphocyte GM CSF induced colony formation in semisolid medium and stimulated thymidine incorporation in liquid culture. All other growth factors and lectins were inactive in both assays. Factor-stimulated thymidine incorporation was detectable 24 hours after stimulation and reached maximal levels 4-6 days after stimulation. A convenient microassay for measuring CSF activity has been developed, enabling a large number of samples to be screened qualitatively in 2 days and permitting CSF activity to be measured quantitatively in 4-5 days. This microassay can supplement the clonal-cell assay method and be especially useful as an initial screening assay for CSF activity. PMID:6606982

  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. Multiple Heavy Metal Tolerance of Soil Bacterial Communities and Its Measurement by a Thymidine Incorporation Technique

    PubMed Central

    Díaz-Raviña, Montserrat; Bååth, Erland; Frostegård, Åsa

    1994-01-01

    A thymidine incorporation technique was used to determine the tolerance of a soil bacterial community to Cu, Cd, Zn, Ni, and Pb. An agricultural soil was artificially contaminated in our laboratory with individual metals at three different concentrations, and the results were compared with the results obtained by using the plate count technique. Thymidine incorporation was found to be a simple and rapid method for measuring tolerance. Data obtained by this technique were very reproducible. A linear relationship was found between changes in community tolerance levels obtained by the thymidine incorporation and plate count techniques (r = 0.732, P < 0.001). An increase in tolerance to the metal added to soil was observed for the bacterial community obtained from each polluted soil compared with the community obtained from unpolluted soil. The only exception was when Pb was added; no indication of Pb tolerance was found. An increase in the tolerance to metals other than the metal originally added to soil was also observed, indicating that there was multiple heavy metal tolerance at the community level. Thus, Cu pollution, in addition to increasing tolerance to Cu, also induced tolerance to Zn, Cd, and Ni. Zn and Cd pollution increased community tolerance to all five metals. Ni amendment increased tolerance to Ni the most but also increased community tolerance to Zn and, to lesser degrees, increased community tolerance to Pb and Cd. In soils polluted with Pb increased tolerance to other metals was found in the following order: Ni > Cd > Zn > Cu. We found significant positive relationships between changes in Cd, Zn, and Pb tolerance and, to a lesser degree, between changes in Pb and Ni tolerance when all metals and amendment levels were compared. The magnitude of the increase in heavy metal tolerance was found to be linearly related to the logarithm of the metal concentration added to the soil. Threshold tolerance concentrations were estimated from these linear

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

    PubMed

    Verhoef, J; Peterson, P K; Quie, P G

    1977-01-01

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

  17. 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.

  18. 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.

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

    SciTech Connect

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

    1988-06-01

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

  20. /sup 3/H arachidonic acid incorporation and metabolism in purified human basophils

    SciTech Connect

    Warner, J.A.; Peters, S.P.; Lichtenstein, L.M.; MacGlashan, D.W. Jr.

    1986-03-01

    A central feature of the allergic response is the generation of arachidonic acid (AA) metabolites by basophils and mast cells. In addition, AA metabolism may play a role in regulating the anti-IgE mediated degranulation of human basophils. To study this biochemistry, purified human basophils (>80%) were labeled with /sup 3/H-AA (0.3 ..mu..M, 25 ..mu..Ci/ml, 2 hours at 37/sup 0/C) and subsequently challenged with anti-IgE. Basophils were found to incorporate 45 +/- 3% of the exogenous AA which distributed into phospholipids (PL) (77.1 +/- 3.5%) and neutral lipids (19.7 +/- 3.3%) with only 5.3 +/- 2.7% remaining as the free acid (n = 7). Phosphatidylcholine (23.9 +/- 1.7%), phosphatidylinositol (22.0 +/- 1.4%) and phosphatidylethanolamine (14.5 +/- 2.7%) accounted for the majority of the AA with the remaining PL containing <3%. Anti-IgE (0.1 ..mu..g/ml) challenge led to the release of histamine (23.8 +/- 4.7%) and /sup 3/H-AA (8.1 +/- 1.7%) (n = 5). HPLC analysis revealed unmetabolized /sup 3/H-AA, /sup 3/H-LTC/sub 4/, /sup 3/H-HETE and an unidentified peak which migrated in the prostaglandin region of the elution profile. The same metabolites were released when the basophils were challenged with antigen. The calcium ionophore A23187 (1..mu..g/ml) also caused the release of histamine (37.4 +/- 4.1%) and /sup 3/H-AA (17.0 +/- 2.9%), while the phorbol ester, TPA caused HR (19.7 +/- 5.8%) but no increase in /sup 3/H AA turnover. Because of limited cell numbers this is the first time the authors have been able to study AA metabolism in human basophils.

  1. Migration and turnover of entero-endocrine and caveolated cells in the epithelium of the descending colon, as shown by radioautography after continuous infusion of 3H-thymidine into mice.

    PubMed

    Tsubouchi, S; Leblond, C P

    1979-12-01

    Adult male mice were given a continuous infusion of about 0.5 muCi of 3H-thymidine per gram body weight per day for periods varying from 1 to 60 days. Semithin sections of descending colon were cut from/plastic-embedded blocks and stained by a method combining silver impregnation and iron hematoxylin, by which argentaffin entero-endocrine cells and caveolated cells could be identified. From radioautographs, the labeling index of these cells was determined. One to three days after the beginning of 3H-thymidine infusion, label is observed in some of the stained entero-endocrine cells in the bottom of the crypts; the apices of these cells reach the crypt lumen and are joined to neighboring cells by terminal bars (junctional complexes). After five to seven days, labeled entero-endocrine cells are seen on the sides of the crypts, where their base stretches along the basement membrane and their apex has lost its terminal bar connections to neighboring cells. Finally, by 13 and 24 days, labeled cells are observed within the epithelium at the mucosal surface. The turnover time, which is taken to be equal to the mean time required for migration from site of origin to site of loss on the mucosal surface, has been estimated at 23.3 days. This is much longer than the 4.6 days required by the two main cell types of the epithelium -- vacuolated-columnar and mucous cells -- to travel the same route. It is likely that, after entero-endocrine cells lose their terminal bar attachment to other epithelial cells, they migrate independently and very slowly. Labeled caveolated cells are first seen in the crypt bottom one day after the beginning of 3H-thymidine infusion. By three to five days, they are on the sides of the crypts; their base is stretched along the basement membrane, but their apex retains its attachment to neighboring cells by terminal bars. By seven days, labeled caveolated cells are on the mucosal surface. Their turnover time has been assessed at 8.2 days. This is, again

  2. 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.

  3. Assay of anti-cancer drugs in tissue culture: conditions affecting their ability to incorporate 3H-leucine after drug treatment.

    PubMed

    Freshney, R I; Paul, J; Kane, I M

    1975-01-01

    An attempt has been made to construct an assay potentially suitable for use with primary cultures of human tumours to measure the survival of exponentially growing monolayer cultures after exposure to anti-neoplastic drugs. Cell survival was assessed using their protein synthetic capacity after removal of drugs. HeLa cells were employed to avoid the ingerent variability and heterogeneity of primary cultures from human tumours, and an assay has been constructed using microtitration trays to provide large numbers of replicate cultures without the requirement of a large number cells. An increase in the duration of the exposure to drug increased sensitivity in nearly all cases examined. Similarly, an increase in the period of culture following drug removal produced increased sensitivity to alkylating agents but allowed recovery from exposure to certain cycle-dependent drugs. Some of the drugs used were shown to be unstable under culture conditions and vinblastine was actively metabolized, although this instability was not necessarily reflected in the time course of the drug's effect. Mustine sensitivity was shown to be reduced by an increase in cell density at a level where density limitation of 3H-thymidine incorporation becomes apparent. These variations and possible methods of minimizing their effects are discussed.

  4. Assay of anti-cancer drugs in tissue culture: conditions affecting their ability to incorporate 3H-leucine after drug treatment.

    PubMed Central

    Freshney, R. I.; Paul, J.; Kane, I. M.

    1975-01-01

    An attempt has been made to construct an assay potentially suitable for use with primary cultures of human tumours to measure the survival of exponentially growing monolayer cultures after exposure to anti-neoplastic drugs. Cell survival was assessed using their protein synthetic capacity after removal of drugs. HeLa cells were employed to avoid the ingerent variability and heterogeneity of primary cultures from human tumours, and an assay has been constructed using microtitration trays to provide large numbers of replicate cultures without the requirement of a large number cells. An increase in the duration of the exposure to drug increased sensitivity in nearly all cases examined. Similarly, an increase in the period of culture following drug removal produced increased sensitivity to alkylating agents but allowed recovery from exposure to certain cycle-dependent drugs. Some of the drugs used were shown to be unstable under culture conditions and vinblastine was actively metabolized, although this instability was not necessarily reflected in the time course of the drug's effect. Mustine sensitivity was shown to be reduced by an increase in cell density at a level where density limitation of 3H-thymidine incorporation becomes apparent. These variations and possible methods of minimizing their effects are discussed. PMID:1156513

  5. Identification of an amphotericin B resistant strain of Candida albicans using a rapid 3H-glucose incorporation microassay.

    PubMed

    Sweeney, J F; Greene, J N; Hiemenz, J W; Wei, S; Rosemurgy, A S; Djeu, J Y

    1996-11-01

    Using a 3H-glucose incorporation assay, antifungal sensitivity testing undertaken on an isolate of Candida albicans cultured from the blood of a bone marrow transplant patient documented resistance to amphotericin B but sensitivity to fluconazole and itraconazole. Information obtained from in vitro antifungal sensitivity testing can be used to direct in vivo antifungal therapy. Widespread application of standardized in vitro antifungal sensitivity testing is needed.

  6. Comparative evaluation of the antiproliferative effect of cyclosporin A and gamma-interferon on normal and HPV-transformed keratinocytes by cell counting, MTT assay and tritiated thymidine incorporation.

    PubMed

    Marionnet, A V; Lizard, G; Chardonnet, Y; Schmitt, D

    1997-02-01

    We compared three techniques, the MTT tetrazolium assay, cell counting, and tritiated thymidine ([3H]TdR) incorporation assay to measure the antiproliferative effect of cyclosporin A (CsA) and interferon-gamma (IFN-gamma) on normal human skin keratinocyte cultures (NHK) used at the second passage and human papilomavirus type 16- and 18-transformed cell lines (EK16 and EK18) exposed continuously to the drugs for 3 days. The three techniques showed that under CsA (0.5 and 8 micrograms/ml) and IFN-gamma (5 and 160 U/ml) treatments the cells remained viable and that the growth of keratinocytes was inhibited. For IFN-gamma, the MTT colorimetric assay consistently underestimated its growth inhibitory activity as compared to cell counting or [3H]TdR incorporation, whatever the cells used. For high doses of CsA, MTT and cell counting gave similar percentages, of inhibitory activity whatever the cells; MTT underestimated this activity as compared to [3H]TdR incorporation only in NHK and EK18 cells, whereas similar results were obtained with EK16 cells. In conclusion, this investigations shows that MTT sensitivity differed with the drug and also according to the keratinocyte cultures. The MTT test is clearly not appropriate for study of IFN-gamma treatment whatever the keratinocytes used. Such discrepancies indicate that the MTT test should be done with care on cultures to measure the effects of drugs on cell growth; the growth inhibition should be carefully considered and it would be best if two different methods were used.

  7. 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.

  8. Perturbation of N-linked oligosaccharide structure results in an altered incorporation of (/sup 3/H)palmitate into specific proteins in Chinese hamster ovary cells

    SciTech Connect

    Wellner, R.B.; Ghosh, P.C.; Roecklein, B.; Wu, H.C.

    1987-09-25

    Increased (/sup 3/H)palmitate incorporation into specific cellular proteins has been reported to occur in Chinese hamster ovary and yeast mutant cells. In this paper we report studies concerning the relationship between N-linked oligosaccharide structure and (/sup 3/H)palmitate incorporation into proteins of Chinese hamster ovary (CHO) cells. We have compared the incorporation of (/sup 3/H)palmitate into proteins of wild-type and four different mutant CHO cell lines defective in various steps of N-linked protein glycosylation. Sodium dodecyl sulfate-gel electrophoretic analysis showed that three of the mutants exhibited increased (/sup 3/H)palmitate incorporation into several CHO cellular proteins (approximately 30,000-38,000 molecular weight) as compared to the wild-type cells. One of the affected mutants which accumulates the Man5Gn2Asn intermediate structure was examined in detail. In agreement with earlier reports, virtually all of the (/sup 3/H) palmitate-labeled proteins of both wild-type and mutant cell lines are membrane-bound. Pretreatment of the mutant cell line with tunicamycin blocked the increased (/sup 3/H)palmitate incorporation into the two specific proteins (both of approximately 30,000 molecular weight) observed in untreated cells; the decreased incorporation of (/sup 3/H)palmitate into the 30,000 molecular weight species was accompanied by a concomitant increase in the incorporation of (/sup 3/H)palmitate into two proteins of approximately 20,000 molecular weight. Pretreatment of wild-type cells with tunicamycin also caused increased (/sup 3/H)palmitate incorporation into the 20,000 molecular weight species.

  9. Adaptation of the 3H-leucine incorporation technique to measure heterotrophic activity associated with biofilm on the blades of the seaweed Sargassum spp.

    PubMed

    Coelho-Souza, Sergio A; Miranda, Marcio R; Salgado, Leonardo T; Coutinho, Ricardo; Guimaraes, Jean R D

    2013-02-01

    The ecological interaction between microorganisms and seaweeds depends on the production of secondary compounds that can influence microbial diversity in the water column and the composition of reef environments. We adapted the (3)H-leucine incorporation technique to measure bacterial activity in biofilms associated with the blades of the macroalgae Sargassum spp. We evaluated (1) if the epiphytic bacteria on the blades were more active in detritus or in the biofilm, (2) substrate saturation and linearity of (3)H-leucine incorporation, (3) the influence of specific metabolic inhibitors during (3)H-leucine incorporation under the presence or absence of natural and artificial light, and (4) the efficiency of radiolabeled protein extraction. Scanning electron microscopy showed heterogeneous distribution of bacteria, diatoms, and polymeric extracellular secretions. Active bacteria were present in both biofilm and detritus on the blades. The highest (3)H-leucine incorporation was obtained when incubating blades not colonized by macroepibionts. Incubations done under field conditions reported higher (3)H-leucine incorporation than in the laboratory. Light quality and sampling manipulation seemed to be the main factors behind this difference. The use of specific metabolic inhibitors confirmed that bacteria are the main group incorporating (3)H-leucine but their association with primary production suggested a symbiotic relationship between bacteria, diatoms, and the seaweed.

  10. 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.

  11. Modification of the 3H-leucine Incorporation Technique for Quantifying Rates of Bacterial Secondary Production on Decaying Wetland Plant Litter: Effectiveness of Microdialysis.

    NASA Astrophysics Data System (ADS)

    Gillies, J. E.; Francoeur, S. N.; Kuehn, K. A.

    2005-05-01

    The radiolabelled 3H-leucine incorporation technique for quantifying rates of bacterial production has increased in popularity since its original description for bacterioplankton communities. Prior studies addressing incorporation conditions (e.g., substrate saturation) for bacterial communities in other habitats, such as decaying plant litter, have reported a wide range of final leucine concentrations (400nM to 50,000nM) to achieve saturation-level uptake. We assessed the application of the 3H-leucine incorporation procedure for measuring bacterial production on decaying wetland plant litter. Substrate saturation experiments (9 concentrations, 10nM to 50,000nM final leucine) were conducted for bacterial communities colonizing submerged litter of three emergent plant species (Typha angustifolia, Schoenoplectus validus, and Phragmites australis). A modified 3H-leucine protocol was developed by coupling previously described incubation and extraction protocols with microdialysis (500MWCO) of the final radiolabelled protein extract. Incorporation of 3H-leucine into protein exhibited a biphasic saturation curve, with lower Km values ranging from 400nM to 1200nM depending on the plant species studied. Upper Km values ranged from 4000nM to 6000nM. Dialysis of the crude protein extract significantly improved counting precision and the signal-to-noise ratio. These results suggest differential uptake by litter associated microbial assemblages, with lower Km values possibly representing bacterial uptake and higher Km values representing non-bacterial uptake.

  12. Platelet-derived growth factor stimulation of (/sup 3/H)-glucosamine incorporation in density-arrested BALB/c-3T3 cells

    SciTech Connect

    Harrington, M.A.; Wharton, W.; Pledger, W.J.

    1987-01-01

    G/sub 0//G/sub 1/ traverse in density-arrested BALB/c-3T3 cells is controlled by multiple serum-derived growth factors. Platelet-derived growth factor (PDGF) initiates a proliferative response, whereas factors present in plasma facilitate progression through G/sub 0//G/sub 1/. In the absence of competence formation, progression factors are unable to stimulate cell cycle traverse. The authors have identified the stimulation of a biochemical process specific to competence formation in BALB/c-3T3 cells. PDGF treated BALB/c-3T3 cells incorporated 5-10 fold more (/sup 3/H)-glucosamine (GlcN) into acid-insoluble material as compared to platelet-poor plasma (PPP) treated cultures. Increased GlcN incorporation occurred in density-arrested BALB/c-3T3 cells in response to treatment with other competence factors, fibroblast growth factor, and Ca/sub 3/ (PO/sub 4/)/sub 2/ and was not due to cell-cycle traverse. Stimulation of (/sup 3/H)-GlcN incorporation by PDGF was time dependent, and increased incorporation of (/sup 3/H)-GlcN into protein required de novo protein synthesis. Several mechanisms through which PDGF could increase GlcN incorporation into cellular material were examined. Results of these studies suggest an increase in the cellular capacity to glycosylate proteins is a response to or a part of competence formation.

  13. IL-1. alpha. increases arachidonyl-CoA: Lysophospholipid acyltransterase activity and stimulates ( sup 3 H) arachidonate incorporation into phospholipids in rat mesangial cells

    SciTech Connect

    Nakazato, Y.; Sedor, J.R. )

    1992-01-01

    The proinflammatory cytokine interleukin-1{alpha} is a potent stimulus of prostaglandin synthesis. The authors have previously shown that IL-1 amplifies mesangial cell prostaglandin synthesis by inducing synthesis of a non-pancreatic phospholipase A{sub 2}. Phospholipase A{sub 2}. Phospholipase A{sub 2} activation results in the formation of lysophospholipids and free fatty acids. They now investigate the effects of IL-1{alpha} on reacylation of lysophospholipids. Incubations with IL-1{alpha} for 24 hours significantly stimulated mesangial cell ({sup 3}H)arachidonic acid incorporation but not ({sup 3}H)oleic acid incorporation into phosphatidylinositol and phosphatidylethanolamine. Lysophospholipid acyltransferase activity was measured in vitro. Cytokine treatment increased enzyme activity when lysophosphatidylcholine, lysophosphatidylethanolamine and lysophosphatidylinositol were used as exogenous substrates. They conclude that IL-1 promotes cellular phospholipid remodeling by stimulating the deacylation and reacylation of phospholipids.

  14. The influence of dietary nucleotides and long-chain polyunsaturated fatty acids on the incorporation of [3H] arachidonic acid on experimental liver cirrhosis.

    PubMed

    Leite, L H; Moreira-Vaz, E; Rosa, G; Pereira, A C; Monteiro, C R; Medeiros, F J; Chagas, V L

    2000-09-01

    The purposes of this study were to determine: a) the incorporation of labeled [3H] arachidonic acid on the intestinal mucosa, the liver and plasma, after 1,3 and 5 hours of administration, b) preferential incorporation by different tissues, c) and the effects on experimental rats with thioacetamide-induced cirrhosis, after four weeks of a dietary supplementation with nucleotides and long-chain polyunsaturated fatty acids. 209 female Wistar rats were divided into two groups (control and TAA group). The TAA group was given 300 mg of thioacetamide/L, in their drinking water for four months. After this period, a sample of 6 rats were taken from each group and examined, to evaluate the biochemical and histological changes of the experimental model, and 36 rats were taken to determine the incorporation of radioactivity by the groups. The rest of the animals were divided into four subgroups. Each group, receiving a supplementary diet with only long-chain polyunsaturated fatty acids and/or nucleotides or neither, for 4 weeks. After four months of thioacetamide, the incorporation of the [3H] arachidonic acid showed: a) an increased within 3 h in the intestinal mucosa, b) a decreased in the liver after 3 to 5 h c) and a drastic decrease in the plasma after 3 to 5 h. With a dietary supplementation of long-chain polyunsaturated fatty acids and nucleotides combined, there was a decrease of accumulate [3H] arachidonic acid in the intestine and a increase in the liver and plasma. The simultaneous supply of dietary polyunsaturated fatty acids and nucleotides was beneficial in the reversal of abnormalities of the lipid metabolism, in this experimental model of liver cirrhosis.

  15. Incorporation of /sup 35/S-sulfate and /sup 3/H-glucosamine into heparan and chondroitin sulfates during the cell cycle of B16-F10 cells

    SciTech Connect

    Blair, O.C.; Sartorelli, A.C.

    1984-05-01

    Changes in glycosaminoglycan composition occurring during the cell cycle were determined in B16-F10 cells sorted flow cytometrically with respect to DNA content. Incorporation of /sup 35/S-sulfate into heparan sulfate and chondroitin sulfate of unsorted and G1,S, and G2 +M sorted cells was determined following chondroitinase ABC or nitrous acid treatment; the incorporation into surface material was measured as the difference between the radioactivity of control and trypsin-treated cells. Incorporation of /sup 35/S-sulfate and /sup 3/H-glucosamine into cetyl pyridinium chloride (CPC)-precipitable material was characterized before and after chondroitinase or nitrous acid treatment by Sephadex G50 chromatography. Long-term (48 h) and short-term (1 h) labeling studies demonstrate that (a) the amount of total cellular chondroitin sulfate is greater than that of heparan sulfate, with larger amounts of unsulfated heparan than chondroitin being present; (b) the rate of turnover of heparan sulfate is greater than that of chondroitin sulfate; (c) greatest short-term incorporation of 3H-glucosamine into CPC-precipitable material occurs during S phase; and (d) the rate of turnover of both heparan sulfate and chondroitin sulfate is decreased in S phase relative to G1 and G2 + M.

  16. Radioactive contamination of the marine environment: Uptake and distribution of3H in Dunaliella bioculata

    NASA Astrophysics Data System (ADS)

    Strack, S.; Bonotto, S.; Kirchmann, R.

    1980-03-01

    The marine flagellate Dunaliella bioculata, which is easily cultivated under laboratory conditions, is a suitable organism for assessing the importance of the radioactive contamination by3H bound to organic molecules. We have studied the uptake of the following tritiated precursors: thymidine-methyl-3H, adenine-2-3H, uridine-5-3H, l-leucine-4-3H, glycine-2-3H, l-arginine-3.4-3H, 1-aspartic acid-2. 3-3H, 1-phenylalanine-2.3-3H, D-glucose-2-3H and D-glucose-6-3H. Under the experimental conditions (2000 lux; incubation time 30 min), all tritiated molecules are taken up by D. bioculata. Their intracellular concentration may reach that of the external medium. However, leucine and adenine accumulate in the algae: their respective concentrations are 10 and 100 times higher than in the culture medium. The molecular distribution of3H has been studied by various biochemical techniques and by sieve chromatography on sepharose 4B. It has been found that more l-leucine-4-3H is incorporated into acid and acetone soluble substances than into proteins. Adenine-2-3H is mainly incorporated into macromolecules of biological significance (RNA, DNA). CsCl gradient centrifugation has shown that the total DNA of Dunaliella is constituted by a major (ϖ=1.707 g/cm3) and by a minor (ϖ=1.693 g/cm3) component.

  17. 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.

  18. Determination of protein-bound palmitate turnover rates using a three-compartment model that formally incorporates [3H]palmitate recycling.

    PubMed

    Qanbar, Riad; Bouvier, Michel

    2004-09-28

    The observation that the palmitoylation state of certain proteins can be biologically modulated led to the proposal that it could, much like phosphorylation, be an important dynamic regulator of protein function. However, based on single-phase exponential decay analysis of data from [(3)H]palmitate pulse/chase experiments, the measured protein-bound palmitate turnover rates were often found to be too slow to account for rapid physiological responses. This paper reports that exponential decay does not adequately describe the results of such experiments because it fails to account for the recycling of [(3)H]palmitate from cellular lipids to palmitoyl CoA. Taking this recycling into account, a three-compartment model was used to deduce the time-dependent changes of cellular [(3)H]palmitoyl CoA and to infer the time course for the incorporation of [(3)H]palmitate into proteins. The validity of the inferences made by the model was checked against data obtained by metabolic labeling of endogenous HEK293 cell proteins. In addition, the model could account for reported anomalies, discrepancies, and apparently paradoxical observations obtained by traditional analysis of data from pulse/chase experiments. Including the recycling of cellular palmitate in the formal description of the system offers a new tool for quantitative assessment of protein-bound palmitate turnover rates. Through the re-evaluation of these rates, the model provides a means for the reassessment of the potential physiological implications of dynamic palmitoylation. The model may also be generally applicable to other areas of research where recycling of tracer is a concern.

  19. Effect of bovine serum albumin on the synthesis of prostaglandin and incorporation of (/sup 3/H)acetate into platelet-activating factor

    SciTech Connect

    Heinsohn, C.; Polgar, P.; Fishman, J.; Taylor, L.

    1987-09-01

    The binding of fatty acids by bovine serum albumin (BSA) is well documented. However, the interaction between the synthesis of prostaglandins (PGs) and the trapping of arachidonate released from cellular lipid stores is not as well understood. In this communication, we relate the trapping of fatty acids to the synthesis of PGs and the incorporation of (/sup 3/H)acetate into platelet-activating factor (PAF). Our results show that, as determined by radioimmunoassay, BSA inhibits bradykinin (BK) (5 ng/ml) and ionophore A23187 (10 microM)-stimulated synthesis of PGs in human embryo lung fibroblasts (IMR-90) in a concentration-dependent manner. Experiments using prelabel with (/sup 3/H)arachidonate followed by extraction and thin-layer chromatography show that, in the presence of 2 mg/ml BSA, IMR-90 release essentially only fatty acid following stimulation with bradykinin. Little if any prostaglandin and no endoperoxide are detected. In the same experiment, in absence of BSA, about 70% of the released label is detected as prostaglandin. alpha-Cyclodextrin, another trapper of fatty acid, inhibits PG synthesis in much the same way. BSA and alpha-cyclodextrin also inhibit prostacyclin synthesis in endothelial cells derived from the calf pulmonary artery. However, the inhibition of PG synthesis in these cells is not as complete as that in the IMR-90. In contrast to the effect of the trappers on PG synthesis, BSA and alpha-cyclodextrin are observed to potentiate BK- and ionophore-stimulated incorporation of (3H)acetate into PAF in the endothelial cells. The labeled PAF is not released from the cells in either the presence or absence of the trappers, leading us to conclude that BSA causes an increase in acetate-labeled cellular PAF by trapping released fatty acid.

  20. 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

  1. Does FDG uptake measure proliferative activity of human cancer cells? In vitro comparison with DNA flow cytometry and tritiated thymidine uptake.

    PubMed

    Higashi, K; Clavo, A C; Wahl, R L

    1993-03-01

    The relationship between 3H-2-fluoro-2-deoxy-D-glucose (FDG) uptake and the proliferative rate of a human ovarian adenocarcinoma cell line (HTB77IP3) was examined in vitro. HTB77IP3 cells were plated and allowed to grow through lag, exponential and plateau phases. Proliferative rate assessed by DNA flow cytometry and 3H-thymidine incorporation was highest in the lag phase and fell significantly as the cells progressed from the exponential through plateau phases. By DNA flow cytometry, the proliferation index (% of S+G2/M phase cells) fell from 65% to 23%. Thymidine uptake per cell also declined, by 82%, from lag to plateau phase. By contrast, 3H-FDG uptake per cell was largely unchanged as the cells progressed through the cell growth cycle. Total 3H-FDG uptake was strongly correlated with the number of viable cancer cells present (r = 0.957). Total thymidine uptake, however, substantially underestimated the number of viable cancer cells present. These in vitro differences in tracer uptake suggest that in this adenocarcinoma cell line, FDG measures a substantially different parameter (viable cell number) than thymidine (proliferative rate) and that these differences may result in disparate findings on PET imaging of cancers using these two tracers. Our data for this in vitro system indicate that FDG uptake does not relate to the proliferative activity of cancer cells. However, FDG uptake is strongly related to the number of viable tumor cells.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. Herpes simplex virus thymidine kinase activity of thymidine kinase-deficient Escherichia coli K-12 mutant transformed by hybrid plasmids.

    PubMed

    Kit, S; Otsuka, H; Qavi, H; Hazen, M

    1981-01-01

    A hybrid plasmid (pAGO) that contains the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene in the form of a 2-kilobase-pair (kbp) Pvu II fragment inserted at the Pvu II site of plasmid pBR322 was used to transform TK- Escherichia coli K-12 strain KY895. pAGO-transformed KY895 cells exhibited partially restored ability to incorporate [3H]dThd into DNA and an HSv-1-specific TK activity. Bacteria cured of plasmid pAGO (or transformed by plasmid pBR322) did not show enhanced incorporation of [3H]dThd into DNA or HSV-1 TK activity. Plasmid pMH1A was derived from pAGO by deletion of 2067 bp of DNA sequence from pBR322 and 105 bp from the HSV-1 TK gene. E. coli K-12 strain KY895 cells transformed by pMH1A did not show enhanced incorporation of [3H]dThd into bacterial DNA, although pMH1A DNA isolated from transformed KY895 cells, like pAGO DNA, did transform TK- mouse fibroblast [LM(TK-)] cells to the TK+ phenotype. The expression of HSV-1 TK activity by E. coli K-12 suggests that intervening sequences may be absent from the coding region of HSV-1 tk or that the coding region of the gene possesses short intervening sequences which do not disrupt the translational reading frame.

  7. 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.

  8. Kinetics of the formation of a G2 block from tritiated thymidine in phytohemagglutinin-stimulated human lymphocytes

    SciTech Connect

    Pollack, A.; Bagwell, C.B.; Irvin, G.L.; Jensen, J.A.

    1980-07-01

    Flow cytometry (FCM) was used to monitor the radiation effects promoted by incorporated tritiated thymidine (/sup 3/H-TdR) on phytohemagglutinin (PHA)-stimulated human peripheral blood lymphocytes stained with propidium iodide (PI). Lymphocyte microcultures were continuously labeled or pulse-labeled for various periods of time with different /sup 3/H-TdR concentrations. Two types of DNA histogram analyses were performed on unperturbed and /sup 3/H)TdR perturbed lymphocytes. The data analyses consisted of statistical analyses between averaged groups of histograms (nonparametric analysis) and cell cycle analyses (parametric analysis) to determine the percentages of cells in G0 + G1, S and G2 + M. The results showed that (a) /sup 3/H-TdR when added to proliferating lymphocytes under certain conditions (both short-term continuous and pulse-labeling) caused a highly significant increase in the proportion of tetraploid (4C) cells by FCM, (b) the increase in the proportion of 4C cells represented a block in G2 and (c) the relative increase in the percentage of 4C cells was proportional to /sup 3/H-TdR incorporation which was proportional to labeling time and concentration. Therefore, it was concluded that short labeling times be used to minimize adverse radiation effects when /sup 3/H-TdR is used to assay substances affecting lymphocyte proliferation or in the estimation of cell cycle time.

  9. 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.

  10. 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.

  11. 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.

  12. Functional expression of the Herpes simplex virus thymidine kinase gene in Escherichia coli K-12.

    PubMed

    Kit, S; Otsuka, H; Qavi, H; Kit, M

    1981-12-01

    The recombinant plasmid pAGO contains the Herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene and consists of a 2-kb PvuII fragment of HSV-1 DNA inserted into the PvuII site of pBR322. A deletion mutant of pAGO, designated pMH110, has been isolated which removes the normal HSV-1 TK gene promoter but places the promoter of the pBR322 tetracycline-resistance (tetr) gene only about 400 bp from the translational start codon of the HSV-1 TK polypeptide. In contrast to pAGO, which transforms mouse LM(TK-) cells to TK+ but is only weakly expressed in TK- bacteria, pMH110 not only efficiently transforms LM(TK-) cells to TK+ but also enables TK- Escherichia coli K-12 cells to form colonies on selective plates containing 5-fluorodeoxyuridine (FdUrd) plus thymidine (dThd) and to exhibit fully restored ability to incorporate [3H]dThd into DNA. The levels of TK activity expressed by bacteria harboring pMH110 were about as high as those expressed by bacteria harboring plasmid pTK3, which contains the wild-type E. coli TK gene. The TK activity expressed in bacteria harboring pMH110 was partially purified and shown to be HSV-1-specific by serological and disc PAGE analyses and by experiments demonstrating that this enzyme phosphorylated [125I]deoxycytidine.

  13. 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.

  14. Addition of thymidine to culture media for accurate examination of thymidine-dependent small-colony variants of methicillin-resistant Staphylococcus aureus: a pilot study.

    PubMed

    Horiuchi, Kazuki; Matsumoto, Takehisa; Ota, Yusuke; Kasuga, Eriko; Negishi, Tatsuya; Yaguchi, Tomomi; Sugano, Mitsutoshi; Honda, Takayuki

    2015-03-01

    Small-colony variants (SCVs) are slow-growing subpopulations of various auxotrophic bacterial strains. Thymidine-dependent SCVs (TD-SCVs) are unable to synthesize thymidine; hence, these variants fail to grow in a medium without thymidine. In this study, we used 10 TD-SCVs of Staphylococcus aureus, of which four strains possessed mecA. We compared the efficacy of a newly modified medium containing thymidine for the detection of TD-SCVs of methicillin-resistant S. aureus (MRSA) to the efficacy of routinely used laboratory media. We observed that none of the 10 TD-SCVs of S. aureus grew in Mueller-Hinton agar, and four TD-SCVs of MRSA failed to grow on all MRSA screening media, except for the ChromID™ MRSA medium. Laboratory tests conducted using medium with thymidine incorporated showed that thymidine did not affect the minimum inhibitory concentrations of oxacillin and cefoxitin for clinical isolates of S. aureus, and was able to detect MRSA, including TD-SCVs. These findings showed that thymidine-incorporated media are able to detect TD-SCVs of MRSA without altering the properties of other clinically isolated MRSA strains.

  15. 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.

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

    PubMed Central

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

    1989-01-01

    The thymidine kinase gene encoded by herpesvirus of turkeys has been identified and characterized. A viral mutant (ATR0) 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 [125I]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. Images PMID:2724415

  17. 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.

  18. 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.

  19. From {sup 3}He{at}C{sub 60} to {sup 3}H{at}C{sub 60}: Hot-atom incorporation of tritium in C{sub 60}

    SciTech Connect

    Khong, A.; Cross, R.J.; Saunders, M.

    2000-05-04

    The introduction of tritium into the cavity of C{sub 60} using hot-atom chemistry was studied. Neutron irradiation of {sup 3}He in {sup 3}He{at}C{sub 60} produced tritium ions and atoms which enter the C{sub 60}. {sup 3}H{at}C{sub 60} was detected with a scintillation counter. The Bingel reaction was carried out on this material under conditions so that the monoadduct formed predominantly, and the products were probed for tritium activity. Analysis with {sup 3}He NMR gave a peak with the characteristic {sup 3}He shift for the malonate product. The product fractions gave only 33% of the original activity; most was recovered in the unreacted C{sub 60} portion. No reaction was observed between C{sub 60} and the stable radical di-tert-butylnitroxide; all tritium activity was recovered in the C{sub 60} peak. These results suggest similarities in properties between {sup 3}H{at}C{sub 60} and pure C{sub 60}, implying that the tritium exists as a free atom inside the molecule.

  20. Thymidine analogues for tracking DNA synthesis.

    PubMed

    Cavanagh, Brenton L; Walker, Tom; Norazit, Anwar; Meedeniya, Adrian C B

    2011-09-15

    Replicating cells undergo DNA synthesis in the highly regulated, S-phase of the cell cycle. Analogues of the pyrimidine deoxynucleoside thymidine may be inserted into replicating DNA, effectively tagging dividing cells allowing their characterisation. Tritiated thymidine, targeted using autoradiography was technically demanding and superseded by 5-bromo-2-deoxyuridine (BrdU) and related halogenated analogues, detected using antibodies. Their detection required the denaturation of DNA, often constraining the outcome of investigations. Despite these limitations BrdU alone has been used to target newly synthesised DNA in over 20,000 reviewed biomedical studies. A recent breakthrough in "tagging DNA synthesis" is the thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU). The alkyne group in EdU is readily detected using a fluorescent azide probe and copper catalysis using 'Huisgen's reaction' (1,3-dipolar cycloaddition or 'click chemistry'). This rapid, two-step biolabelling approach allows the tagging and imaging of DNA within cells whilst preserving the structural and molecular integrity of the cells. The bio-orthogonal detection of EdU allows its application in more experimental assays than previously possible with other "unnatural bases". These include physiological, anatomical and molecular biological experimentation in multiple fields including, stem cell research, cancer biology, and parasitology. The full potential of EdU and related molecules in biomedical research remains to be explored.

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

    PubMed

    Sun, Ren; Wang, Liya

    2014-10-07

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

  2. 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.

  3. Muon Capture on ^3H

    NASA Astrophysics Data System (ADS)

    Golak, Jacek; Skibiński, Roman; Witała, Henryk; Topolnicki, Kacper; Kamada, Hiroyuki; Nogga, Andreas; Marcucci, Laura E.

    2017-01-01

    The μ ^- + ^3H → ν _μ + n + n + n capture reaction is studied under full inclusion of final-state interactions with the AV18 nucleon-nucleon potential and the Urbana IX three-nucleon force. We employ the single nucleon weak current operator comprising the dominant relativistic corrections to obtain first estimates of the total capture rates based on realistic forces. Our results are compared with older theoretical predictions.

  4. Malignant transformation induced by incorporated radionuclides in BALB/3T3 mouse embryo fibroblasts.

    PubMed Central

    LeMotte, P K; Adelstein, S J; Little, J B

    1982-01-01

    The induction of lethality and malignant transformation by 5-[125I]iododeoxyuridine and [3H]thymidine incorporated into cellular DNA and by x-irradiation was studied in vitro in BALB/3T3 cells. Under these conditions, 125I radiation is highly localized to small regions of the DNA at the site of each decay and produces DNA double-strand breaks with high efficiency. Incorporated 125I was found to be 12-16 times as lethal per decay as incorporated 3H. For the induction of malignant transformation, however, 125I was approximately 25 times as effective per decay as 3H. When the frequencies of transformation induced at various levels of survival by 125I, 3H, and x-rays were compared, lethality was found to correlate closely with transformation at doses that yielded significant cell killing. An exception occurred at low doses, where 125I appeared much more efficient than x-irradiation in inducing transformation; transformation frequencies equal to those induced by 3-5 Gy of x-rays resulted from 125I exposures that yielded little or no cell killing. PMID:6961448

  5. 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.

  6. Low dose exposure of diethylnitrosamine affects mice liver thymidine kinase.

    PubMed

    Pariat, T; Sharan, R N

    1995-11-17

    Swiss albino mice exposed to 5 and 10 mg diethylnitrosamine kg-1 body weight by intravenous route up to four weeks showed cyto- and genotoxic effects. Distortion of cell and nucleus shapes and extensive necrosis were observed. Thymidine kinase activity in the liver declined in diethylnitrosamine dose and duration dependent manners. The adult-form of thymidine kinase isozyme declined continuously during this period. Simultaneously, two isozymic forms of thymidine kinase, with small anodic migrations in an electrophoretic field, were gradually induced. Significance of theses changes in diethylnitrosamine induced precarcinogenic toxicity has been discussed.

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

    PubMed

    Suzuki, Toshinori; Kitabatake, Akihiko; Koide, Yuki

    2016-01-01

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

  8. 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.

  9. Transition state analysis of the arsenolytic depyrimidination of thymidine by human thymidine phosphorylase.

    PubMed

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

    2011-03-01

    Human thymidine phosphorylase (hTP) is responsible for thymidine (dT) homeostasis, promotes angiogenesis, and is involved in metabolic inactivation of antiproliferative agents that inhibit thymidylate synthase. Understanding its transition state structure is on the path to design transition state analogues. Arsenolysis of dT by hTP permits kinetic isotope effect (KIE) analysis of the reaction by forming thymine and the chemically unstable 2-deoxyribose 1-arsenate. The transition state for the arsenolytic reaction was characterized using multiple KIEs and computational analysis. Transition state analysis revealed a concerted bimolecular (A(N)D(N)) mechanism. A transition state constrained to match the intrinsic KIE values was found using density functional theory (B3LYP/6-31G*). An active site histidine is implicated as the catalytic base responsible for activation of the arsenate nucleophile and stabilization of the thymine leaving group during the isotopically sensitive step. At the transition state, the deoxyribose ring exhibits significant oxocarbenium ion character with bond breaking (r(C-N) = 2.45 Å) nearly complete and minimal bond making to the attacking nucleophile (r(C-O) = 2.95 Å). The transition state model predicts a deoxyribose conformation with a 2'-endo ring geometry. Transition state structure for the slow hydrolytic reaction of hTP involves a stepwise mechanism [Schwartz, P. A., Vetticatt, M. J., and Schramm, V. L. (2010) J. Am. Chem. Soc. 132, 13425-13433], in contrast to the concerted mechanism described here for arsenolysis.

  10. Phage display of ScFv peptides recognizing the thymidine(6–4)thymidine photoproduct

    PubMed Central

    Zavala, Anamaria G.; Lancaster, Thaddeus; Groopman, John D.; Strickland, Paul T.; Chandrasegaran, Srinivasan

    2000-01-01

    Solar ultraviolet (UV) radiation induces DNA photoproducts in skin cells and is the predominant cause of human skin cancers. To understand human susceptibility to skin cancer and to facilitate the development of prevention measures, highly specific reagents to detect and quantitate UV-induced DNA adducts in human skin will be needed. One approach towards this end is the use of monoclonal antibody-based molecular dosimetry methods. To facilitate the development of photoproduct-specific antibody reagents we have: (i) cloned and sequenced a single chain variable fragment (ScFv) gene coding for one such high affinity monoclonal antibody, αUVssDNA-1 (mAb C3B6), recognizing the thymidine(6–4)thymidine photoproduct; (ii) expressed and displayed the cloned ScFv gene on the surface of phage; (iii) selected functional recombinant phage by panning; (iv) purified the ScFv peptide; (v) shown that the purified ScFv peptide binds to UV-irradiated polythymidylic acid but not unirradiated polythymidylic acid. This is the first demonstration of the use of phage display to select a ScFv recognizing DNA damage. In addition, this is the initial step towards immortalizing the antibody gene for genetic manipulation, structure–function studies and application to human investigations. PMID:10710441

  11. Systematic differences in time of cerebellar-neuron origin derived from bromodeoxyuridine immunoperoxidase staining protocols and tritiated thymidine autoradiography: A comparative study.

    PubMed

    Martí, Joaquín; Santa-Cruz, M C; Serra, Roger; Hervás, José P

    2015-12-01

    As exogenous markers of DNA synthesis, 5-bromo-2'-deoxyuridine (BrdU) and tritiated thymidine ([(3)H]TdR) have revolutionized our ability to identify proliferating neuroblasts and follow their fate during the development of the central nervous system. The effect of the incorporation of these molecules into DNA on cell proliferation, migration and differentiation is frequently neglected (Duque and Rakic, 2011. J. Neurosci. 31, 15205-15217). By a progressively delayed cumulative labeling method, the current paper analyzes the development of the cerebellum in mice exposed to either BrdU or [(3)H]TdR as embryos and collected at postnatal day 90. We observed that, in comparison to the saline group, several parameters of the cerebellum such as length of the cerebellar cortex, the area of the molecular layer, Purkinje cell (PCs) number, the areas of the cerebellar nuclei, and the number of the deep cerebellar nuclei (DCN) neurons were lower in the BrdU injected group. No consequence of [(3)H]TdR administration was observed. On the other hand, we also studied whether immunohistochemical methods, including BrdU antibodies from different vendors (Sigma and Dako), partial DNA denaturation procedures and trypsin pretreatments, alter the neurogenetic timetables of PC and DCN neurons that resulted from analysis of these tissue specimens. Our analysis revealed that the generative programs of these macroneurons were unrelated to differences in the sensibility of BrdU antibodies but were dependent on the partial denaturation of DNA and trypsin digestion protocols. Finally, we also compare the generation and spatial distribution of PC and DCN neurons in mice exposed to either BrdU or [(3)H]TdR to assess whether the results obtained by these two markers are quantitatively similar. The data presented here show that systematic differences exist in the pattern of neurogenesis and the spatial location of cerebellar neurons between mice injected with BrdU or [(3)H]TdR. These findings have

  12. 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.

  13. Synthesis and biological evaluation of novel oxadiazole derivatives: a new class of thymidine phosphorylase inhibitors as potential anti-tumor agents.

    PubMed

    Shahzad, Sohail Anjum; Yar, Muhammad; Bajda, Marek; Jadoon, Bushra; Khan, Zulfiqar Ali; Naqvi, Syed Ali Raza; Shaikh, Ahson Jabbar; Hayat, Khizar; Mahmmod, Adeem; Mahmood, Nasir; Filipek, Sławomir

    2014-02-01

    Based on the fact that the thymidine phosphorylase inhibitors are considered potential anti-tumor agents, a range of novel oxadiazole derivatives 3a-3u was designed and synthesized by a simple and facile synthetic route. The biological assay revealed that majority of compounds displayed modest inhibitory activity against thymidine phosphorylase at low micromolar concentrations (IC50 173.23±3.04 to 14.40±2.45μM). In the current study the most active compounds were 3h and 3q with IC50 values 14.40±2.45 and 17.60±1.07μM, respectively. Molecular docking studies were performed on the most active compounds (3h, 3k, 3o-3q) to show their binding mode.

  14. Effect of {sup 3}H decays on DNA partition during cell division

    SciTech Connect

    Macieira-Coelho, A.

    1994-01-01

    Normal human embryonic lung fibroblasts were grown in the presence of different concentrations of tritium-labeled thymidine ([{sup 3}H]dThd) for various periods of time, and the flow of cells from metaphase to anaphase was followed. Mitotic indices were evaluated as a function of the percentage of cells in metaphase and anaphase. They revealed that the presence of [{sup 3}H]dThd induces a disturbance of the metaphase-anaphase transition. The DNA content of cells in metaphase and anaphase was measured with cytophotometry after the cells were grown in the presence of the radioactive precursor. It was found that the latter interfered with the flow of cells from metaphase to anaphase, impairing the symmetric distribution of DNA between daughter cells. 8 refs., 2 figs., 1 tab.

  15. 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)].

  16. Thymidine kinase mutants obtained by random sequence selection.

    PubMed

    Munir, K M; French, D C; Loeb, L A

    1993-05-01

    Knowledge of the catalytic properties and structural information regarding the amino acid residues that comprise the active site of an enzyme allows one, in principle, to use site-specific mutagenesis to construct genes that encode enzymes with altered functions. However, such information about most enzymes is not known and the effects of specific amino acid substitutions are not generally predictable. An alternative approach is to substitute random nucleotides for key codons in a gene and to use genetic selection to identify new and interesting enzyme variants. We describe here the construction, selection, and characterization of herpes simplex virus type 1 thymidine kinase mutants either with different catalytic properties or with enhanced thermostability. From a library containing 2 x 10(6) plasmid-encoded herpes thymidine kinase genes, each with a different nucleotide sequence at the putative nucleoside binding site, we obtained 1540 active mutants. Using this library and one previously constructed, we identified by secondary selection Escherichia coli harboring thymidine kinase mutant clones that were unable to grow in the presence of concentrations of 3'-azido-3'-deoxythymidine (AZT) that permits colony formation by E. coli harboring the wild-type plasmid. Two of the mutant enzymes exhibited a reduced Km for AZT, one of which displayed a higher catalytic efficiency for AZT over thymidine relative to that of the wild type. We also identified one mutant with enhanced thermostability. These mutants may have clinical potential as the promise of gene therapy is increasingly becoming a reality.

  17. 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.

  18. Elevation of serum thymidine kinase 1 in a bacterial infection: canine pyometra.

    PubMed

    Sharif, H; Hagman, R; Wang, L; Eriksson, S

    2013-01-01

    Pyometra is a bacterial infection of the uterus that is common in dogs and is potentially life-threatening if delayed in diagnosis and/or treatment. Thymidine kinase 1 (TK1) is a cytosolic enzyme involved in DNA precursor synthesis, and it is also present in serum from patients with malignant diseases. TK1 has been used as a cell proliferation biomarker for many years in human medicine and recently in dogs. However, little is known regarding serum TK1 levels in individuals with bacterial infection. The objective of this study was to determine the activity of serum TK1 in dogs with pyometra and compare it with hematologic and biochemical parameters, e.g., acute phase proteins and inflammatory mediators such as C-reactive protein and Prostaglandin F(2α). Serum and plasma TK1 activity of 40 healthy female dogs and 54 dogs with pyometra were analyzed using an optimized [(3)H]-thymidine phosphorylation assay. TK1 activities in serum or plasma were significantly higher in dogs with pyometra as compared with healthy female dogs (mean ± SD: 4.0 ± 7.3 pmol/min/mL in the pyometra group and 1.07 ± 0.34 pmol/min/mL in healthy control group). However, there was no difference in TK1 activity between systemic inflammatory response syndrome (SIRS) positive (n = 38) and SIRS negative (n = 16) pyometra cases. Furthermore, the plasma TK1 activity decreased in six and increased in one pyometra patients (n = 10), 24 h after ovariohysterectomy. No significant correlations (P > 0.05) were found between TK1 activity and hematological or other biochemical parameters. In conclusion, the TK1 activity was significantly elevated in dogs with pyometra. Further studies are needed to evaluate the mechanism and role of serum TK1 activity in bacterial infections and its possible diagnostic or prognostic value.

  19. 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

  20. Multicolor flow cytometry analysis of the proliferations of T-lymphocyte subsets in vitro by EdU incorporation.

    PubMed

    Sun, Yanli; Sun, Yu; Lin, Guigao; Zhang, Rui; Zhang, Kuo; Xie, Jiehong; Wang, Lunan; Li, Jinming

    2012-10-01

    EdU (5-ethynyl-2'-deoxyuridine) incorporation has proved advantageous in the studies of cell kinetics, DNA synthesis, and cellular proliferation in vitro and in vivo compared to [(3) H]thymidine incorporation and BrdU (5-bromo-2'-deoxyuridine) incorporation. Here, we describe a method that combines EdU incorporation and immunostaining with flow cytometric analysis to detect the proliferations of T lymphocyte subsets in vitro and optimized the assay's conditions. We found that the number of EdU(+) cells were associated with EdU concentration, incubation time, and the volume of Click reaction solution, the best EdU concentration 10-50 μM, the optimal incubation time 8-12 h and the proper volume of Click volume 100 μl for labeling 1 × 10(6) lymphocytes. Fixation was better to be performed before permeabilization, not together with. Furthermore, the permeabilization detergent reagent, PBS with 0.05% saponin was better than Tris buffer saline (TBS) with 0.1% Triton X-100. In addition, sufficient wash with PBS with 0.05% saponin has no influence on the staining of EdU(+) cells. Also, the lymphocytes incorporating EdU could be stored at 4°C, -80°C, and in liquid nitrogen up to 21 days. The present study will aid in optimization of flow cytometry assay to detect the proliferations of T cell subsets by EdU incorporation and the labeling of cell surface antigens.

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

    NASA Astrophysics Data System (ADS)

    Frecer, Vladimir; Seneci, Pierfausto; Miertus, Stanislav

    2011-01-01

    Thymidine monophosphate kinase (TMPKmt) 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 TMPKmt binding site. The three-dimensional structure of the TMPKmt complexed with 5-hydroxymethyl-dUMP, an analog of dTMP, was employed to develop a QSAR model, to parameterize a scoring function specific for the TMPKmt 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.

  2. Sequence of the canine herpesvirus thymidine kinase gene: taxon-preferred amino acid residues in the alphaherpesviral thymidine kinases.

    PubMed

    Rémond, M; Sheldrick, P; Lebreton, F; Foulon, T

    1995-12-01

    Multiple sequence alignments of evolutionarily related proteins are finding increasing use as indicators of critical amino acid residues necessary for structural stability or involved in functional domains responsible for catalytic activities. In the past, a number of alignments have provided such information for the herpesviral thymidine kinases, for which three-dimensional structures are not yet available. We have sequenced the thymidine kinase gene of a canine herpesvirus, and with a multiple alignment have identified amino acids preferentially conserved in either of two taxons, the genera Varicellovirus and Simplexvirus, of the subfamily Alphaherpesvirinae. Since some regions of the thymidine kinases show otherwise elevated levels of substitutional tolerance, these conserved amino acids are candidates for critical residues which have become fixed through selection during the evolutionary divergence of these enzymes. Several pairs with distinctive patterns of distribution among the various viruses occur in or near highly conserved sequence motifs previously proposed to form the catalytic site, and we speculate that they may represent interacting, co-ordinately variable residues.

  3. Comparison of /sup 3/H-galactose and /sup 3/H-glucose as precursors of hepatic glycogen in control-fed rats

    SciTech Connect

    Michaels, J.E.; Garfield, S.A.; Hung, J.T.; Cardell, R.R. Jr.

    1989-05-01

    Labeling of hepatic glycogen derived from 3H-galactose and 3H-glucose was compared shortly after intravenous injection in control-fed rats. The rats were allowed to accumulate 5-8% glycogen prior to receiving label. Fifteen minutes to 2 hours after labeling, liver was excised and processed for routine light (LM) and electron microscopic (EM) radioautography (RAG) or biochemical analysis. After injection of 3H-galactose, LM-RAGs revealed that the percentage of heavily labeled hepatocytes increased from 37% after 15 minutes to 68% after 1 hour but showed no further increase after 2 hours. alpha-Amylase treatment removed most glycogen and incorporated label; thus few silver grains were observed, indicating little incorporation of label except into glycogen. EM-RAGs demonstrated that most label occurred where glycogen was located. Biochemical analysis showed initially a high blood level of label that rapidly plateaued at a reduced level by 5 minutes. Concomitantly, glycogen labeling determined by liquid scintillation counting reflected the increases observed in the RAGs. After injection of 3H-glucose, LM-RAGs revealed that only 12% of the hepatocytes were heavily labeled at 1 hour and 20% at 2 hours. In tissue treated with alpha-amylase, glycogen was depleted and label was close to background level at each interval observed. EM-RAGs showed most grains associated with glycogen deposits. Biochemically, blood levels of label persisted at a high level for 30 minutes and tissue levels increased slowly over the 2-hour period. This study shows that incorporation from 3H-galactose was more rapid than incorporation of 3H-glucose; however, label derived from both carbohydrates appeared to be incorporated mainly into glycogen.

  4. Thymidine phosphorylase, 2-deoxy-D-ribose and angiogenesis.

    PubMed Central

    Brown, N S; Bicknell, R

    1998-01-01

    Angiogenesis is the term used to describe the formation of new blood vessels from the existing vasculature. In order to attract new vessels, a tissue must release an endothelial-cell chemoattractant. 2-Deoxy-D-ribose is produced in vivo by the catalytic action of thymidine phosphorylase (TP) on thymidine and has recently been identified as an endothelial-cell chemoattractant and angiogenesis-inducing factor. TP, previously known only for its role in nucleotide salvage, is now known to be angiogenic. TP expression is elevated in many solid tumours and in chronically inflamed tissues, both known areas of active angiogenesis. There is evidence that TP is also involved in physiological angiogenesis such as endometrial angiogenesis during the menstrual cycle. The majority of known endothelial-cell chemoattractants are polypeptides that bind to endothelial-cell-surface receptors. In contrast, 2-deoxy-D-ribose appears to lack a cell-surface receptor. Glucose is another sugar that acts as an endothelial-cell chemoattractant. The migratory activity of glucose is blocked by ouabain. It is possible that 2-deoxy-D-ribose and glucose stimulate endothelial-cell migration via a similar mechanistic pathway. PMID:9693094

  5. 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.

  6. Adrenergic receptor and catecholamine distribution in rat cerebral cortex: binding studies with [3H]prazosin, [3H]idazoxan and [3H]dihydroalprenolol.

    PubMed

    Diop, L; Brière, R; Grondin, L; Reader, T A

    1987-02-03

    The tritiated adrenergic antagonists [3H]dihydroalprenolol ([3H]DHA; beta-receptors), [3H]prazosin ([3H]PRZ; alpha 1-receptors), and [3H]idazoxan ([3H]IDA; alpha 2-receptors) were used to determine the distribution of these sites in 5 defined areas of the adult rat cerebral cortex. The highest density of [3H]PRZ binding was found in the prefrontal cortex, with a lower and homogeneous distribution for the frontal, parietal, occipital and temporal areas. The [3H]IDA binding sites were fairly uniform for all areas, except for the temporal cortex where it was very dense. In contrast, beta-adrenoceptors labelled by [3H]DHA were very homogeneous for all the regions examined. The functional significance of the distribution of alpha 1, alpha 2 and beta-adrenoceptors is discussed in relation to the catecholamine innervation and monoamine contents measured by high performance liquid chromatography.

  7. [3H]Ethynylbicycloorthobenzoate ([3H]EBOB) binding in recombinant GABAA receptors.

    PubMed

    Yagle, Monica A; Martin, Michael W; de Fiebre, Christopher M; de Fiebre, NancyEllen C; Drewe, John A; Dillon, Glenn H

    2003-12-01

    Ethynylbicycloorthobenzoate (EBOB) is a recently developed ligand that binds to the convulsant site of the GABAA receptor. While a few studies have examined the binding of [3H]EBOB in vertebrate brain tissue and insect preparations, none have examined [3H]EBOB binding in preparations that express known configurations of the GABAA receptor. We have thus examined [3H]EBOB binding in HEK293 cells stably expressing human alpha1beta2gamma2 and alpha2beta2gamma2 GABAA receptors, and the effects of CNS convulsants on its binding. The ability of the CNS convulsants to displace the prototypical convulsant site ligand, [35S]TBPS, was also assessed. Saturation analysis revealed [3H]EBOB binding at a single site, with a K(d) of approximately 9 nM in alpha1beta2gamma2 and alpha2beta2gamma2 receptors. Binding of both [3H]EBOB and [35S]TBPS was inhibited by dieldrin, lindane, tert-butylbicycloorthobenzoate (TBOB), PTX, TBPS, and pentylenetetrazol (PTZ) at one site in a concentration-dependent fashion. Affinities were in the high nM to low microM range for all compounds except PTZ (low mM range), and the rank order of potency for these convulsants to displace [3H]EBOB and [35S]TBPS was the same. Low [GABA] stimulated [3H]EBOB binding, while higher [GABA] (greater than 10 microM) inhibited [3H]EBOB binding. Overall, our data demonstrate that [3H]EBOB binds to a single, high affinity site in alpha1beta2gamma2 and alpha2beta2gamma2 GABAA receptors, and modulation of its binding is similar to that seen with [35S]TBPS. [3H]EBOB has a number of desirable traits that may make it preferable to [35S]TBPS for analysis of the convulsant site of the GABAA receptor.

  8. The equine herpes virus 4 thymidine kinase is a better suicide gene than the human herpes virus 1 thymidine kinase.

    PubMed

    Loubière, L; Tiraby, M; Cazaux, C; Brisson, E; Grisoni, M; Zhao-Emonet, J; Tiraby, G; Klatzmann, D

    1999-09-01

    The herpes simplex virus type 1 thymidine kinase suicide gene (HSV1tk) together with ganciclovir (GCV) have been successfully used for in vivo treatment of various experimental tumors, and many clinical trials using this system have been launched. With the aim to improve this therapeutic system, we compared the potential efficacy of different herpes virus derived thymidine kinases (HSV1, varicella-zoster virus, equine herpes virus type-4 and Epstein-Barr virus) as suicide genes in association with the nucleoside analogs acyclovir, ganciclovir and bromovinyldeoxyur- idine. Using various murine and human cell lines expressing these viral tk, we show that HSV1- and EHV4tk are the more efficient suicide genes for the different nucleoside analogs tested. Moreover, EHV4tk expressing murine and human cells were three- to 12-fold more sensitive to GCV than HSV1tk expressing cells. This was correlated with the presence of five-fold higher amounts of the toxic triphosphated-GCV in EHV4- versus HSV1tk expressing cells. Altogether, these experiments underline the potential advantages of the EHV4tk as a suicide gene.

  9. Cell-cycle analyses using thymidine analogues in fission yeast.

    PubMed

    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.

  10. N-H Stretching Excitations in Adenosine-Thymidine Base Pairs in Solution: Base Pair Geometries, Infrared Line Shapes and Ultrafast Vibrational Dynamics

    PubMed Central

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

    2013-01-01

    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 NH2 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

  11. 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

  12. Successive /sup 3/H and /sup 14/C labeling of DNA in a stimulus-response system

    SciTech Connect

    Lindgren, A.L.; Riley, E.F.

    1987-11-01

    Quiescent (G0) cells of the central zone region of the rat lens epithelium were recruited into the cell cycle by a wound stimulus. Cells were pulsed with labeled DNA precursor at several different times after the initiation of the DNA synthesis response to wounding and allowed to progress into the mitotic phase. Analysis of mitotic figures resulted in PLM (percentage labeled mitoses) curves that indicated a G2 duration of about 6 h. Double isotopic labeling ((/sup 3/H)thymidine followed by (/sup 14/C)thymidine) was utilized to demonstrate the completion of DNA synthesis in earliest responders. Cells completed DNA synthesis in less time (3-5 h) than reflected by the approximately 8-h widths of PLM curves. This discrepancy is attributed to the uptake and retention of labeled precursor by the stimulus-responsive cells while they are still in a pre-S phase condition. Based on a comparison of transit times through G2 and of labeling times to midpoint appearances of labeled mitotic figures, earlier responders do not appear to have faster rates of cell cycle progression than cells responding 2-4 h later. G2 transit time is also comparable for central zone lens cells responding to the relatively strong stimulus of wounding and for the nonperturbed cells previously studied in the germinative zone of the lens epithelium.

  13. Localization of thymidine phosphorylase in advanced gastric and colorectal cancer.

    PubMed

    Kobayashi, Michiya; Okamoto, Ken; Akimori, Toyokazu; Tochika, Naoshige; Yoshimoto, Tadashi; Okabayashi, Takehiro; Sugimoto, Takeki; Araki, Keijiro

    2004-01-01

    Thymidine phosphorylase (TP) is known to be more concentrated in human cancer tissues than in adjacent normal tissue based on findings using enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. However, the ultrastructural localization of TP in cancer tissues has not previously been demonstrated. We investigated the localization of TP in gastric cancer and colorectal cancer tissue by ELISA, immunohistochemistry, and immunoelectron microscopy. Between April 1997 and May 2000, we obtained surgically resected specimens from 42, 46, and 36 cases of advanced gastric, colon, and rectal cancer, respectively. ELISA demonstrated that the TP level was higher in cancer tissues than in adjacent normal tissue. Immunohistochemically, cancer cells were positive for the enzyme in some cases. However, in a number of cases immunopositive inflammatory cells were also present in cancerous tissues. At the electron microscope level, TP was diffusely distributed in the cytoplasm of cancer cells and in the mitochondria of the neutrophil in gastric cancer tissue. In rectal cancer tissues, cytoplasmic granules in macrophages in cancer tissues were immunoreactive for the TP. These findings suggest that TP is produced by macrophages and exists in neutrophils and cancer cells.

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

    PubMed Central

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

    2015-01-01

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

  15. 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.

  16. 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

  17. 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.

  18. Binding of [3H]-prazosin and [3H]-dihydroergocryptine to rat cardiac alpha-adrenoceptors.

    PubMed Central

    Guicheney, P.; Meyer, P.

    1981-01-01

    1 [3H]-prazosin binds specifically to a single class of alpha-adrenoceptors in rat cardiac membranes (KD25 degrees C = 0.2 nM). 2 That these receptors are of the alpha 1-type was indicated by competition studies, i.e. alpha 1-antagonists such as prazosin and (2-(2,6-dimethoxyphenoxyethyl) aminomethyl-1, 4-benzodioxane (WB 4101) were more potent than the alpha 2-antagonists, yohimbine and piperoxan in inhibiting [3H]-prazosin binding. 3 A comparative study of [3H]-prazosin binding and [3H]-dihydroergocryptine binding to cardiac membranes showed that both [3H]-prazosin and [3H]-dihydroergocryptine (at low concentrations) bind to alpha 2-adrenoceptors, while [3H]-dihydroergocryptine (at higher concentrations) also binds to another class of sites. PMID:6269682

  19. Delivery of imatinib-incorporated nanoparticles into lungs suppresses the development of monocrotaline-induced pulmonary arterial hypertension.

    PubMed

    Akagi, Satoshi; Nakamura, Kazufumi; Miura, Daiji; Saito, Yukihiro; Matsubara, Hiromi; Ogawa, Aiko; Matoba, Tetsuya; Egashira, Kensuke; Ito, Hiroshi

    2015-05-13

    Platelet-derived growth factor (PDGF) is implicated in the pathogenesis of pulmonary arterial hypertension (PAH). Imatinib, a PDGF-receptor tyrosine kinase inhibitor, improved hemodynamics, but serious side effects and drug discontinuation are common when treating PAH. A drug delivery system using nanoparticles (NPs) enables the reduction of side effects while maintaining the effects of the drug. We examined the efficacy of imatinib-incorporated NPs (Ima-NPs) in a rat model and in human PAH-pulmonary arterial smooth muscle cells (PASMCs). Rats received a single intratracheal administration of PBS, FITC-NPs, or Ima-NPs immediately after monocrotaline injection. Three weeks after monocrotaline injection, intratracheal administration of Ima-NPs suppressed the development of pulmonary hypertension, small pulmonary artery remodeling, and right ventricular hypertrophy in the rat model of monocrotaline-induced PAH. We also examined the effects of imatinib and Ima-NPs on PDGF-induced proliferation of human PAH-PASMCs by (3)H-thymidine incorporation. Imatinib and Ima-NPs significantly inhibited proliferation after 24 hours of treatment. Ima-NPs significantly inhibited proliferation compared with imatinib at 24 hours after removal of these drugs. Delivery of Ima-NPs into lungs suppressed the development of MCT-induced PAH by sustained antiproliferative effects on PAS-MCs.

  20. 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.

  1. Effects of delta9-THC on the synaptosomal uptake of 3H-tryptophan and 3H-choline.

    PubMed

    Johnson, K M; Dewey, W L

    1978-01-01

    The in vitro addition of (-)-delta9-tetrahydrocannabinol (delta9-THC) resulted in a dose-responsive inhibition of the high-affinity specific synaptosomal uptake of both 3H-tryptophan and 3H-choline in mouse forebrain crude synaptosomal preparations. The approximate concentrations of delta9-THC required to cause a 50% inhibition of the uptake of 3H-tryptophan and 3H-choline were 33 and 16 muM, respectively. Kinetic analysis showed that inhibition of both compounds were consistent with a noncompetitive mechanism. The pretreatment of mice with doses of 10, 30 or 100 mg/kg delta9-THC had no effect on the subsequent in vitro synaptosomal uptake of either 3H-tryptophan or 3H-choline into forebrain synaptosomes.

  2. 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.

  3. 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.

  4. 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.

  5. Characterization and performance of Pt/SBA-15 for low-temperature SCR of NO by C3H6.

    PubMed

    Liu, Xinyong; Jiang, Zhi; Chen, Mingxia; Shi, Jianwei; Shangguan, Wenfeng; Teraoka, Yasutake

    2013-05-01

    Pt supported on mesoporous silica SBA-15 was investigated as a catalyst for low temperature selective catalytic reduction (SCR) of NO by C3H6 in the presence of excess oxygen. The prepared catalysts were characterized by means of XRD, BET surface area, TEM, NO-TPD, NO/C3H6-TPO, NH3-TPD, XPS and 27Al MAS NMR. The effects of Pt loading amount, O2/C3H6 concentration, and incorporation of Al into SBA-15 have been studied. It was found that the removal efficiency increased significantly after Pt loading, but an optimal loading amount was observed. In particular, under an atmosphere of 150 ppm NO, 150 ppm C3H6, and 18 vol.% O2, 0.5% Pt/SBA-15 showed remarkably high catalytic performance giving 80.1% NOx reduction and 87.04% C3H6 conversion simultaneously at 140 degrees C. The enhanced SCR activity of Pt/SBA-15 is associated with its outstanding oxidation activities of NO to NO2 and C3H6 to CO2 in low temperature range. The research results also suggested that higher concentration of O2 and higher concentration of C3H6 favored NO removal. The incorporation of Al into SBA-15 improved catalytic performance, which could be ascribed to the enhancement of catalyst surface acidity caused by tetrahedrally coordinated AlO4. Moreover, the catalysts could be easily reused and possessed good stability.

  6. Addition of cysteamine to thymine and thymidine monophosphate, initiated by. gamma. -irradiation

    SciTech Connect

    Grachev, S.A.; Kropachev, E.V.; Litvyakova, G.I.

    1986-04-20

    On radiolysis of deaerated solutions containing thymine and cysteamine, or thymidine monophosphate and cysteamine, coupling products of these compounds are formed resulting from the addition of cysteamine to the double bond of the base. The radiochemical yields of the coupling products are dependent on the ratio of the concentration thymine (T) to that of cysteamine (RSH), or of thymidine monophosphate (TMP) to cysteamine in the irradiated solutions, and are at a maximum when (T)/(RSH) = 5-10, and (TMP)/(RSH) = 15-20. The mode of formation of coupling products involves reaction of H-, OH-, and e/sub aq//sup -/-adducts of thymine or thymidine monophosphate with cysteamine thiyl radicals.

  7. Binding of [3H]idazoxan and of its methoxy derivative [3H] RX821002 in human fat cells: [3H]idazoxan but not [3H] RX821002 labels additional non-alpha 2-adrenergic binding sites.

    PubMed

    Langin, D; Paris, H; Lafontan, M

    1990-06-01

    Binding studies were carried out in human fat cell membranes with two alpha 2-adrenergic antagonists, [3H]idazoxan and its methoxy derivative [3H]RX821002. Inhibition studies with epinephrine enantiomers indicate that [3H]RX821002 only binds to alpha 2-adrenoceptors, whereas [3H]idazoxan labels alpha 2-adrenoceptors and additional nonadrenergic sites (NAIBS). NAIBS and alpha 2-adrenoceptors display different affinities towards drugs from various chemical families. Imidazoline and some guanidine derivatives exhibit a high affinity for NAIBS. Pharmacological studies of human NAIBS indicate that they are slightly different from those previously reported in the rabbit, suggesting the existence of several subtypes of NAIBS. Furthermore, NAIBS are different from the previously described "imidazoline-preferring sites." [3H]idazoxan and [3H]RX821002 saturation analyses were performed in human adipocytes from different anatomical locations, in order to compare the number of NAIBS and alpha 2-adrenoceptors. Although there was an important variation in NAIBS and alpha 2-adrenoceptor numbers in the studied samples, a very poor correlation was obtained between the Bmax values of the two sites. Moreover, alkylation of alpha 2-adrenoceptors by phenoxybenzamine produces a 90% reduction in accessible [3H]RX821002 binding sites, without modification of [3H]idazoxan binding. These data show that NAIBS are not closely related to the alpha 2-adrenergic molecule. In addition, benextramine appears to be a reversible competitor at NAIBS. [3H]idazoxan binding, but not [3H]RX821002 binding, is sensitive to K+, suggesting that the domains involved in the ligand-NAIBS interaction are different from those involved in the ligand-alpha 2-adrenoceptor interaction.

  8. Double labeling of human leukemic cells using /sup 3/H-cytarabine and monoclonal antibody against bromodeoxyuridine

    SciTech Connect

    Raza, A.; Preisler, H.D.

    1985-02-01

    A new technique using immunofluorescence and autoradiography is described, in which the DNA of cells in S phase are labeled with two different probes. This method makes it possible to study the relationship between DNA synthesis and the uptake and/or incorporation of chemotherapeutic agents into normal or neoplastic cells. An example is provided in which the incorporation of /sup 3/H-cytarabine into DNA is demonstrated to occur only in cells which were synthesizing DNA during exposure to /sup 3/H-cytarabine. Other radioactively labeled probes can be used as well.

  9. /sup 3/H-imipramine uptake into rat striatal slices and imipramine-induced /sup 3/H-dopamine efflux

    SciTech Connect

    Saito, R.; Kawasaki, K.; Ono, N.; Kamiya, H.

    1983-04-01

    The effect of imipramine on spontaneous efflux of radiolabelled dopamine (DA) from slices of rat striatum was examined by a superfusion method. Imipramine at concentrations of 10 - 100 microM enhanced the efflux of DA accumulated in a high-affinity uptake system in a concentration-dependent manner. This efflux of /sup 3/H-DA was not affected by conditions (Ca/sup 2 +/-free medium, 100 microM bretylium and 30 microM tetrodotoxin) which inhibited the release of /sup 3/H-DA by electrical stimulation. Furthermore, this imipramine-induced /sup 3/H-DA efflux was temperature-dependent. The uptake of /sup 3/H-imipramine into striatal slices was determined. This uptake was concentration- and temperature-dependent and increased linearly. These results are discussed in relation to the hypothesis that /sup 3/H-DA efflux by imipramine is connected with uptake of imipramine.

  10. Assimilation of 3H in photosynthesizing leaves exposed to HTO.

    PubMed

    Guenot, J; Belot, Y

    1984-12-01

    Potato and wine grape plants were exposed for a period of 4 h to tritiated water vapor, and simultaneously to 14CO2 which served as a tracer for photosynthetic assimilation. During and after exposure, foliar samples were collected in which the exchangeable and nonexchangeable fractions of organic 3H were determined, taking care that the exchangeable fraction should not be lost to atmosphere. It was demonstrated that about 20% of the organic H in vegetation could be exchanged with 3H of tissue water, and that nonexchangeable 3H was fixed by photosynthesis. The kinetic behavior of the 2 forms was very different.

  11. [Enzymatic activity of thymidine kinase of herpes simlex virus strain resistant to H-phosphonates of Acv].

    PubMed

    Gus'kova, A A; Skoblov, M Iu; Andronova, V L; Galegov, G A; Kochetkov, S N; Skoblov, Iu S

    2011-01-01

    Cloned laboratory mutants of herpes simplex virus type I resistant to acycloguanosine H-phosphonate have been investigated. For all clones were shown that mutations resulted to increasing of sensitivity to acting of sidofovir. Thymidine kinase of mutant viruses partially preserves the ability to phosphorilate thymidine, but loses the ability to phosphorilate BVDU.

  12. Extensive labeling with [3H]ethanolamine of a hydrophilic protein of animal cells.

    PubMed

    Tisdale, E J; Tartakoff, A M

    1988-06-15

    Murine T-lymphomas and Thy-1- mutants were labeled overnight with [3H]ethanolamine to detect proteins which possess a glycophospholipid anchor. When labeled cells were treated with 10% trichloroacetic acid and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, both Thy-1 and a second intensely labeled protein (46 kDa) were observed. The presence of the radiolabeled 46-kDa protein in wild type and class E Thy-1 negative cells (cells in which Thy-1 is synthesized but cannot be labeled with [3H]ethanolamine) suggested incorporation into a distinct moiety. Labeling of the 46-kDa protein with [3H]ethanolamine is rapidly inhibited by cycloheximide. Further characterization of the 46-kDa protein by subcellular fractionation and Triton X-114 partitioning indicated that the protein is located in the cytosol. The protein is basic and does not bind to either concanavalin A or wheat germ agglutinin. Labeling of a 46-kDa protein has also been demonstrated in Chinese hamster ovary, COS, rat myeloma, cloned human T-lymphocytes, and HeLa cells. Pronase digestion of the [3H]ethanolamine-labeled 46-kDa protein of wild type lymphoma cells generated a nonbasic and polar labeled fragment which is labile to strong acid and base ([3H]ethanolamine is liberated), insensitive to periodate oxidation and alkaline phosphatase, and does not bind to concanavalin A or wheat germ agglutinin. Judging from methylation studies, the labeled ethanolamine residue does not contain a free amino group. Based on these results, we report a novel post-translational modification of selected protein(s) by the covalent addition of [3H]ethanolamine.

  13. Renal Aldosterone Receptors: Studies with [3H]Aldosterone and the Anti-Mineralocorticoid [3H]Spirolactone (SC-26304)

    PubMed Central

    Marver, Diana; Stewart, John; Funder, John W.; Feldman, David; Edelman, Isidore S.

    1974-01-01

    In vivo, a spirolactone (SC-26304) inhibited the effects of aldosterone on urinary K+:Na+ ratios and the binding of [3H]aldosterone to renal cytoplasmic and nuclear receptors. Cytoplasmic binding of [3H]aldosterone and [3H]spirolactone (SC-26304) was similar in magnitude and involved the same set of sites. Under three sets of conditions—(i) in the intact rat, (ii) in kidney slices, and (iii) in reconstitution studies (mixing prelabeled cytoplasm with either purified renal nuclei or chromatin), [3H]spirolactone (SC-26304) did not yield specific nuclear complexes in contrast to the reproducible generation of these complexes with [3H]aldosterone. In glycerol density gradients, cytoplasmic [3H]aldosterone receptor complexes sedimented at 8.5 S and 4 S in low concentrations of salt and at 4.5 S in high concentrations of salt. Cytoplasmic [3H]spirolactone (SC-26304) receptor complexes sedimented at 3 S in low concentrations of salt and 4 S in high concentrations of salt. These results are discussed in terms of an allosteric model of the receptor system. Images PMID:4364539

  14. (/sup 3/H) clonidine binding to rat hippocampal membranes

    SciTech Connect

    George, C.R.

    1982-02-01

    Alpha adrenergic receptor subtypes in rat hippocampal membranes were studied, using (/sup 3/H) clonidine as the radioactive ligand. On the basis of competitive binding studies, using the selective antagonist-prazosin, WB-4101, and yohimbine, (/sup 3/H) clonidine appeared to bind to a population of presynaptic sites that are pharmacologically similar to receptors previously classified as alpha 2. A computerized model that linearized and produced the best possible fit to the experimental data points indicated that (/sup 3/H) clonidine binds to a single population of receptors possessing equal affinity for the ligand. Binding data also indicated that rat hippo-campus contains significantly fewer (/sup 3/H)clonidine binding sites than rat cortex.

  15. Theoretical exploration of hydrogen loss from Al3H9.

    PubMed

    Nold, Christopher P; Head, John D

    2012-05-03

    The Al(3)H(9) and Al(3)H(7) potential energy surfaces were explored using quantum chemistry calculations to investigate the H(2) loss mechanism from Al(3)H(9), which provide new insights into hydrogen production from bulk alane, [AlH(3)](x), a possible energy storage material. We present results of B3LYP/6-311++G(d,p) calculations for the various Al(3)H(9) and Al(3)H(7) optimized local minima and transition state structures along with some reaction pathways for their interconversion. We find the energy for Al(3)H(9) decomposition into Al(2)H(6) and AlH(3) is slightly lower than that for H(2) loss and Al(3)H(7) formation, but the calculations show that H(2) loss from Al(3)H(9) is a lower energy process than for losing hydrogen from either Al(2)H(6) or AlH(3). We found four transition state structures and reaction pathways for Al(3)H(9) → Al(3)H(7) + H(2), where the lowest energy activation barrier is around 25-73 kJ/mol greater than the experimental value for H(2) loss from bulk alane. Intrinsic reaction coordinate calculations show that the H(2) loss pathway involves considerable rearrangement of the H atom positions around a single Al center. Three of the pathways start with the formation of an AlH(3) moiety, which then enables a terminal H on the AlH(3) to get within 1.1 to 1.2 Å of a nearby bridging H atom. The bridging and terminal H atoms eventually combine to form H(2) and leave Al(3)H(9). One implication of these H(2) loss reaction pathways is that, since the H atoms in bulk alanes are all at bridging positions, if a similar H(2) loss mechanism were to apply to bulk alane, then H(2) loss would most likely occur on the bulk alane surface or at a defect site where there should be more terminal H atoms available for reaction with nearby bridging H atoms.

  16. (/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.

  17. 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.

  18. Phytosphingosine biosynthesis differs from sphingosine in fish leukocytes and involves a transfer of methyl groups from [3H-methyl]methionine precursor.

    PubMed

    Bodennec, J; Zwingelstein, G; Koul, O; Brichon, G; Portoukalian, J

    1998-09-08

    We have studied the incorporation of radioactivity from either [3-3H]serine as the direct or [3H-methyl]methionine as the indirect precursor into sphingoid bases of free ceramides in lymphocytes from fish. Radioactivity from serine was incorporated mostly in the sphingosine moiety of ceramides. In contrast, the radioactivity from methionine was exclusively incorporated into phytosphingosine base (i.e., 4-hydroxy-sphinganine) and the incorporation increased by about twofold in the presence of folic acid or niacinamide. Identity of the long-chain bases, phytosphingosine and sphingosine, was established chemically by thin-layer chromatography, chemical degradation, and gas-liquid chromatography.

  19. Stimulation of [3H] GABA and beta-[3H] alanine release from rat brain slices by cis-4-aminocrotonic acid.

    PubMed

    Chebib, M; Johnston, G A

    1997-02-01

    cis-4-Aminocrotonic acid (CACA; 100 microM), an analogue of GABA in a folded conformation, stimulated the passive release of [3H] GABA from slices of rat cerebellum, cerebral cortex, retina, and spinal cord and of beta-[3H]alanine from slices of cerebellum and spinal cord without influencing potassium-evoked release. In contrast, CACA (100 microM) did not stimulate the passive release of [3H]taurine from slices of cerebellum and spinal cord or of D-[3H]aspartate from slices of cerebellum and did not influence potassium-evoked release of [3H]-taurine from the cerebellum and spinal cord and D-[3H]-aspartate from the cerebellum. These results suggest that the effects of CACA on GABA and beta-alanine release are due to CACA acting as a substrate for a beta-alanine-sensitive GABA transport system, consistent with CACA inhibiting the uptake of beta-[3H]alanine into slices of rat cerebellum and cerebral cortex. The observed Ki for CACA against beta-[3H]alanine uptake in the cerebellum was 750 +/- 60 microM. CACA appears to be 10-fold weaker as a substrate for the transporter system than as an agonist for the GABAc receptor. The effects of CACA on GABA and beta-alanine release provide indirect evidence for a GABA transporter in cerebellum, cerebral cortex, retina, and spinal cord that transports GABA, beta-alanine, CACA, and nipecotic acid that has a similar pharmacological profile to that of the GABA transporter, GAT-3, cloned from rat CNS. The structural similarities of GABA, beta-alanine, CACA, and nipecotic acid are demonstrated by computer-aided molecular modeling, providing information on the possible conformations of these substances being transported by a common carrier protein.

  20. 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.

  1. Rabbit blastocysts accumulate [3H]prostaglandins in vitro.

    PubMed

    Jones, M A; Harper, M J

    1984-08-01

    Rabbit blastocysts obtained on days 5, 6, and 6.8 of pregnancy were incubated in vitro in Tyrode's buffer with 3H-labeled prostaglandins (PGs). Accumulation of PGs was studied, using Whatman GF/F filters to separate bound and free ligands. The uptake and efflux of [3H]PGs were studied as a function of PG type, incubation time, temperature, and effect of metabolic inhibitors as well as age and number of blastocysts. Blastocysts of the same age accumulated approximately the same amount of [3H]PGE2 and [3H]PGF2 alpha from their environment; however, there was no apparent saturation over a PG concentration range of 1-1000 nM. Both the uptake and efflux of PG were age dependent, with older blastocysts accumulating more PGs. Approximately 90% of the [3H]PGs appear to be transported into the blastocoelic fluid, with little PG remaining in the blastomeres. PG accumulation was relatively insensitive to azide, ouabain, cyanide, or bromcresol green, but was affected by incubation at 0 C or the addition of indomethacin (10 micrograms/ml). No catabolism of the accumulated PGs was observed. The release of PGE2 in general did not differ from that of PGF2 alpha, except on day 6.8 of pregnancy when PGE2 was released more rapidly than on day 6. We conclude that rabbit blastocysts can accumulate PGs from their environment, which may imply a storage potential in the blastocyst and release before implantation.

  2. Base-modified thymidines capable of terminating DNA synthesis are novel bioactive compounds with activity in cancer cells

    PubMed Central

    Borland, Kayla M.; AbdulSalam, Safnas F.; Solivio, Morwena J.; Burke, Matthew P.; Wolfkiel, Patrick R.; Lawson, Sean M.; Stockman, Courtney A.; Andersen, Joel M.; Smith, Skyler; Tolstolutskaya, Julia N.; Gurjar, Purujit N.; Bercz, Aron P.; Merino, Edward J.; Litosh, Vladislav A.

    2015-01-01

    Current FDA-approved chemotherapeutic antimetabolites elicit severe side effects that warrant their improvement; therefore, we designed compounds with mechanisms of action focusing on inhibiting DNA replication rather than targeting multiple pathways. We previously discovered that 5-(α-substituted-2-nitrobenzyloxy)methyluridine-5′-triphosphates were exquisite DNA synthesis terminators; therefore, we synthesized a library of 35 thymidine analogs and evaluated their activity using an MTT cell viability assay of MCF7 breast cancer cells chosen for their vulnerability to these nucleoside derivatives. Compound 3a, having an α-tert-butyl-2-nitro-4-(phenyl)alkynylbenzyloxy group, showed an IC50 of 9 ± 1 μM. The compound is more selective for cancer cells than for fibroblast cells compared with 5-fluorouracil. Treatment of MCF7 cells with 3a elicits the DNA damage response as indicated by phosphorylation of γ-H2A. A primer extension assay of the 5′-triphosphate of 3a revealed that 3aTP is more likely to inhibit DNA polymerase than to lead to termination events upon incorporation into the DNA replication fork. PMID:25778768

  3. Extra thymidine stacks into the d(CTGGTGCGG).d(CCGCCCAG) duplex. An NMR and model-building study.

    PubMed Central

    van den Hoogen, Y T; van Beuzekom, A A; van den Elst, H; van der Marel, G A; van Boom, J H; Altona, C

    1988-01-01

    NMR and model-building studies were carried out on the duplex d(CTGGTGCGG).d(CCGCCCAG), referred to as (9+8)-mer, which contains an unpaired thymidine residue. Resonances of the base and of several sugar protons of the (9+8)-mer were assigned by means of a NOESY experiment. Interresidue NOEs between dG(4) and dT(5) as well as between dT(5) and dG(6) provided evidence that the extra dT is stacked into the duplex. Thermodynamic analysis of the chemical shift vs temperature profiles yielded an average TmD value of 334 K and delta HD of -289 kJmol-1 for the duplex in equilibrium random-coil transition. The shapes of the shift profiles as well as the thermodynamic parameters obtained for the extra dT residue and its neighbours again indicate that the unpaired dT base is incorporated inside an otherwise intact duplex. This conclusion is further supported by (a) the observation of an imino-proton resonance of the unpaired dT; (b) the relatively small dispersion in 31P chemical shifts (approximately 0.5 ppm) for the (9+8)-mer, which indicates the absence of t/g or g/t combinations for the phosphate diester torsion angles alpha/zeta. An energy-minimized model of the (9+8)-mer, which fits the present collection of experimental data, is presented. PMID:3368313

  4. Extra thymidine stacks into the d(CTGGTGCGG).d(CCGCCCAG) duplex. An NMR and model-building study.

    PubMed

    van den Hoogen, Y T; van Beuzekom, A A; van den Elst, H; van der Marel, G A; van Boom, J H; Altona, C

    1988-04-11

    NMR and model-building studies were carried out on the duplex d(CTGGTGCGG).d(CCGCCCAG), referred to as (9+8)-mer, which contains an unpaired thymidine residue. Resonances of the base and of several sugar protons of the (9+8)-mer were assigned by means of a NOESY experiment. Interresidue NOEs between dG(4) and dT(5) as well as between dT(5) and dG(6) provided evidence that the extra dT is stacked into the duplex. Thermodynamic analysis of the chemical shift vs temperature profiles yielded an average TmD value of 334 K and delta HD of -289 kJmol-1 for the duplex in equilibrium random-coil transition. The shapes of the shift profiles as well as the thermodynamic parameters obtained for the extra dT residue and its neighbours again indicate that the unpaired dT base is incorporated inside an otherwise intact duplex. This conclusion is further supported by (a) the observation of an imino-proton resonance of the unpaired dT; (b) the relatively small dispersion in 31P chemical shifts (approximately 0.5 ppm) for the (9+8)-mer, which indicates the absence of t/g or g/t combinations for the phosphate diester torsion angles alpha/zeta. An energy-minimized model of the (9+8)-mer, which fits the present collection of experimental data, is presented.

  5. Imaging of Sleeping Beauty-Modified CD19-Specific T Cells Expressing HSV1-Thymidine Kinase by Positron Emission Tomography

    PubMed Central

    Najjar, Amer M.; Manuri, Pallavi R; Olivares, Simon; Flores, Leo; Mi, Tiejuan; Huls, Helen; Shpall, Elizabeth J.; Champlin, Richard E.; Turkman, Nashaat; Paolillo, Vincenzo; Roszik, Jason; Rabinovich, Brian; Lee, Dean A.; Alauddin, Mian; Gelovani, Juri; Cooper, Laurence J.N.

    2016-01-01

    Purpose We have incorporated a positron emission tomography (PET) functionality in T cells expressing a CD19-specific chimeric antigen receptor (CAR) to non-invasively monitor the adoptively transferred cells. Procedures We engineered T cells to express CD19-specific CAR, firefly luciferase (ffLuc), and herpes simplex virus type-1 thymidine kinase (TK) using the non-viral-based Sleeping Beauty (SB) transposon/transposase system adapted for human application. Electroporated primary T cells were propagated on CD19+ artificial antigen-presenting cells. Results After 4 weeks, 90 % of cultured cells exhibited specific killing of CD19+ targets in vitro, could be ablated by ganciclovir, and were detected in vivo by bioluminescent imaging and PET following injection of 2′-deoxy-2′-[18F]fluoro-5-ethyl-1-β-D-arabinofuranosyl-uracil ([18F]FEAU). Conclusion This is the first report demonstrating the use of SB transposition to generate T cells which may be detected using PET laying the foundation for imaging the distribution and trafficking of T cells in patients treated for B cell malignancies. PMID:27246312

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

    PubMed

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

    2015-12-01

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

  7. The rational of catalytic activity of herpes simplex virus thymidine kinase. a combined biochemical and quantum chemical study.

    PubMed

    Sulpizi, M; Schelling, P; Folkers, G; Carloni, P; Scapozza, L

    2001-06-15

    Most antiherpes therapies exploit the large substrate acceptance of herpes simplex virus type 1 thymidine kinase (TK(HSV1)) relative to the human isoenzyme. The enzyme selectively phosphorylates nucleoside analogs that can either inhibit viral DNA polymerase or cause toxic effects when incorporated into viral DNA. To relate structural properties of TK(HSV1) ligands to their chemical reactivity we have carried out ab initio quantum chemistry calculations within the density functional theory framework in combination with biochemical studies. Calculations have focused on a set of ligands carrying a representative set of the large spectrum of sugar-mimicking moieties and for which structural information of the TK(HSV1)-ligand complex is available. The k(cat) values of these ligands have been measured under the same experimental conditions using an UV spectrophotometric assay. The calculations point to the crucial role of electric dipole moment of ligands and its interaction with the negatively charged residue Glu(225). A striking correlation is found between the energetics associated with this interaction and the k(cat) values measured under homogeneous conditions. This finding uncovers a fundamental aspect of the mechanism governing substrate diversity and catalytic turnover and thus represents a significant step toward the rational design of novel and powerful prodrugs for antiviral and TK(HSV1)-linked suicide gene therapies.

  8. 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.

  9. Effects of linalool on [(3)H]MK801 and [(3)H] muscimol binding in mouse cortical membranes.

    PubMed

    Brum, L F; Elisabetsky, E; Souza, D

    2001-08-01

    Linalool is a monoterpene compound reported to be a major component of essential oils of several aromatic species. Several linalool-producing species are used in traditional medical systems for sedative purposes, including the interruption and prevention of seizures. Previous studies in mice revealed that linalool modulates glutamatergic (competitive antagonism of L-[(3)H]glutamate binding, delayed intraperitoneal NMDA-induced convulsions and blockade of intracerebroventricular Quin-induced convulsions) and GABAergic transmission (protection against pentylenetetrazol and picrotoxin-induced convulsions). To further clarify the anticonvulsive mechanisms of linalool, we studied the effects of linalool on binding of [(3)H]MK801 (NMDA antagonist) and [(3)H]muscimol (GABA(A) agonist) to mouse cortical membranes. Linalool showed a dose dependent non-competitive inhibition of [(3)H]MK801 binding (IC(50) = 2.97 mM) but no effect on [(3)H]muscimol binding. The data suggest that the anticonvulsant mode of action of linalool includes a direct interaction with the NMDA receptor complex. The data do not, however, support a direct interaction of linalool with GABA(A) receptors, although changes in GABA-mediated neuronal inhibition or effects on GABA release and uptake cannot be ruled out.

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

    DOE PAGES

    Brune, C. R.; Caggiano, J. A.; Sayre, D. B.; ...

    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

  11. [Pharmacokinetics and biodistribution of 3H-norcantharidin in mice].

    PubMed

    Wei, Chun-Min; Wang, Ben-Jie; Ma, Ya; Sun, Zi-Ping; Li, Xiao-Li; Guo, Rui-Chen

    2007-05-01

    A single dose of 3H-norcantharidin solution was intragastrically given, blood, tissues, urine and feces were collected as scheduled, and radioactivity in these samples was determined by tritium tracing method to investigate the pharmacokinetics, tissue distribution and excretion of norcantharidin in Kunming mice. The pharmacokinetic characteristics of norcantharidin were evaluated by DAS version 2.0. The blood concentration reached to maximum 0. 5 h after intragastric administration. The radioactivity in tissues was high in small intestine, gallbladder, stomach, adrenal gland, kidney, heart and uterus 15 minutes after administration, descending with time, and high in gallbladder, adrenal gland and uterus 3 hours post dosing. The 24 h accumulative excretion ratio of urine and feces were 65.40% and 1.33% respectively. 3H-norcantharidin was easily absorbed after orally given to mice, the radioactivity was high and existed for a long-time in gallbladder, adrenal gland and uterus, and low but also existed for a long-time in large intestine, thymus and fat tissue. 3H-norcantharidin was declined quickly in small intestine, stomach, kidney and heart, and occurred rarely in brain. Norcantharidin was excreted mainly by urinary route and seldom in feces, which may be the cause of the urinary stimulation side effects observed. Because the radioactivity measured were the sum of 3H labeled norcantharidin and its metabolites, further studies on the disposition of norcantharidin in mammal animals, on the separation or identification of metabolites and, if any, on their activities, are fairly needed.

  12. Dual-channel detection of metallothioneins and mercury based on a mercury-mediated aptamer beacon using thymidine-mercury-thymidine complex as a quencher.

    PubMed

    Chen, Si-Han; Wang, Yong-Sheng; Chen, Yun-Sheng; Tang, Xian; Cao, Jin-Xiu; Li, Ming-Hui; Wang, Xiao-Feng; Zhu, Yu-Feng; Huang, Yan-Qin

    2015-01-01

    A novel dual-channel strategy for the detection of metallothioneins (MTs) and Hg(2+) has been developed based on a mercury-mediated aptamer beacon (MAB) using thymidine-mercury-thymidine complex as a quencher for the first time. In the presence of Hg(2+), the T-rich oligonucleotide with a 6-carboxyfluorescein (TRO-FAM) can form an aptamer beacon via the formation of T-Hg(2+)-T base pairs, which results in a fluorescence quenching of the sensing system owing to the fluorescence resonance energy transfer (FRET) from the fluorophore of FAM to the terminated T-Hg(2+)-T base pair. The addition of MTs into this solution leads to the disruption of the T-Hg(2+)-T complex, resulting in an increase of the fluorescent signal of the system. In the optimizing condition, ΔF was directly proportional to the concentrations ranging from 5.63 nM to 0.275 μM for MTs, and 14.2 nM to 0.30 μM for Hg(2+) with the detection limits of 1.69 nM and 4.28 nM, respectively. The proposed dual-channel method avoids the label steps of a quencher in common molecular beacon strategies, without tedious procedure or the requirement of sophisticated equipment, and is rapid, inexpensive and sensitive.

  13. Rabbit blastocysts accumulate (/sup 3/H)prostaglandins in vitro

    SciTech Connect

    Jones, M.A.; Harper, M.J.

    1984-08-01

    Rabbit blastocysts obtained on days 5, 6, and 6.8 of pregnancy were incubated in vitro in Tyrode's buffer with /sup 3/H-labeled prostaglandins (PGs). Accumulation of PGs was studied, using Whatman GF/F filters to separate bound and free ligands. The uptake and efflux of (/sup 3/H)PGs were studied as a function of PG type, incubation time, temperature, and effect of metabolic inhibitors as well as age and number of blastocysts. Blastocysts of the same age accumulated approximately the same amount of (/sup 3/H)PGE2 and (/sup 3/H)PGF2 alpha from their environment; however, there was no apparent saturation over a PG concentration range of 1-1000 nM. Both the uptake and efflux of PG were age dependent, with older blastocysts accumulating more PGs. Approximately 90% of the (/sup 3/H)PGs appear to be transported into the blastocoelic fluid, with little PG remaining in the blastomeres. PG accumulation was relatively insensitive to azide, ouabain, cyanide, or bromcresol green, but was affected by incubation at 0 C or the addition of indomethacin (10 micrograms/ml). No catabolism of the accumulated PGs was observed. The release of PGE2 in general did not differ from that of PGF2 alpha, except on day 6.8 of pregnancy when PGE2 was released more rapidly than on day 6. The authors conclude that rabbit blastocysts can accumulate PGs from their environment, which may imply a storage potential in the blastocyst and release before implantation.

  14. 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

  15. (/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.

  16. Monitoring DNA replication in fission yeast by incorporation of 5-ethynyl-2'-deoxyuridine.

    PubMed

    Hua, Hui; Kearsey, Stephen E

    2011-05-01

    We report procedures to allow incorporation and detection of 5-ethynyl-2'-deoxyuridine (EdU) in fission yeast, a thymidine analogue which has some technical advantages over use of bromodeoxyuridine. Low concentrations of EdU (1 µM) are sufficient to allow detection of incorporation in cells expressing thymidine kinase and human equilibrative nucleoside transporter 1 (hENT1). However EdU is toxic and activates the rad3-dependent checkpoint, resulting in cell cycle arrest, potentially limiting its applications for procedures which require labelling over more than one cell cycle. Limited DNA synthesis, when elongation is largely blocked by hydroxyurea, can be readily detected by EdU incorporation using fluorescence microscopy. Thus EdU should be useful for detecting early stages of S phase, or DNA synthesis associated with DNA repair and recombination.

  17. 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.

  18. 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.

  19. /sup 3/H)forskolin. Direct photoaffinity labeling of the erythrocyte D-glucose transporter

    SciTech Connect

    Shanahan, M.F.; Morris, D.P.; Edwards, B.M.

    1987-05-05

    Irradiation of erythrocyte ghosts in the presence of (/sup 3/H)forskolin resulted in a concentration-dependent, covalent incorporation of radiolabel into several of the major membrane protein bands. Most of the incorporation occurred in four regions of the gel. Peak 1 (216 kDa) was a sharp peak near the top of the gel in the region corresponding to spectrin. Peak 2 appeared to be associated with band 3 (89 kDa), while a third peak occurred around the position of band 4.2 (76 kDa). The fourth region of labeling was a broad area between 43-75 kDa which corresponds to the region of the glucose transporter. Forskolin labeling of this region was inhibited by cytochalasin B and D-glucose, but not L-glucose. Extraction of extrinsic membrane proteins resulted in a loss of radiolabeled protein from the 216- and 76-kDa regions. Treatment of membranes labeled with either cytochalasin B or forskolin with endo-beta-galactosidase resulted in identical shifts of the 43 to 75-kDa peaks to 42 kDa. Similarly, trypsinization of membranes photolabeled with either cytochalasin B or forskolin resulted in the generation of a 17-kDa radiolabeled fragment in both cases. Photoincorporation of (/sup 3/H)cytochalasin B into the glucose transporter was blocked in a concentration-dependent manner by unlabeled forskolin.

  20. Conformational changes in the H3. H4 histone complex

    SciTech Connect

    Feldman, L.; Beaudette, N.V.; Stollar, B.D.; Fasman, G.D.

    1980-08-10

    The H3.H4 complex has been extracted from calf thymus chromatin in either 0.05 M NaOAc, pH 5.0, or in 2.0 M KCl, 0.1 M KPO/sub 4/, pH 6.7, and both forms have been shown to consist solely of histones H3 and H4 when examined on sodium dodecyl sulfate-polyacrylamide gels. Serological and circular dichroism analyses indicate the existence of structural differences between the two forms. Dilution of the high salt form of the complex into low salt produces a time-dependent conformational change which is related to reduction in the ..cap alpha.. helix content of the complex and which exposes antigenic sites on the complex. The existence of multiple forms of the H3.H4 complex may be related to the dynamic equilibrium of nucleosome structure in vivo.

  1. Silver-Catalyzed C(sp(3))-H Chlorination.

    PubMed

    Ozawa, Jun; Kanai, Motomu

    2017-03-17

    A silver-catalyzed chlorination of benzylic, tertiary, and secondary C(sp(3))-H bonds was developed. The reaction proceeded with as low as 0.2 mol % catalyst loading at room temperature under air atmosphere with synthetically useful functional group compatibility. The regioselectivity and reactivity tendencies suggest that the chlorination proceeded through a radical pathway, but an intermediate alkylsilver species cannot be ruled out.

  2. (/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.

  3. 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.

  4. Localization of 3H-diethylstilbestrol in skeletal muscle

    SciTech Connect

    Gruber, B.; Cohen, L.

    1981-11-01

    The localization of diethylstilbestrol (DES) in skeletal muscle was studied in CF1 mice and perfused rat hindlimbs. There was a slow accumulation of 3H-DES in mouse muscle from 4 to 24 hours following i.p. injection even though plasma DES was decreasing. Twenty-four hours after injection of 50 microCi 3H-DES (714 pmole) mouse gastrocnemius contained 8.9 x 10(-17) mole unaltered 3H-DES per mg muscle. Extrapolating to the entire skeletal muscle mass of the animal, this represents 0.15% of the radioactivity injected. The radioactivity in muscle was completely extracted with 95% ethanol or ether: ethanol (3:1), and both unaltered DES and DES-metabolites were present in the extracts. The fraction of radioactivity due to unaltered DES 4 hours after injection was 0.51 +/- 0.09 in muscle and 0.30 +/- 0.11 in plasma. Significant extrahepatic metabolism of DES was demonstrated in perfused isolated rat hindlimbs by the presence of DES-metabolites in the perfusate. The radioactivity extracted from the perfused muscle itself was unaltered DES. These results indicate that skeletal muscle is an important site of DES localization in rodents.

  5. Effect of sequence of administration on the pharmacokinetic interaction between the anticholinergic drug biperiden and [3H]quinuclidinyl benzylate or [3H]N-methylscopolamine in rats.

    PubMed

    Ishizaki, J; Yokogawa, K; Nakashima, E; Takayasu, T; Ohshima, T; Ichimura, F

    1998-02-01

    In rats the pharmacokinetic interactions between the anticholinergic drug biperiden and [3H]quinuclidinyl benzylate ([3H]QNB) or [3H]N-methylscopolamine ([3H]NMS) is affected by the sequence in which the drugs are administered. Drug concentrations in various tissues were determined after intravenous administration of [3H]QNB or [3H]NMS (325 ng kg(-1)). Biperiden (6.4 mg kg(-1)) was administered either 5 min before, concomitantly with or 20 min after injection of [3H]QNB or [3H]NMS. When biperiden was administered concomitantly with or before [3H]QNB, distribution of [3H]QNB among the regions of the brain and other tissues was reduced; at 4 h the ratio of the distribution of [3H]QNB for experimental animals to that for control animals ranged from 0.15 to 0.9. When biperiden was administered after [3H]QNB, the distribution of [3H]QNB in the brain and other tissues was significantly higher than for the other two treatments (P < 0.01). However, for [3H]NMS the sequence of administration had no effect on the distribution of the drug in the brain and other tissues except for the kidney. In-vitro, in crude synaptosomal membranes, the amount of [3H]QNB at 2 h relative to the control concentration at equilibrium was 87% when biperiden was added before [3H]QNB and 56% when biperiden was added after [3H]QNB. In both instances the concentration of [3H]NMS reached equilibrium within 30 min. These findings suggest that the difference between the rate constant of association and dissociation at the possible site of action gives rise to the effect of the sequence of administration on the pharmacokinetic interaction.

  6. In vivo binding of (/sup 3/H)Ro 15-1788 in mice: comparison with the in vivo binding of (/sup 3/H)flunitrazepam

    SciTech Connect

    Potier, M.C.; de Carvalho, L.P.; Dodd, R.H.; Brown, C.L.; Rossier, J.

    1988-01-01

    Benzodiazepine binding sites have generally been labelled with benzodiazepine agonists: (/sup 3/H)flunitrazepam and (/sup 3/H)diazepam in vivo. The authors studied the in vivo binding of the antagonist (/sup 3/H)Ro 15-1788 in mice and compared it to the in vivo binding of (/sup 3/H)flunitrazepam. For this in vivo labelling, mice were injected with labelled and unlabelled ligands. Animals were then sacrificed and bound radioactivity was measured after homogenization of the excised brain and filtration of the homogenate. (/sup 3/H)Ro 15-1788 is a better tool than (/sup 3/H)flunitrazepam for in vivo labelling of benzodiazepine receptors since 1) it labels specifically the central type binding sites, 2) injection of 4 times less (/sup 3/H)Ro 15-1788 than (/sup 3/H)flunitrazepam produced the same amount of bound radioactivity, 3) 70-90% of the total (/sup 3/H)Ro 15-1788 present in the brain is membrane bound instead of 45-55% with (/sup 3/H)flunitrazepam, 4) maximal binding of (/sup 3/H)Ro 15-1788 is reached within 3 min, 5) only 5% of the membrane bound (/sup 3/H)Ro 15-1788 is nonspecific instead of 15% for (/sup 3/H)flunitrazepam.

  7. Ability of melanins to protect against the radiolysis of thymine and thymidine.

    PubMed

    Hill, H Z; Huselton, C; Pilas, B; Hill, G J

    1987-01-01

    Individuals with black skin rarely get skin cancer, and melanomas, tumors arising from pigmented cells, are generally resistant to radiation therapy. The role of melanin in these two phenomena has not been defined, but oxygen-radical species have been implicated in both effects. These studies were undertaken to determine the ability of various melanins to compete for ionizing radiation-produced radicals which destroy nucleic acid bases. The ability of Sigma eumelanin (S-eumelanin) to protect against the radiolysis of thymidine in buffered solutions was compared to the protective ability of seven amino acids, including melanin precursors; bovine serum albumin, as a model protein; ficoll, as a model polysaccharide; and DNA. Both proteins and polysaccharides are known to scavenge hydroxyl radicals in cells. The concentration of thymidine after exposure to gamma radiation was determined by High Performance Liquid Chromatography (HPLC) analysis after removal of insoluble melanin by acid precipitation. S-eumelanin was more effective at competing with thymidine for free radicals than bovine serum albumin, Ficoll, or DNA, but less effective than certain of the small molecules. Several of the above compounds were also examined for ability to protect against thymine radiolysis. In addition, melanins from other sources were compared to S-eumelanin. Of these, enzymatically synthesized phaeomelanin was the most effective. The results indicate that melanins can compete for base- and nucleoside-damaging free radicals more effectively than other cellular macromolecules. Of the small molecules, the phenolic compounds had the greatest scavenging ability. In vivo, melanins are found in melanosomes bound to protein. Therefore, the relevance of these findings to the photo- and radiobiology of melanins in vivo has yet to be determined.

  8. 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}.

  9. Toxicokinetics of ( sup 3 H)microcystin-LR in mice

    SciTech Connect

    Robinson, N.A.; Matson, C.F.; Miura, G.A.; Lynch, T.G.; Pace, J.G. )

    1990-02-26

    Toxicokinetics of ({sup 3}H)microcystin-LR, an algal hepatotoxin, was measured in CD-1 mice (iv, 35 {mu}g/kg, sublethal). The distribution and biological half-lives in plasma were 0.8 and 7 minutes, respectively. Liver rapidly accumulated toxin; uptake half-life was 7 minutes. After 60 minutes, liver, kidney, intestine and carcass retained 67{+-}4, 0.8{+-}0.1, 8.6{+-}0.7 and 6{+-}2% of dose, respectively. By 8 days, 18{+-}1 and 7.6{+-}0.6% of the administered radiolabel appeared in feces and urine, while liver and carcass retained 50{+-}2 and 8{+-}3%, respectively. In urine (6 and 12 hours) and feces (6, 12 and 24 hours) {approximately}60% of the tritium was associated with the parent compound. Hepatic radiolabel was tightly bound to cytosolic protein(s). One hour after injection, 27{+-}3% of the hepatic radiolabel was parent compound and 73{+-}3% was a more-polar component (peak 1). By 8 days, there was a redistribution of hepatic radioactivity: 69{+-}2% with a parent and 4{+-}0.5% with peak 1. The authors conclude that ({sup 3}H)MCYST rapidly accumulated in liver, was metabolized and was associated with cytosolic protein.

  10. Tissue distribution of 3H-terbutaline in rabbits.

    PubMed

    Hsu, C H; Robinson, C P; Basmadjian, G P

    1994-01-01

    Terbutaline is a widely used, selective beta 2-adrenergic agonist whose penetration into brain has not been demonstrated in laboratory animals. Although its tissue uptake has been reported in some animals, no uptake into brain has been demonstrated. A single dose of 20 microCi of 3H-terbutaline along with 10 mg/kg of unlabeled terbutaline was injected into a rabbit marginal ear vein. The distribution of 3H-terbutaline in several tissues was determined 0.5, 1, 3, or 6 hr later. Radioactivity in the brain was well-maintained over the 6 hr observation period. In most tissues, radioactivity peaked in less than 1 hr, then declined. Radioactivity in the urine was high at all time periods and was highest at 3 hr. Thus, terbutaline or a metabolite(s) does cross the blood-brain barrier in rabbits, and the radioactivity in the rabbit brain does not decrease during the 6 hours following terbutaline injection.

  11. 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.

  12. The 3H-3He Charge Radii Difference

    SciTech Connect

    Myers, Luke S.; Arrington, John R.; Higinbotham, Douglas 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.

  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. 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%

  15. EdU, a new thymidine analogue for labelling proliferating cells in the nervous system.

    PubMed

    Chehrehasa, Fatemah; Meedeniya, Adrian C B; Dwyer, Patrick; Abrahamsen, Greger; Mackay-Sim, Alan

    2009-02-15

    Labelling and identifying proliferating cells is central to understanding neurogenesis and neural lineages in vivo and in vitro. We present here a novel thymidine analogue, ethynyl deoxyuridine (EdU) for labelling dividing cells, detected with a fluorescent azide which forms a covalent bond via the "click" chemistry reaction (the Huisgen 1,3-dipolar cycloaddition reaction of an organic azide to a terminal acetylene). Unlike the commonly used BrdU, EdU detection requires no heat or acid treatment. It is quick and easy and compatible with multiple probes for fluorescence immunochemistry, facilitating the characterisation of proliferating cells at high resolution.

  16. 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.

  17. Inhibition of Thymidine Kinase Activity and Deoxyribonucleic Acid Synthesis in L Cells Infected with the Meningopneumonitis Agent

    PubMed Central

    Lin, Hsiu-San

    1968-01-01

    The activities of enzymes related to deoxyribonucleic acid (DNA) synthesis were studied in uninfected L cells and in L cells infected with Chlamydia psittaci (strain meningopneumonitis). The meningopneumonitis agent multiplied normally but failed to induce the synthesis of thymidine kinase in LM (TK−) cells which contain no thymidine kinase in the uninfected state. It was concluded that this microorganism has no thymidine kinase of its own and that it does not depend on the functioning of the host enzyme for synthesizing its DNA. Exposure of clone 5b L cells to the meningopneumonitis agent was followed by a decline in their thymidine kinase activity to nearly zero levels, whereas the levels of uridine kinase and thymidylate synthetase remained unchanged. Inhibition of thymidine kinase activity in L cells occurred soon after infection and required new protein synthesis by the meningopneumonitis agent. This inhibition occurred before inhibition of host DNA synthesis, but it was not an essential prelude to the latter inhibition. On the basis of this and previous investigations and in light of present knowledge of the mammalian cell cycle, it was postulated that the meningopneumonitis agent inhibits macromolecular synthesis in L cells by preventing the initiation of a new cell cycle. PMID:5724972

  18. 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.

  19. 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.

  20. Comparison of (/sup 3/H)pirenzepine and (/sup 3/H)quinuclidinylbenzilate binding to muscarinic cholinergic receptors in rat brain

    SciTech Connect

    Luthin, G.R.; Wolfe, B.B.

    1984-03-01

    The properties of (/sup 3/H)quinuclidinylbenzilate ( (/sup 3/H)QNB) binding and (/sup 3/H)pirenzepine ( (/sup 3/H)PZ) binding to various regions of rat brain were compared. (/sup 3/H)PZ appeared to bind with high affinity to a single site, with a Kd value of approximately 15 nM in the cerebral cortex. The rank order of potencies of muscarinic drugs to inhibit binding of either (/sup 3/H)QNB or (/sup 3/H)PZ was QNB greater than atropine . scopolamine greater than pirenzepine greater than oxotremorine greater than bethanechol. Muscarinic antagonists (except PZ) inhibited both (/sup 3/H)PZ and (/sup 3/H)QNB binding with Hill coefficients of approximately 1. PZ inhibited (/sup 3/H)QNB binding in cortex with a Hill coefficient of 0.7, but inhibited (/sup 3/H)PZ binding with a Hill coefficient of 1.0. Hill coefficients for agonists were less than 1. The density of (/sup 3/H)PZ binding sites was approximately half the density of (/sup 3/H)QNB binding sites in cortex, striatum and hippocampus. In pons-medulla and cerebellum, the densities of (/sup 3/H)PZ binding sites were 20 and 0%, respectively, relative to the densities of (/sup 3/H)QNB binding sites. When unlabeled PZ was used to compete for (/sup 3/H)QNB binding, the relative number of high-affinity PZ binding sites in cortex, pons and cerebellum agreed with the relative number of (/sup 3/H)PZ binding sites in those regions. The binding of (/sup 3/H)PZ and (/sup 3/H)QNB was nonadditive in cortex. GTP inhibited high-affinity oxotremorine binding, but not PZ binding. Together, these data suggest that (/sup 3/H)PZ binds to a subset of (/sup 3/H)QNB binding sites. Whether this subset reflects the existence of subtypes of muscarinic receptors or is a consequence of coupling to another membrane protein remains to be seen.

  1. Infrared Predissociation Spectroscopy of the Hydrocarbon Cations C_3H^+, C_2H^+, and C_3H_2^+

    NASA Astrophysics Data System (ADS)

    Brünken, Sandra; Lipparini, Filippo; Gauss, Jürgen; Stoffels, Alexander; Redlich, Britta; van der Meer, Lex; Berden, Giel; Oomens, Jos; Schlemmer, Stephan

    2016-06-01

    Reactive hydrocarbon cations play an important role in the astrochemistry of the interstellar medium, but spectroscopic data, needed for their identification in astronomical observations, is sparse. Here we report the first gas-phase vibrational spectra of the linear C_3H^+ (^1 Σ), the radical cation C_2H^+ (^3 Π), and the linear-/cyclic-C_3H_2^+ (^2 Π /^2A_1, resp.). Broadband spectra were recorded by Ne- and He-messenger infrared-predissociation (IR-PD) action spectroscopy in a cryogenic (4-11 K) ion trap instrument (FELion) in the 250-3500 {wn} range using a free electron laser and a MIR-OPO at the FELIX (Free-Electron Laser for Infrared eXperiments) laboratory. The band positions (determined with a precision of 1-2 wn) covering the C-H and C-C stretching as well as several bending modes are compared to high-level (CCSD(T) with large basis sets) quantum-chemical calculations with an emphasis on anharmonic effects and on the influence of the rare-gas messenger atom. The experimental and theoretical data provide a solid basis for subsequent IR high-resolution studies, with the ultimate goal to predict and measure accurate rotational spectra for a radio-astronomical search of these molecular ions in space.

  2. The tissue-specific distribution of 3H-seocalcitol (EB 1089) and 3H-calcitriol in rats.

    PubMed

    Kissmeyer, Anne-Marie; Nielsen, Jeanet Løgsted; Binderup, Lise

    2004-05-01

    Seocalcitol (EB 1089) is under development for the treatment of hepato-cellular carcinoma (HCC). The tissue distribution of 3H-seocalcitol was investigated in comparison to 3H-calcitriol in rats. Quantitative whole-body autoradiography was used to quantify the tissue distribution. The greatest difference in distribution between the two compounds was observed in the bloodstream. For most tissues the ratio seocalcitol/calcitriol varied between 0.2 and 3.1. The concentration of radioactivity in the liver was almost the same for the two compounds. For seocalcitol the concentration in the liver was 10 times higher than in serum. Assuming that the liver/serum concentration ratio is the same in rats and humans, the concentration of seocalcitol in the human liver is expected to be higher than the concentration resulting in more than 50% inhibition of cancer cell proliferation, and thus pharmacologically effective in HCC. It is questionable whether calcitriol would be present in the human liver in sufficient concentrations to be effective for the treatment of HCC, as the antiproliferative activity of calcitriol is generally more than 10-fold lower compared to that of seocalcitol and as calcitriol can only be administered at a dose that is ca. three-fold lower than the dose of seocalcitol.

  3. 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.

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

    PubMed

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

    2013-09-01

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

  5. Increased cytotoxicity and bystander effect of 5-fluorouracil and 5′-deoxy-5-fluorouridine in human colorectal cancer cells transfected with thymidine phosphorylase

    PubMed Central

    Evrard, A; Cuq, P; Ciccolini, J; Vian, L; Cano, J-P

    1999-01-01

    5-Fluorouracil (5-FU) and 5′-deoxy-5-fluorouridine (5′-DFUR), a prodrug of 5-FU, are anticancer agents activated by thymidine phosphorylase (TP). Transfecting the human TP cDNA into cancer cells in order to sensitize them to these pyrimidine antimetabolites may be an important approach in human cancer gene therapy research. In this study, an expression vector containing the human TP cDNA (pcTP5) was transfected into LS174T human colon carcinoma cells. Eight stable transfectants were randomly selected and analysed. The cytotoxic effects of 5-FU and 5′-DFUR were higher in TP-transfected cells as compared to wild-type cells. The maximal decreases in the IC50 were 80-fold for 5-FU and 40-fold for 5′-DFUR. The increase in sensitivity to these pyrimidines of TP-transfected cells significantly correlated with the increase in both TP activity and TP expression. Transfected clone LS174T-c2 but not wild-type cells exhibited formation of [3H]FdUMP from [3H]5-FU. In addition the LS174T-c2 clone enhanced the cytotoxic effect of 5′-DFUR, but also that of 5-FU, towards co-cultured parental cells. For both anti-cancer agents, this bystander effect did not require cell–cell contact. These results show that both 5-FU or 5′-DFUR could be used together with a TP-suicide vector in cancer gene therapy. © 1999 Cancer Research Campaign PMID:10468288

  6. The hydrocarbon ring C3H2 is ubiquitous in the Galaxy

    NASA Technical Reports Server (NTRS)

    Matthews, H. E.; Irvine, W. M.

    1985-01-01

    The discovery of a strong microwave (1.6 cm-wavelength) spectral line, the carrier of which is common and widespread throughout the Galaxy is reported. A survey of a large number of sources shows that the line appears in emission in cold dust clouds, in absorption in the direction of the Galactic center, and exhibits complex profiles toward H II regions. Toward Cas A and distant H II regions, intervening 'spiral arm' clouds produce absorption. For almost all cases, the absorption features show a striking 1:1 radial velocity correspondence with those seen, e.g., in H2CO spectra of the same objects. The data indicate that the line arises between low-lying energy states of a rather polar molecule. Recent work by Thaddeus, Vrtilek, and Gottlieb (1985) incorporating the present data, shows that the line in question is the 1(10)-1(01) transition of the small hydrocarbon ring C3H2.

  7. Mitochondrial Neurogastrointestinal Encephalomyopathy Caused by Thymidine Phosphorylase Enzyme Deficiency: From Pathogenesis to Emerging Therapeutic Options

    PubMed Central

    Yadak, Rana; Sillevis Smitt, Peter; van Gisbergen, Marike W.; van Til, Niek P.; de Coo, Irenaeus F. M.

    2017-01-01

    Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a progressive metabolic disorder caused by thymidine phosphorylase (TP) enzyme deficiency. The lack of TP results in systemic accumulation of deoxyribonucleosides thymidine (dThd) and deoxyuridine (dUrd). In these patients, clinical features include mental regression, ophthalmoplegia, and fatal gastrointestinal complications. The accumulation of nucleosides also causes imbalances in mitochondrial DNA (mtDNA) deoxyribonucleoside triphosphates (dNTPs), which may play a direct or indirect role in the mtDNA depletion/deletion abnormalities, although the exact underlying mechanism remains unknown. The available therapeutic approaches include dialysis and enzyme replacement therapy, both can only transiently reverse the biochemical imbalance. Allogeneic hematopoietic stem cell transplantation is shown to be able to restore normal enzyme activity and improve clinical manifestations in MNGIE patients. However, transplant related complications and disease progression result in a high mortality rate. New therapeutic approaches, such as adeno-associated viral vector and hematopoietic stem cell gene therapy have been tested in Tymp-/-Upp1-/- mice, a murine model for MNGIE. This review provides background information on disease manifestations of MNGIE with a focus on current management and treatment options. It also outlines the pre-clinical approaches toward future treatment of the disease. PMID:28261062

  8. Effects of Mitomycin C and Thymidine Deprivation on Lysogenic and Sensitive Strains of Bacillus megaterium1

    PubMed Central

    Maisch, W. F.; Wachsman, J. T.

    1967-01-01

    A triple auxotroph of Bacillus megaterium strain KM was lysogenized with a phage suspension from B. megaterium 899a. The lysogenic and phage-sensitive derivatives of KM were found to die at the same exponential rate during thymineless incubation, despite the fact that the lysogenic strain became induced. The lysogenic strain was also induced by mitomycin C, and died at an exponential rate which was approximately twice that of the sensitive strain. With both strains, the lethality of mitomycin C was the same in the presence and absence of thymidine; thymidine was required for maximal phage production. Mitomycin C preferentially inhibited deoxyribonucleic acid (DNA) synthesis of both strains for the first 60 min. The (DNA) synthetic ability of the lysogenic strain was subsequently restored, due to phage production. Since there was no evidence that sensitive strains of KM contained other inducible elements (prophage or probacteriocins), it is concluded that both thymineless death and mitomycin C death can occur via mechanisms not involving induction. PMID:4963773

  9. 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.

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

    PubMed Central

    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

    2016-01-01

    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 upregulated the methylation of RUNX2 and osterix, leading to decreased bone formation. In osteoclast progenitors, TP upregulated the methylation of IRF8, thereby enhanced expression of NFATc1, leading to increased bone resorption. TP reversibly catalyzes thymidine into thymine and 2DDR. Myeloma-secreted 2DDR bound to integrin αVβ3/α5β1 in the progenitors, activated PI3K/Akt signaling, and increased DNMT3A 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. As TP overexpression is common in bone-metastatic tumors, our findings could have additional mechanistic implications. PMID:27559096

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

    PubMed

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

    2013-02-15

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

  12. 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.

  13. Clinical and treatment effects on /sup 3/H-clonidine and /sup 3/H-imipramine binding in elderly depressed patients

    SciTech Connect

    Georgotas, A.; Schweitzer, J.; McCue, R.E.; Armour, M.; Friedhoff, A.J.

    1987-06-01

    /sup 3/H-clonidine and /sup 3/H-imipramine binding were measured in depressed patients, 55 years and older. There was no significant difference in either /sup 3/H-clonidine or /sup 3/H-imipramine binding between depressed patients and age- and sex-matched controls. There was no significant correlation between /sup 3/H-clonidine or /sup 3/H-imipramine binding and severity of depression before treatment. There was a significant negative correlation between the K/sub D/ of /sup 3/H-imipramine binding sites and Hamilton score over seven weeks of antidepressant treatment. There was no significant difference between receptor data of responders and nonresponders to antidepressant treatment. 19 references, 2 tables.

  14. 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.

  15. 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.

  16. Dihydropyridine receptor of L-type Ca sup 2+ channels: Identification of binding domains for ( sup 3 H)(+)-PN200-110 and ( sup 3 H)azidopine within the. alpha. 1 subunit

    SciTech Connect

    Striessnig, J.; Murphy, B.J.; Catterall, W.A. )

    1991-12-01

    To identify the binding domain for dihydropyridine Ca{sup 2{plus}} antagonists, skeletal muscle Ca{sup 2{plus}} channels were photolabeled with ({sup 3}H)({plus})-PN200-110 and ({sup 3}H) azidopine. Regions of {alpha}1 photolabeled by these ligands were then identified by antibody mapping of proteolytic fragments. Approximately 50% of the specific labeling by both ligands was incorporated in domain III. ({sup 3}H)Azidopine labeled peptide Gln-989-Arg-1022, which contains a portion of the connecting loop between transmembrane segments IIIS5 and IIIS6 (IIIS5/S6), and peptide Ala-1023-Lys-1077, which contains IIIS6 itself and some adjacent amino acid residues. In contrast, ({sup 3}H)({plus})-PN200-110 labeling occurred almost exclusively in the fragment containing IIIS6. A second site labeled by both ligands was identified in transmembrane segment S6 of domain IV and adjacent residues. It is proposed, based on physiological studies, that these three peptide segments interact to form a receptor site accessible from the extracellular surface of the Ca{sup 2{plus}} channel.

  17. CAF-1-induced oligomerization of histones H3/H4 and mutually exclusive interactions with Asf1 guide H3/H4 transitions among histone chaperones and DNA.

    PubMed

    Liu, Wallace H; Roemer, Sarah C; Port, Alex M; Churchill, Mair E A

    2012-12-01

    Anti-silencing function 1 (Asf1) and Chromatin Assembly Factor 1 (CAF-1) chaperone histones H3/H4 during the assembly of nucleosomes on newly replicated DNA. To understand the mechanism of histone H3/H4 transfer among Asf1, CAF-1 and DNA from a thermodynamic perspective, we developed and employed biophysical approaches using full-length proteins in the budding yeast system. We find that the C-terminal tail of Asf1 enhances the interaction of Asf1 with CAF-1. Surprisingly, although H3/H4 also enhances the interaction of Asf1 with the CAF-1 subunit Cac2, H3/H4 forms a tight complex with CAF-1 exclusive of Asf1, with an affinity weaker than Asf1-H3/H4 or H3/H4-DNA interactions. Unlike Asf1, monomeric CAF-1 binds to multiple H3/H4 dimers, which ultimately promotes the formation of (H3/H4)(2) tetramers on DNA. Thus, transition of H3/H4 from the Asf1-associated dimer to the DNA-associated tetramer is promoted by CAF-1-induced H3/H4 oligomerization.

  18. Electron and Positron Scattering from C3H6 Isomers

    NASA Astrophysics Data System (ADS)

    Makochekanwa, Casten; Sueoka, Osamu; Kimura, Mineo; Hoshino, Masamitsu; Tanaka, Takahiro; Kitajima, Masashi; Tanaka, Hiroshi

    2004-09-01

    Hydrocarbons play an important role in high temperature plasmas in Tokamak fusion devices in plasma processing and many other fields [1]. In this paper we report experiments for 0.4-1000 eV electron and 0.2-1000 eV positron total cross sections (TCS) measured using a linear time-of-flight apparatus [2], and electron differential cross sections (DCS) for elastic, vibrational and electronic excitations covering the ranges 1.5 to 100 eV and 15 deg to 130 deg, measured using the crossed beam and relative flow method [3]. The continuum multiple scattering (CMS) [4] calculations have also been performed for the theoretical analysis of the observed features in our cross sections. We observe the isomer effect in both electron and positron TCSs and DCSs. The presence of a dipole moment in propene molecules shows up in enhanced forward scattering in DCSs, leading to larger TCSs and integral cross sections compared to cyclopropane at energies less than 20 eV. However, both electron and positron TCSs for these two molecules nearly equal each other above 100 eV, i.e. the molecular size effect. [1] W. L. Moragn, Adv. At. Mol. Opt. Phys. 43, 79 (2000). [2] O. Sueoka, S. Mori and A. Hamada, J. Phys. B 27, 1453 (1994). [3] H. Tanaka, L. Boesten, D. Matsunaga and T. Kudo, J. Phys. B 21, 1255 (1988). [4] M. Kimura and H. Sato, Comments At. Mol. Phys. 26, 333 (1991).

  19. 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.

  20. Comparison of dopamine receptor sites labeled by (/sup 3/H)-S-sulpiride and (/sup 3/H)-spiperone in striatum

    SciTech Connect

    Zahniser, N.R.; Dubocovich, M.L.

    1983-12-01

    Binding of the radiolabeled active isomer of the neuroleptic sulpiride, (/sup 3/H)-S-sulpiride, to rat and rabbit striatal membranes was characterized. Regardless of whether the specific binding of (/sup 3/H)-S-sulpiride was defined with spiperone or the active isomers of butaclamol or flupenthixol, a single homogeneous++ population of binding sites (rat: Kd . 5.6 nM, maximum binding . 590 fmol/mg of protein; rabbit: Kd . 8.3 nM, maximum binding . 540 fmol/mg of protein) was detected. The pharmacological profile of these sites was characteristic of that described for the dopaminergic D-2 receptor subtype. To determine whether (/sup 3/H)-S-sulpiride and (/sup 3/H)spiperone label common sites in the striatum, the binding of these two radioligands was compared under similar assay conditions. When specific binding of (/sup 3/H)spiperone was defined with S-sulpiride, (/sup 3/H)spiperone labeled the same number of binding sites as (/sup 3/H)-S-sulpiride despite the fact that the affinity of the sites for (/sup 3/H)spiperone was 80- to 90-fold higher than for (/sup 3/H)-S-sulpiride. When specific binding of (/sup 3/H)spiperone was defined with either (+)-butaclamol or (alpha)-flupenthixol, however, approximately 30% more sites were labeled. The predominant site labeled by (/sup 3/H)spiperone also possessed the characteristics of the D-2 receptor. It is concluded that (/sup 3/H)-S-sulpiride under the conditions used is a selective radioligand with which dopamine receptors of the D-2 subtype can be directly measured and localized. (/sup 3/H)Spiperone can be used to detect the same sites only if specific binding is defined with S-sulpiride.

  1. Development of a Novel High-Density [3H]Hypoxanthine Scintillation Proximity Assay To Assess Plasmodium falciparum Growth

    PubMed Central

    Caballero, Iván; Colmenarejo, Gonzalo; Sanz, Laura M.; Álvarez-Ruiz, Emilio; Gamo, Francisco-Javier; Cid, Concepción

    2016-01-01

    The discovery and development of new antimalarial drugs are becoming imperative because of the spread of resistance to current clinical treatments. The lack of robustly validated antimalarial targets and the difficulties with the building in of whole-cell activity in screening hits are hampering target-based approaches. However, phenotypic screens of structurally diverse molecule libraries are offering new opportunities for the identification of novel antimalarials. Several methodologies can be used to determine the whole-cell in vitro potencies of antimalarial hits. The [3H]hypoxanthine incorporation assay is considered the “gold standard” assay for measurement of the activity of antimalarial compounds against intraerythrocytic forms of Plasmodium falciparum. However, the method has important limitations, as the assay is not amenable for high-throughput screening since it remains associated with the 96-well plate format. We have overcome this drawback by adapting the [3H]hypoxanthine incorporation method to a 384-well high-density format by coupling a homogeneous scintillation proximity assay (SPA) and thus eliminating the limiting filtration step. This SPA has been validated using a diverse set of 1,000 molecules, including both a representative set from the Tres Cantos Antimalarial Set (TCAMS) of compounds and molecules inactive against whole cells. The results were compared with those from the P. falciparum lactate dehydrogenase whole-cell assay, another method that is well established as a surrogate for parasite growth and is amenable for high-throughput screening. The results obtained demonstrate that the SPA-based [3H]hypoxanthine incorporation assay is a suitable design that is adaptable to high-throughput antimalarial drug screening and that maintains the features, robustness, and reliability of the standard filtration hypoxanthine incorporation method. PMID:27458216

  2. 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.

  3. 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

  4. Si^3+ +H collisions: role of rotational couplings

    NASA Astrophysics Data System (ADS)

    Joseph, D. C.; Saha, B. C.

    2012-06-01

    State selective charge exchange cross sections are calculated using both the quantal and the semi classical molecular orbital close coupling approaches in the adiabatic representation. In addition to radial coupling, all angular couplings are also incorporated in our close coupling calculations. The multi-reference single- and double-excitation configuration interaction (MRD-CI) method [1] is employed to describe the adiabatic electronic states of (SiH)^3+ system. Details of our findings will be reported at the conference.[4pt] [1] R. J. Buenker, in Current Aspects of Quantum Chemistry, edited by R. Carbo, in Physical and Theoretical Chemistry Vol. 21 (Elsevier, Amsterdam, 1981).

  5. Light microscopic autoradiographic localization of (/sup 3/H)pirenzepine and (/sup 3/H)(/sup -/)quinuclidinyl benzilate binding in human stellate ganglia

    SciTech Connect

    Yamamura, H.I.; Watson, M.; Wamsley, J.K.; Johnson, P.C.; Roeske, W.R.

    1984-08-13

    The LKB Ultrofilm method of autoradiography has been utilized to anatomically localize putative M/sub 1/ and M/sub 2/ muscarinic receptor subtypes in human stellate ganglia. Ten micron sections were labeled in vitro with either 1 nM of the classical antagonist (/sup 3/H)(/sup -/)quinuclidinyl benzilate ((/sup 3/H)(/sup -/)QNB) or 20 nM of the non-classical antagonist (/sup 3/H)pirenzepine ((/sup 3/H)PZ), using 1 ..mu..M atropine sulfate to define non-specific binding for both ligands. The results indicate that (/sup 3/H)(/sup -/)QNB and (/sup 3/H)PZ binding sites are distributed within the principal ganglion cells and nerve bundles.

  6. X-ray analysis of azido-thymidine diphosphate binding to nucleoside diphosphate kinase.

    PubMed

    Xu, Y; Sellam, O; Moréra, S; Sarfati, S; Biondi, R; Véron, M; Janin, J

    1997-07-08

    To be effective as antiviral agent, AZT (3'-azido-3'-deoxythymidine) must be converted to a triphosphate derivative by cellular kinases. The conversion is inefficient and, to understand why AZT diphosphate is a poor substrate of nucleoside diphosphate (NDP) kinase, we determined a 2.3-A x-ray structure of a complex with the N119A point mutant of Dictyostelium NDP kinase. It shows that the analog binds at the same site and, except for the sugar ring pucker which is different, binds in the same way as the natural substrate thymidine diphosphate. However, the azido group that replaces the 3'OH of the deoxyribose in AZT displaces a lysine side chain involved in catalysis. Moreover, it is unable to make an internal hydrogen bond to the oxygen bridging the beta- and gamma-phosphate, which plays an important part in phosphate transfer.

  7. X-ray analysis of azido-thymidine diphosphate binding to nucleoside diphosphate kinase

    PubMed Central

    Xu, Yingwu; Sellam, Olivier; Moréra, Solange; Sarfati, Simon; Biondi, Ricardo; Véron, Michel; Janin, Joël

    1997-01-01

    To be effective as antiviral agent, AZT (3′-azido-3′-deoxythymidine) must be converted to a triphosphate derivative by cellular kinases. The conversion is inefficient and, to understand why AZT diphosphate is a poor substrate of nucleoside diphosphate (NDP) kinase, we determined a 2.3-Å x-ray structure of a complex with the N119A point mutant of Dictyostelium NDP kinase. It shows that the analog binds at the same site and, except for the sugar ring pucker which is different, binds in the same way as the natural substrate thymidine diphosphate. However, the azido group that replaces the 3′OH of the deoxyribose in AZT displaces a lysine side chain involved in catalysis. Moreover, it is unable to make an internal hydrogen bond to the oxygen bridging the β- and γ-phosphate, which plays an important part in phosphate transfer. PMID:9207061

  8. 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

  9. 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.

  10. A pseudoreceptor modelling study of the varicella-zoster virus and human thymidine kinase binding sites

    NASA Astrophysics Data System (ADS)

    Greenidge, Paulette A.; Merz, Alfred; Folkers, Gerd

    1995-12-01

    A representative range of pyrimidine nucleoside analogues that are known to inhibit herpes simplex virus (HSV) replication have been used to construct receptor binding site models for the varicella-zoster virus (VZV), thymidine kinase (TK) and human TK1. Given a set of interacting ligands, superimposed in such a manner as to define a pharmacophore, the pseudoreceptor modelling technique Yak provides a means of building binding site models of macromolecules for which no three-dimensional experimental structures are available. Once the models have been evaluated by their ability to reproduce experimental binding data [Vedani et al., J. Am. Chem. Soc., 117 (1995) 4987], they can be used for predictive purposes. Calculated and experimental values of relative binding affinity are compared. Our models suggest that the substitution of one residue may be sufficient to determine ligand subtype affinity.

  11. Say what? The activity of the polyamine biosynthesis inhibitor difluoromethylornithine (DFMO) in chemoprevention is a result of reduced thymidine pools?

    PubMed Central

    Casero, Robert A.

    2013-01-01

    Summary In the current issue of Cancer Discovery, Witherspoon, Lipkin and colleagues use an unbiased metabolite profiling approach to study the effects of polyamine depletion by DFMO in colon cancer cells. Their surprising findings indicate that it is a decrease in thymidine pools resulting from altered tetrahydrofolate availability rather than decreases in polyamines that produce cytostasis. PMID:24019331

  12. Say what? The activity of the polyamine biosynthesis inhibitor difluoromethylornithine in chemoprevention is a result of reduced thymidine pools?

    PubMed

    Casero, Robert A

    2013-09-01

    In this issue of Cancer Discovery, Witherspoon and colleagues use an unbiased metabolite profiling approach to study the effects of polyamine depletion by 2-difluoromethylornithine in colon cancer cells. Their surprising findings indicate that it is a decrease in thymidine pools resulting from altered tetrahydrofolate availability rather than decreases in polyamines that produces cytostasis.

  13. Production of cells without deoxyribonucleic acid during thymidine starvation of lexA- cultures of Escherichia coli K-12.

    PubMed Central

    Howe, W E; Mount, D W

    1975-01-01

    When thymidine-requiring lexA- strains were starved for thymidine, the kinetics of survival were similar to those of a nearly isogenic lexA+ strain. The size distribution of cells in the lexA- and lexA+ cultures were, however, quite different. Whereas most of the cells in the starved lexA+ cultures grew into long filamentous forms (longer than 4.0 mum), many of the lexA- cells were found to have a normal rod shape (4.0 mum or shorter). It was shown that lexA- cells undergo more divisions during thymidine starvation than lexA+ cells. Furthermore, using an autoradiographic method to analyze deoxyribonucleic acid (DNA) distribution in the starved cells, we demonstrated that cells without DNA are produced in both normal and starved lexA- cultures at a much higher frequency than in lexA+ cultures. Some of these cells may be produced by breakdown of DNA, but we favor the hypothesis that they result from an abnormal cell division process. Since lexA mutations are dominant, we conclude that a diffusible product decreases the synthesis or activity of an inhibitor of cell division in lexA- strains when DNA synthesis is blocked by thymidine starvation. Images PMID:1104571

  14. Different effects of serotonin antagonists on /sup 3/H-mianserin and /sup 3/H-ketanserin recognition sites

    SciTech Connect

    Gandolfi, O.; Barbaccia, M.L.; Costa, E.

    1985-02-25

    In minces prepared from the frontal cortex of rats treated with ketanserin (10 mg/kg i.p.) or mianserin (5 mg/kg i.p.) twice daily for 21 days, the Vmax of the adenylate cyclase stimulated by NE (100 ..mu..M) is attenuated, suggesting that ketanserin and mianserin share with a number of antidepressants the ability to attenuate the adenylate cyclase stimulation by NE. Ketanserin, given with the above mentioned dose schedule for 7 consecutive days, reduced the Bmax of 5HT/sub 2/ recognition sites but failed to change either the Bmax or the apparent Kd of /sup 3/H-mianserin binding. A significant decrease in the Bmax of 5HT/sub 2/ binding sites is elicited also by a single injection of mianserin. This drug also down-regulates its own binding when given twice daily for 3 weeks. From this and other information, it is concluded that ketanserin and mianserin bind to distinct recognition sites. The possibility that 5HT/sub 2/ and mianserin recognition sites are functionally related and that serotonergic synapses are modulated by multiple chemical signals might be considered. 34 references, 3 figures.

  15. Structural basis for non-competitive product inhibition in human thymidine phosphorylase: implications for drug design.

    PubMed

    El Omari, Kamel; Bronckaers, Annelies; Liekens, Sandra; Pérez-Pérez, Maria-Jésus; Balzarini, Jan; Stammers, David K

    2006-10-15

    HTP (human thymidine phosphorylase), also known as PD-ECGF (platelet-derived endothelial cell growth factor) or gliostatin, has an important role in nucleoside metabolism. HTP is implicated in angiogenesis and apoptosis and therefore is a prime target for drug design, including antitumour therapies. An HTP structure in a closed conformation complexed with an inhibitor has previously been solved. Earlier kinetic studies revealed an ordered release of thymine followed by ribose phosphate and product inhibition by both ligands. We have determined the structure of HTP from crystals grown in the presence of thymidine, which, surprisingly, resulted in bound thymine with HTP in a closed dead-end complex. Thus thymine appears to be able to reassociate with HTP after its initial ordered release before ribose phosphate and induces the closed conformation, hence explaining the mechanism of non-competitive product inhibition. In the active site in one of the four HTP molecules within the crystal asymmetric unit, additional electron density is present. This density has not been previously seen in any pyrimidine nucleoside phosphorylase and it defines a subsite that may be exploitable in drug design. Finally, because our crystals did not require proteolysed HTP to grow, the structure reveals a loop (residues 406-415), disordered in the previous HTP structure. This loop extends across the active-site cleft and appears to stabilize the dimer interface and the closed conformation by hydrogen-bonding. The present study will assist in the design of HTP inhibitors that could lead to drugs for anti-angiogenesis as well as for the potentiation of other nucleoside drugs.

  16. 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.

  17. Computer-aided active-site-directed modeling of the Herpes Simplex Virus 1 and human thymidine kinase

    NASA Astrophysics Data System (ADS)

    Folkers, Gerd; Trumpp-Kallmeyer, Susanne; Gutbrod, Oliver; Krickl, Sabine; Fetzer, Jürgen; Keil, Günther M.

    1991-10-01

    Thymidine kinase (TK), which is induced by Herpes Simplex Virus 1 (HSV1), plays a key role in the antiviral activity of guanine derivatives such as aciclovir (ACV). In contrast, ACV shows only low affinity to the corresponding host cell enzyme. In order to define the differences in substrate binding of the two enzymes on molecular level, models for the three-dimensional (3-D) structures of the active sites of HSV1-TK and human TK were developed. The reconstruction of the active sites started from primary and secondary structure analysis of various kinases. The results were validated to homologous enzymes with known 3-D structures. The models predict that both enzymes consist of a central core β-sheet structure, connected by loops and α-helices very similar to the overall structure of other nucleotide binding enzymes. The phosphate binding is made up of a highly conserved glycine-rich loop at the N-terminus of the proteins and a conserved region at the C-terminus. The thymidine recognition site was found about 100 amino acids downstream from the phosphate binding loop. The differing substrate specificity of human and HSV1-TK can be explained by amino-acid substitutions in the homologous regions. To achieve a better understanding of the structure of the active site and how the thymidine kinase proteins interact with their substrates, the corresponding complexes of thymidine and dihydroxypropoxyguanine (DHPG) with HSV1 and human TK were built. For the docking of the guanine derivative, the X-ray structure of Elongation Factor Tu (EF-Tu), co-crystallized with guanosine diphosphate, was taken as reference. Fitting of thymidine into the active sites was done with respect to similar interactions found in thymidylate kinase. To complement the analysis of the 3-D structures of the two kinases and the substrate enzyme interactions, site-directed mutagenesis of the thymidine recognition site of HSV1-TK has been undertaken, changing Asp162 in the thymidine recognition site

  18. Probing the interactions of the solvated electron with DNA by molecular dynamics simulations: II. bromodeoxyuridine-thymidine mismatched DNA.

    PubMed

    Gantchev, Tsvetan G; Hunting, Darel J

    2009-01-01

    The interaction of solvated electrons (e(-)(aq)) with DNA results in various types of DNA lesions. The in vitro and in vivo sensitisation of DNA to (e(-)(aq))-induced damage is achieved by incorporation of the electron-affinity radiosensitiser bromodeoxyuridine (BUdR) in place of thymidine. However, in DNA duplexes containing single-stranded regions (bulged BUdR-DNA), the type of lesion is different and the efficiency of damage is enhanced. In particular, DNA interstrand crosslinks (ICL) form at high efficiency in bulged DNA but are not detectable in completely duplex DNA. Knowledge about the processes and interactions leading to these differences is obscure. Previously, we addressed the problem by applying molecular modelling and molecular dynamics (MD) simulations to a system of normal (BUdR.A)-DNA and a hydrated electron, where the excess electron was modelled as a localised e(-)(H2O6) anionic cluster. The goal of the present study was to apply the same MD simulation to a wobble DNA-e(-)(aq) system, containing a pyrimidine-pyrimidine mismatched base pair, BUdR.T. The results show an overall dynamic pattern similar to that of the e(-)(aq) motion around normal DNA. However, the number of configuration states when e(-)(aq)) was particularly close to DNA is different. Moreover, in the (BUdR.T)-wobble DNA system, the electron frequently approaches the brominated strand, including BUdR, which was not observed with the normal (BUdR.A)-DNA. The structure and exchange of water at the sites of e(-)(aq) immobilisation near DNA were also characterised. The structural dynamics of the wobble DNA is prone to more extensive perturbations, including frequent formation of cross-strand (cs) interatomic contacts. The structural deviations correlated with e(-)(aq) approaching DNA from the major groove side, with sodium ions trapped deep in the minor groove. Altogether, the obtained results confirm and/or throw light on dynamic-structure determinants possibly responsible for the

  19. 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

    2015-08-05

    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).

  20. sigma opiates and certain antipsychotic drugs mutually inhibit (+)-(/sup 3/H)SKF 10,047 and (/sup 3/H)haloperidol binding in guinea pig brain membranes

    SciTech Connect

    Tam, S.W.; Cook, L.

    1984-09-01

    The relationship between binding of antipsychotic drugs and sigma psychotomimetic opiates to binding sites for the sigma agonist (+)-(/sup 3/H)SKF 10,047 (N-allylnormetazocine) and to dopamine D/sub 2/ sites was investigated. In guinea pig brain membranes, (+)-(/sup 3/H)SKF 10,047 bound to single class of sites with a K/sub d/ of 4 x 10/sup -8/ M and a B/sub max/ of 333 fmol/mg of protein. This binding was different from ..mu.., kappa, or delta opiate receptor binding. It was inhibited by opiates that produce psychotomimetic activities but not by opiates that lack such activities. Some antipsychotic drugs inhibited (+)-(/sup 3/H)SKF 10,047 binding with high to moderate affinities in the following order of potency: haloperidol > perphenazine > fluphenazine > acetophenazine > trifluoperazine > molindone greater than or equal to pimozide greater than or equal to thioridazine greater than or equal to chlorpromazine greater than or equal to triflupromazine. However, there were other antipsychotic drugs such as spiperone and clozapine that showed low affinity for the (+)-(/sup 3/H)SKF 10,047 binding sites. Affinities of antipsychotic drugs for (+)-(/sup 3/H)SKF 10,047 binding sites did not correlate with those for (/sup 3/H)spiperone (dopamine D/sub 2/) sites. (/sup 3/H)-Haloperidol binding in whole brain membranes was also inhibited by the sigma opiates pentazocine, cyclazocine, and (+)-(/sup 3/H)SKF 10,047. In the striatum, about half of the saturable (/sup 3/H)haloperidol binding was to (/sup 3/H)spiperone (D/sub 2/) sites and the other half was to sites similar to (+)-(/sup 3/H)SKF 10,047 binding sites. 15 references, 4 figures, 1 table.

  1. Toll-Like Receptor 4-Defective C3H/HeJ Mice Are Not More Susceptible than Other C3H Substrains to Infection with Mycobacterium tuberculosis

    PubMed Central

    Kamath, Arati B.; Alt, Jennifer; Debbabi, Hajer; Behar, Samuel M.

    2003-01-01

    Mycobacterium tuberculosis produces a variety of molecules capable of activating Toll-like receptors, a family of pattern recognition receptors expressed by macrophages and a variety of other cells. To determine whether Toll-like receptor 4 (TLR4) was critical in resistance to M. tuberculosis infection, we compared the morbidity and mortality of TLR4-defective C3H/HeJ mice to those of TLR4-sufficient C3H mouse substrains. TLR4-defective C3H/HeJ mice and TLR4-sufficient C3H/HeSnJ, C3HeB/FeJ, and C3H/HeOuJ mice were infected by the aerosol route with M. tuberculosis. TLR4-defective C3H/HeJ mice had levels of cytokines in their bronchoalveolar lavage fluids and in vitro mycobacterial antigen-specific recall responses similar to those of other C3H mouse substrains. In addition, bacterial replication and long-term survival of mice following infection appeared to be independent of TLR4. Interestingly, C3HeB/FeJ mice were significantly more susceptible to M. tuberculosis infection, indicating that genetic heterogeneity among inbred C3H mouse substrains modifies resistance to infection. Therefore, cautious interpretation is required when the C3H/HeJ strain is used as a model of a TLR4-defective mouse strain, as there are significant allelic differences between C3H/HeJ and other C3H mouse substrains in response to M. tuberculosis infection. With this caveat, our data indicate that TLR4 may not be required for optimal immunity of mice to M. tuberculosis. PMID:12819102

  2. Synthesis of the tellurium-derivatized phosphoramidites and their incorporation into DNA oligonucleotides.

    PubMed

    Jiang, Sibo; Sheng, Jia; Huang, Zhen

    2011-12-01

    In this unit, an efficient method for the synthesis of 2'-tellerium-modified phosphoramidite and its incorporation into oligonucleotide are presented. We choose 5'-O-DMTr-2,2'-anhydro-uridine and -thymidine nucleosides (S.1, S.2) as starting materials due to their easy preparation. The 5'-O-DMTr-2,2'-anhydro-uridine and -thymidine can be converted to the corresponding 2'-tellerium-derivatized nucleosides by treating with the telluride nucleophiles. Subsequently, the 2'-Te-nucleosides can be transformed into 3'-phosphoramidites, which are the building blocks for DNA/RNA synthesis. The DNA synthesis, purification, and applications of oligonucleotides containing 2'-Te-U or 2'-Te-T are described in the protocol.

  3. Bupropion inhibits nicotine-evoked [(3)H]overflow from rat striatal slices preloaded with [(3)H]dopamine and from rat hippocampal slices preloaded with [(3)H]norepinephrine.

    PubMed

    Miller, Dennis K; Sumithran, Sangeetha P; Dwoskin, Linda P

    2002-09-01

    Bupropion, an efficacious antidepressant and smoking cessation agent, inhibits dopamine and norepinephrine transporters (DAT and NET, respectively). Recently, bupropion has been reported to noncompetitively inhibit alpha3beta2, alpha3beta4, and alpha4beta2 nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes or established cell lines. The present study evaluated bupropion-induced inhibition of native alpha3beta2* and alpha3beta4* nAChRs using functional neurotransmitter release assays, nicotine-evoked [(3)H]overflow from superfused rat striatal slices preloaded with [(3)H]dopamine ([(3)H]DA), and nicotine-evoked [(3)H]overflow from hippocampal slices preloaded with [(3)H]norepinephrine ([(3)H]NE). The mechanism of inhibition was evaluated using Schild analysis. To eliminate the interaction of bupropion with DAT or NET, nomifensine or desipramine, respectively, was included in the superfusion buffer. A high bupropion concentration (100 microM) elicited intrinsic activity in the [(3)H]DA release assay. However, none of the concentrations (1 nM-100 microM) examined evoked [(3)H]NE overflow and, thus, were without intrinsic activity in this assay. Moreover, bupropion inhibited both nicotine-evoked [(3)H]DA overflow (IC(50) = 1.27 microM) and nicotine-evoked [(3)H]NE overflow (IC(50) = 323 nM) at bupropion concentrations well below those eliciting intrinsic activity. Results from Schild analyses suggest that bupropion competitively inhibits nicotine-evoked [(3)H]DA overflow, whereas evidence for receptor reserve was obtained upon assessment of bupropion inhibition of nicotine-evoked [(3)H]NE overflow. Thus, bupropion acts as an antagonist at alpha3beta2* and alpha3beta4* nAChRs in rat striatum and hippocampus, respectively, across the same concentration range that inhibits DAT and NET function. The combination of nAChR and transporter inhibition produced by bupropion may contribute to its clinical efficacy as a smoking cessation agent.

  4. Cortical laminar tau deposits and activated astrocytes in Alzheimer’s disease visualised by 3H-THK5117 and 3H-deprenyl autoradiography

    PubMed Central

    Lemoine, Laetitia; Saint-Aubert, Laure; Nennesmo, Inger; Gillberg, Per-Göran; Nordberg, Agneta

    2017-01-01

    Hyperphosphorylated tau protein deposits and, inflammatory processes are characteristic components of Alzheimer disease (AD) pathology. We here aimed to visualize in vitro the distribution of tau deposits and activated astrocytes across the cortical layers in autopsy AD brain tissue using the radiotracers 3H-THK5117 and 3H-deprenyl. 3H-THK5117 and 3H-deprenyl autoradiographies were carried out on frozen brain sections from three AD patients and one healthy control. 3H-THK5117 showed a distinct laminar cortical binding similar to 3H-deprenyl autoradiography, with an extensive binding in the superficial and deep layers of the temporal neocortices, whereas the middle frontal gyrus showed an even binding throughout the layers. Globally, eventhough some differences could be observed, AT8 (tau) and GFAP (astrocyte) immunostaining showed a laminar pattern comparable to their corresponding radiotracers within each AD case. Some variability was observed between the AD cases reflecting differences in disease phenotype. The similar laminar cortical brain distribution of tau deposits and activated astrocytes supports the hypothesis of a close pathological interconnection. The difference in regional binding patterns of 3H-THK5117 and AT8 antibody staining suggest additional tau binding sites detectable by 3H-THK5117. PMID:28374768

  5. Effect of hemoglobin on the uptake of /sup 3/H-norepinephrine and /sup 3/H-choline chloride into porcine cerebral arteries

    SciTech Connect

    Linnik, M.D.; Lee, T.J.F.

    1986-03-01

    Prolonged constriction of cerebral arteries often follows subarachnoid hemorrhage (SAH). SAH exposes hemoglobin (Hb) to cerebral arteries and Hb has been demonstrated to induce vasoconstriction as well as alter cerebrovascular neurogenic response characteristics. The effect of Hb on uptake of /sup 3/H-norepinephrine (/sup 3/H-NE) and /sup 3/H-choline chloride (/sup 3/H-ChCl) into porcine cerebral arteries was therefore examined. 0.5 to 50 ..mu..M porcine Hb caused a dose-dependent inhibition of /sup 3/H-NE uptake into the anterior (ANT), internal carotid (IC) and middle cerebral (MC) arteries of the pig. IC/sub 50/ values for uptake inhibition were: ANT, 31 ..mu..M; IC, 34 ..mu..M; MC, 37 ..mu..M. Porcine serum albumin (PSA) in the same concentration range also caused a decrease in /sup 3/H-NE uptake. An examination of protein-ligand interactions using column chromatography demonstrated binding of /sup 3/H-NE by both Hb and PSA. This protein binding may be responsible for part of the uptake inhibition. Hb and PSA had little effect on /sup 3/H-ChCl uptake into these arteries.

  6. The effect of phencyclidine on [3H]GABA and [3H]flunitrazepam binding in the brain of naive and handling-habituated rats.

    PubMed

    Lillrank, S M; Oja, S S; Saransaari, P

    1995-01-01

    The effects of handling and handling combined with phencyclidine (PCP) treatment on GABAergic neurotransmission were studied in Sprague-Dawley rats. The animal material consisted of handling-habituated (HH, for 11 d), acutely handled (naive, N), handling-habituated and PCP-treated (10 mg kg-1 i.p., HH + PCP) and acutely handled (naive) PCP-treated (N + PCP) and unhandled 'control' rats. The binding of [3H]GABA and [3H]flunitrazepam (FLU) was studied with membranes and the release of [3H]GABA with slices prepared from the striatum and frontal cortex. In the striatum the maximal binding capacity (Bmax) and the binding constant (KD) of [3H]GABA were the same in N and HH rats, but in the frontal cortex KD was lower in N rats. KD constants of [3H]FLU were significantly lower in both brain areas in N rats than in HH rats. After PCP treatment both Bmax and KD for [3H]FLU increased in these two brain areas in handling-habituated rats, whereas Bmax of [3H]GABA diminished. Neither handling nor PCP had any effect on [3H]GABA release from striatal and frontal cortical slices. Handling prior to killing thus affects differently the GABAergic parameters studied and modulates the PCP-induced effects.

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

    PubMed

    Wallace, David B; Viljoen, Gerrit J

    2005-04-27

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

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

    PubMed Central

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

    1987-01-01

    Induction of thymidine kinase parallels the onset of DNA synthesis. To investigate the transcriptional regulation of the thymidine kinase gene, we have examined whether specific nuclear factors interact in a cell-cycle-dependent manner with sequences upstream of this gene. Two inverted CCAAT boxes near the transcriptional initiation sites were observed to form complexes with nuclear DNA-binding proteins. The nature of the complexes changes dramatically as the cells approach DNA synthesis and correlates well with the previously reported transcriptional increase of the thymidine kinase gene. Images PMID:3479796

  9. 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.

  10. Specific labeling of O6-alkylguanine-DNA alkyltransferase by reaction with O6-(p-hydroxy[3H]methylbenzyl)guanine.

    PubMed

    Ciocco, G M; Moschel, R C; Chae, M Y; McLaughlin, P J; Zagon, I S; Pegg, A E

    1995-09-15

    O6-Alkylguanine-DNA alkyltransferase (AGT) is an important DNA repair protein that removes adducts from the O6 position of guanine by transferring them to a cysteine residue within its amino acid sequence. Mammalian AGTs are readily inactivated by incubation with O6-benzyl-guanine (BG), which is an alternative substrate for the protein. To examine this inactivation in more detail and to develop a procedure for the specific labeling of human AGT, we synthesized a BG derivative, O6-(p-hydroxy[3H]methylbenzyl_guanine ([3H]HMBG) and examined its interaction with AGT in HT29 cell extracts and in HT29 cells. Incubation of human AGT with [3H]HMBG led to the incorporation of radioactivity in the protein in a time- and temperature-dependent manner. This reaction was specific, since neither AGT that was first inactivated by reaction with BG nor proteins other than AGT were labeled. Digestion of the labeled AGT with trypsin showed that a single peptide contained the label. Sequencing of this peptide indicated that the label was bound to cysteine-145. These results demonstrate that AGT accepts HMBG as a substrate and becomes inactivated by transfer of a p-hydroxymethylbenzyl residue to the cysteine-145 acceptor site. When [3H]HMBG was added to cultures of HT29 cells which are Mer+ and express active AGT, radioactivity was incorporated in a macromolecule and could be detected by autoradiography. No such labeling occurred with BE or CHO cells, which are Mer- and lack AGT. Examination of the interaction of [3H]HMBG with mutant AGT proteins that differ greatly in their abilities to react with BG showed that there was a strong correlation between the reaction with BG and the labeling with [3H]HMBG. These results indicate that [3H]HMBG is a potentially useful reagent for the detection and localization of AGT activity and for the investigation of its mechanism of action.

  11. Characterization of antralin: an endogenous protein inhibitor of radioligand binding to (/sup 3/H)RO 5-4864 and (/sup 3/H)nitrendipine binding sites

    SciTech Connect

    Mantione, C.R.; Goldman, M.E.; Bolger, G.T.; Lueddens, H.W.M.; Paul, S.M.; Skolnick, P.

    1986-03-01

    The presence of substances isolated from rat antral stomach that inhibit the binding of (/sup 3/H)Ro 5-4864 (4'-chlorodiazepam) to peripheral-type benzodiazepine receptors (PBR), but do not affect the binding of (/sup 3/H)diazepam to brain-type benzodiazepine receptors have been described. These substances, now obtained by isolation on Sep-Pak C/sub 18/ cartridges, also had no effect on (/sup 3/)PK 11195 binding which labels an antagonist site associated with PBR. Neither was any significant inhibition of (/sup 3/H)Ro 15-1788, (/sup 3/H)..beta..CCM, or (/sup 3/H)dihydroalprenolol binding observed. However, the Sep-Pak eluates were found to produce a concentration-dependent inhibition of (/sup 3/H)nitrendipine binding equivalent to that seen with (/sup 3/H)Ro 5-4864. This material, termed antralin, reduced the apparent K/sub d/ of both radioligands without significantly affecting B/sub max/. Its activity was destroyed by both heat treatment and pronase, and partially reduced by trypsin. Furthermore, its activity was enhanced by Ca/sup + +/ (0.1 mM). Antralin levels were unevenly distributed in rat tissues. These findings suggest that antralin is a protein that may regulate both PBR and dihydropyridine Ca/sup + +/-channel antagonist binding sites in vivo.

  12. 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.

  13. The effect of delta9-tetrahydrocannabinol on the conversion of [3H]trypotphan to 5-[3H] hydroxytryptamine in the mouse brain.

    PubMed

    Johnson, K M; Dewey, W L

    1978-10-01

    The effects of a 30-min pretreatment with varying doses of delta9-tetrahydrocannibinol (delta9-THC) on the synthesis of 5-[3H]hydroxytryptamine (5-[3H]HT) from an intravenous 10-min pulse of L-[3H]tryptophan ([3H]try) were measured in the mouse brain. We also determined the effects of delta9-THC on several parameters believed to influence the synthesis of brain 5-HT, including total and free plasma tryptophan and the high-affinity synaptosomal uptake of tryptophan. Delta9-THC was found to increase the amount of [3H]try accumulated by the brain as well as the amount of 5-[3H]HT synthesized. This effect was greater at intermediate doses than at the highest dose tested. However, delta9-THC was determined to have no effect on the actual rate of conversion of [3H]try into 5-[3H]HT at any dose tested. Delta9-THC had no effect on either plasma tyrosine ot free tryptophan levels. However, delta9-THC decreased total plasma tryptophan at low and intermediate doses, but had no effect at the highest dose tested. Synaptosomal uptake of [3H]try was unaffected by pretreatment with delta9-THC at any dose tested. These data suggest that delta9-THC increases the synthesis of 5-HT, not by altering the actual rate of conversion of tryptophan to 5-HT, but by altering, via an unknown mechanism, the quantity of tryptophan available for conversion to 5-HT.

  14. 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.

  15. /sup 3/H-retinol derived photopigment in chick pineal membranes

    SciTech Connect

    Wallingford, J.; Zatz, M.

    1986-05-01

    Pineal glands display a day-night rhythm in the synthesis and secretion of melatonin. Dispersed chick pinealocytes retain their ability to respond to light in vitro for at least a week. Pinealocytes incubated overnight with /sup 3/H-retinol in the dark incorporate radioactivity predominantly into retinyl esters. To identify the chick pineal photopigment, SDS-PAGE was performed on radiolabelled preparations of pinealocytes and (intraocularly injected) rat retina. When intact cells or membrane preparations of cultured cells were incubated with NaCNBH/sub 3/, in the dark, a single radioactive peak with an apparent molecular weight of 32,000 daltons was observed. Rat retina preparations revealed a major peak at approximately 40,000 daltons. Protease inhibitors were present in the workup, and radioactivity corresponding to the smaller peak from pineal was not observed in retina. There was no radioactive peak when NaCNBH/sub 3/ was omitted. When samples were boiled in SDS the radioactivity shifted to the origin. These data suggest a protein in pinealocyte membranes which binds retinoid via a Schiff's base. Exposure to light of deoxycholate solubilized pineal membranes reduced the radioactivity associated with the protein. These findings raise the possibility that this protein is the pinealocyte's photopigment. Photopigments smaller than those observed in mammals have been reported in invertebrates.

  16. 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

  17. 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.

  18. Interpretation of DNA vibration modes. II--The adenosine and thymidine residues involved in oligonucleotides and polynucleotides.

    PubMed

    Letellier, R; Ghomi, M; Taillandier, E

    1987-02-01

    Normal coordinate analysis of the adenosine and thymidine residues involved in the right- and left-handed conformations of oligonucleotides and polynucleotides has been performed. The valence force field, employed in this work, allowed recently to reproduce the vibrational spectra of 2'-deoxythymidine and 2'-deoxyadenosine. The calculated wavenumbers based on a non-redundant set of internal coordinates have been compared to the Raman and infrared peak positions arising from A, B, C, D and Z conformations, in the 1550-1250 cm-1 and 800-600 cm-1 spectral regions: i.e. characteristic of adenosine and thymidine residues. Moreover, a systematic study has been performed on the evolution of the vibrational wavenumbers as a function of the glycosidic angle (chi) and the sugar pucker conformation.

  19. 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.

  20. Synthesis, thymidine phosphorylase inhibition and molecular modeling studies of 1,3,4-oxadiazole-2-thione derivatives.

    PubMed

    Shahzad, Sohail Anjum; Yar, Muhammad; Bajda, Marek; Shahzadi, Lubna; Khan, Zulfiqar Ali; Naqvi, Syed Ali Raza; Mutahir, Sadaf; Mahmood, Nasir; Khan, Khalid Mohammed

    2015-06-01

    Thymidine phosphorylase (TP) inhibitors have attracted great attention due to their ability to suppress the tumors formation. In our ongoing research, a series of 1,3,4-oxadiazole-2-thione (1-12) has been synthesized under simple reaction conditions in good to excellent yields (86-98%) and their TP inhibition potential has also been evaluated. The majority of synthesized compounds showed moderate thymidine phosphorylase inhibitory activity with IC50 values ranging from 38.24±1.28 to 258.43±0.43μM, and 7-deazaxanthine (7DX) was used as a reference compound (IC50 38.68±4.42). The TP activity was very much dependent on the C-5 substituents; among this series the compound 6 bearing 4-hydroxyphenyl group was found to be the most active with IC50 38.24±1.28μM. Molecular docking studies revealed their binding mode.

  1. Histone chaperone spt16 promotes redeposition of the original h3-h4 histones evicted by elongating RNA polymerase.

    PubMed

    Jamai, Adil; Puglisi, Andrea; Strubin, Michel

    2009-08-14

    Nucleosomes are surprisingly dynamic structures in vivo, showing transcription-independent exchange of histones H2A-H2B genome-wide and exchange of H3-H4 mainly within the promoters of transcribed genes. In addition, nucleosomes are disrupted in front of and reassembled behind the elongating RNA polymerase. Here we show that inactivation of histone chaperone Spt16 in yeast results in rapid loss of H2B and H3 from transcribed genes but also from inactive genes. In all cases, histone loss is blocked by a transcription inhibitor, indicating a transcription-dependent event. Thus, nucleosomes are efficiently evicted by the polymerase but do not reform in the absence of Spt16. Yet exchange of nucleosomal H2B with free histones occurs normally, and, unexpectedly, incorporation of new H3 increases at all loci tested. This points to Spt16 restoring normal nucleosome structure by redepositing the displaced H3-H4 histones, thereby preventing incorporation of new histones and perhaps changes in histone modification patterns associated with ongoing transcription.

  2. Recent discovery of non-nucleobase thymidine phosphorylase inhibitors targeting cancer.

    PubMed

    Bera, Hriday; Chigurupati, Sridevi

    2016-11-29

    Thymidine phosphorylase (TP, EC 2.4.2.4), an enzyme involved in pyrimidine salvage pathway, is identical to platelet-derived endothelial cell growth factor (PD-ECGF) and gliostatin. It is extremely upregulated in a variety of solid tumours. The TP amplification is associated with concomitant overexpression of many angiogenic factors such as matrix metalloproteases (MMPs), interleukins (ILs), vascular endothelial growth factor (VEGF) etc., resulting in promotion of angiogenesis and cancer metastasis. In addition, overshooting TP level protects tumour cells from apoptosis and helps cell survival. Thus, TP is identified as a prime target for developing novel anticancer therapies. Pioneering research activities investigated a large number of TP inhibitors, most of which are pyrimidine or purine analogues. Recently, an array of structurally diverse non-nucleobase derivatives was designed, synthesized and established as promising TP inhibitors. This review, following an outline on the TP structure and functions, gives an overview of the recent advancement of various non-nucleobase TP inhibitors as novel anti-cancer agents.

  3. Time-resolved infrared spectroscopy of the lowest triplet state of thymine and thymidine

    PubMed Central

    Hare, Patrick M.; Middleton, Chris T.; Mertel, Kristin I.

    2008-01-01

    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 – 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=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. PMID:19936322

  4. Development of a cancer-marker activated enzymatic switch from the herpes simplex virus thymidine kinase.

    PubMed

    Shelat, Nirav Y; Parhi, Sidhartha; Ostermeier, Marc

    2017-02-01

    Discovery of new cancer biomarkers and advances in targeted gene delivery mechanisms have made gene-directed enzyme prodrug therapy (GDEPT) an attractive method for treating cancer. Recent focus has been placed on increasing target specificity of gene delivery systems and reducing toxicity in non-cancer cells in order to make GDEPT viable. To help address this challenge, we have developed an enzymatic switch that confers higher prodrug toxicity in the presence of a cancer marker. The enzymatic switch was derived from the herpes simplex virus thymidine kinase (HSV-TK) fused to the CH1 domain of the p300 protein. The CH1 domain binds to the C-terminal transactivation domain (C-TAD) of the cancer marker hypoxia inducible factor 1α. The switch was developed using a directed evolution approach that evaluated a large library of HSV-TK/CH1 fusions using a negative selection for azidothymidine (AZT) toxicity and a positive selection for dT phosphorylation. The identified switch, dubbed TICKLE (Trigger-Induced Cell-Killing Lethal-Enzyme), confers a 4-fold increase in AZT toxicity in the presence of C-TAD. The broad substrate specificity exhibited by HSV-TK makes TICKLE an appealing prospect for testing in medical imaging and cancer therapy, while establishing a foundation for further engineering of nucleoside kinase protein switches.

  5. 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.

  6. Comparative aspects of the proliferation marker thymidine kinase 1 in human and canine tumour diseases.

    PubMed

    von Euler, H; Eriksson, S

    2011-03-01

    As cell proliferation is one of the hallmarks of cancer, various types of proliferation markers are used as important tools in diagnosis, prognosis, treatment decision-making and follow-up in clinical oncology. The S phase-specific protein thymidine kinase 1 (TK1) can be used in immunohistochemistry for RNA/protein expression in tissue specimens and for activity or protein/peptide levels in serum from patients. TK1 has been used mainly in haematologic malignancies in humans, but also found beneficial in canine malignancies. As the protein sequence homology is high between humans and dogs, findings in canine models will have a high comparative value in further human research as well. In the present review, we will focus on the recent results concerning TK1's S phase-correlated expression, increased serum levels of TK1 in patients with malignancies and the relevance for veterinary and comparative oncology. Finally, the benefit of recently developed specific anti-TK1 antibodies suitable for immunologic assay is discussed.

  7. Role of the promoter in the regulation of the thymidine kinase gene

    SciTech Connect

    Travali, S.; Lipson, K.E.; Jaskulski, D.; Lauret, E.; Baserga, R.

    1988-04-01

    To identify the regulatory elements of the human thymidine kinase (TK) gene, the authors established stable cell lines carrying different chimeric constructs of the TK gene. Their results can be summarized as follows. (i) When the TK coding sequence is under the control of the calcyclin promoter (a promoter that is activated when G/sub o/ cells are stimulated by growth factors), TK mRNA levels are higher in G/sub 1/-arrested cells than in proliferating cells: (ii) when the TK coding sequence is under the control of the promoter of heat shock protein HSP70, steady-state levels of TK mRNA are highest after heat shock, regardless of the position of the cells in the cell cycle; (iii) the bacterial CAT gene under the control of the human TK promoter is maximally expressed in the S phase; (iv) the TK cDNA driven by the simian virus 40 promoter is also maximally expressed in the S phase; and (v) TK enzyme activity is always at a maximum in the S phase, even when the levels of TK mRNA are highest in nonproliferating cells. The authors conclude that although the TK coding sequence may also play some role, the TK promoter has an important role in the cell cycle regulation of TK mRNA levels.

  8. Herpes simplex virus thymidine kinase gene therapy for rat malignant brain tumors.

    PubMed

    Vincent, A J; Vogels, R; Someren, G V; Esandi, M C; Noteboom, J L; Avezaat, C J; Vecht, C; Bekkum, D W; Valerio, D; Bout, A; Hoogerbrugge, P M

    1996-01-20

    Transfer of a herpes simplex virus-derived thymidine kinase (HSV-tk) gene into brain tumor cells and subsequent ganciclovir (GCV) treatment has been shown by others to be an effective treatment in rats with intracerebrally inoculated 9L gliosarcomas. Mechanism of action and reproducibility are, however, still a matter of debate. We have used the same model to test the therapeutic effects of both retrovirus- and adenovirus-mediated transfer of the HSV-tk gene followed by GCV treatment. Survival time of rats with intracerebral 9L tumors was significantly prolonged after a single administration of adenovirus carrying a HSV-tk gene as compared to controls. Retrovirus-mediated gene transfer also resulted in significantly prolonged survival time when recombinant retrovirus-producing cells were transplanted. Direct injection of the recombinant retrovirus, HSV-tk-expressing cells, virus-producing cells without GCV administration and recombinant retrovirus-lacZ or interleukin-2 (IL-2)-producing cells did not result in tumor cell kill. In the present study, no significant difference in survival of 9L brain tumor carrying rats was found after treatment with adenovirus as compared to retrovirus-mediated HSV-tk-mediated gene transfer and subsequent GCV treatment.

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

    PubMed Central

    Tieng, Vannary; Cherpin, Ophelie; Gutzwiller, Eveline; Zambon, Alexander C; Delgado, Christophe; Salmon, Patrick; Dubois-Dauphin, Michel; Krause, Karl-Heinz

    2016-01-01

    Pluripotent stem cell (PSC)-based cell therapy is an attractive concept for neurodegenerative diseases, but can lead to tumor formation. This is particularly relevant as proliferating neural precursors rather than postmitotic mature neurons need to be transplanted. Thus, safety mechanisms to eliminate proliferating cells are needed. Here, we propose a suicide gene approach, based on cell cycle-dependent promoter Ki67-driven expression of herpes simplex virus thymidine kinase (HSV-TK). We generated a PSC line expressing this construct and induced neural differentiation. In vitro, proliferating PSC and early neural precursor cells (NPC) were killed by exposure to ganciclovir. In vivo, transplantation of PSC led to tumor formation, which was prevented by early ganciclovir treatment. Transplanted NPC did not lead to tumor formation and their survival and neural maturation were not affected by ganciclovir. In conclusion, the cell cycle promoter-driven suicide gene approach described in this study allows killing of proliferating undifferentiated precursor cells without expression of the suicide gene in mature neurons. This approach could also be of use for other stem cell-based therapies where the final target consists of postmitotic cells. PMID:27990449

  10. Thymidine phosphorylase gene variant, platelet counts and survival in gastrointestinal cancer patients treated by fluoropyrimidines

    PubMed Central

    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

  11. 3D-Pharmacophore mapping of thymidine-based inhibitors of TMPK as potential antituberculosis agents

    NASA Astrophysics Data System (ADS)

    Andrade, Carolina Horta; Pasqualoto, Kerly F. M.; Ferreira, Elizabeth I.; Hopfinger, Anton J.

    2010-02-01

    Tuberculosis (TB) is the primary cause of mortality among infectious diseases. Mycobacterium tuberculosis monophosphate kinase (TMPKmt) is essential to DNA replication. Thus, this enzyme represents a promising target for developing new drugs against TB. In the present study, the receptor-independent, RI, 4D-QSAR method has been used to develop QSAR models and corresponding 3D-pharmacophores for a set of 81 thymidine analogues, and two corresponding subsets, reported as inhibitors of TMPKmt . The resulting optimized models are not only statistically significant with r 2 ranging from 0.83 to 0.92 and q 2 from 0.78 to 0.88, but also are robustly predictive based on test set predictions. The most and the least potent inhibitors in their respective postulated active conformations, derived from each of the models, were docked in the active site of the TMPKmt crystal structure. There is a solid consistency between the 3D-pharmacophore sites defined by the QSAR models and interactions with binding site residues. Moreover, the QSAR models provide insights regarding a probable mechanism of action of the analogues.

  12. 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.

  13. Inhibition by interferon of biochemical transformation induced by cloned herpesvirus thymidine kinase genes.

    PubMed

    Otsuka, H; Qavi, H; Kit, S

    1982-10-01

    To learn whether interferon could prevent the biochemical transformations induced by cloned herpesvirus thymidine kinase (TK) genes, LM(TK-) mouse fibroblast cultures were pretreated for 24 h with 2.4-40 international units (I.U.)/ml mouse alpha + beta interferon, and subsequently transformed to the TK+ phenotype with recombinant plasmids containing the herpes simplex virus type 1 (HSV-1) TK gene (pAGO and pMH110) and the marmoset herpesvirus (MarHV) TK gene (pMAR035). Mouse alpha + beta interferon inhibited transformation and the inhibition was interferon dose-dependent. Transformation was also inhibited when LM(TK-) cells were pretreated for 2-5 h with 40 I.U./ml interferon. Maximal inhibitions of TK+ colony formation were observed following a 9-20 h pretreatment period with interferon. In contrast, 40 I.U./ml interferon treatment for 20 h did not reduce the rate or extent of LM(TK-) cell growth. Experiments in which cultures were first treated with plasmid pAGO and only afterwards treated with interferon also showed that, as the interferon concentration used, interferon did not inhibit the outgrowth of transformated colonies. Enzyme assays showed that pretreatment with interferon inhibited the induction of TK activity in cells that had been transfected with pAGO DNA.

  14. Stimulation of chick embryo cartilage sulfate and thymidine uptake: comparison of human serum, purified somatomedins, and other growth factors.

    PubMed

    Jennings, J; Buchanan, F; Freeman, D; Garland, J T

    1980-11-01

    We have compared the stimulation of sulfate and thymidine uptake into 10-day-old embryonic chick cartilage by normal human serum, partially purified somatomedins (Sm) A and B, homogeneous insulin-like growth factors (IGFs) I and II, and several other substances. With the exception of epidermal growth factor, all growth factors ((GFs) were assayed in the absence of other protein. Pelvic rudiments were preincubated in buffer for 6 h and then incubated for 24 h with the GF or serum, with labels added for the final 6 h. Human serum enhanced cartilage uptake of both thymidine and sulfate. There was a dose-dependent stimulation of thymidine uptake by Sm A or B (0.05--2 microgram/ml) and IGF I or II (0.5--20 ng/ml). Unlike serum, neither Sms nor IGFs increased SO4 uptake under these conditions. Bovine GH (10--500 ng/ml), albumin (100-1000 ng/ml), fibroblast GF (1--100 ng/ml), and epidermal GF (1--100 ng/ml) were inactive for both thymidine and sulfate. When a shorter incubation was used (7 h), Sm A enhanced SO4 uptake, and discrimination was increased by preincubation of the rudiments in buffer for 24 h. With this procedure, IGF I (0.5 ng/ml) was nearly equipotent to 5% serum. On a weight basis, IGF I was more active than either Sm A or IGF II. The data suggest that assay conditions are crucial for demonstration of Sm activity. Appropriate conditions may be different for isolated GF than for a complex medium such as serum. The results further suggest that with certain protocols, the responsiveness of chick embryo cartilage is qualitatively similar to that of hypophysectomized rat cartilage.

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

    PubMed Central

    Lashkov, Alexander A.; Gabdulkhakov, Azat G.; Prokofev, Igor I.; Seregina, Tatyana A.; Sotnichenko, Sergey E.; Lyashenko, Andrey V.; Shtil, Alexander A.; Mironov, Alexander S.; Betzel, Christian; Mikhailov, Al’bert M.

    2012-01-01

    A high-resolution structure of the complex of Vibrio cholerae uridine phosphorylase (VchUPh) with its physiological ligand thymidine is important in order to determine the mechanism of the substrate specificity of the enzyme and for the rational design of pharmacological modulators. Here, the expression and purification of VchUPh and the crystallization of its complex with thymidine are reported. Conditions for crystallization were determined with an automated Cartesian Dispensing System using The Classics, MbClass and MbClass II Suites crystallization kits. Crystals of the VchUPh–thymidine complex (of dimensions ∼200–350 µm) were grown by the sitting-drop vapour-diffusion method in ∼7 d at 291 K. The crystallization solution consisted of 1.5 µl VchUPh (15 mg ml−1), 1 µl 0.1 M thymidine and 1.5 µl reservoir solution [15%(w/v) PEG 4000, 0.2 M MgCl2.6H2O in 0.1 M Tris–HCl pH 8.5]. The crystals diffracted to 2.12 Å resolution and belonged to space group P21 (No. 4), with unit-cell parameters a = 91.80, b = 95.91, c = 91.89 Å, β = 119.96°. The Matthews coefficient was calculated as 2.18 Å3 Da−1; the corresponding solvent content was 43.74%. PMID:23143257

  16. 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}.

  17. Nqrs Data for C3H12INO7 [C3H7NO2·HIO3·2(H2O)] (Subst. No. 0646)

    NASA Astrophysics Data System (ADS)

    Chihara, H.; Nakamura, N.

    This document is part of Subvolume A `Substances Containing Ag … C10H15' of Volume 48 `Nuclear Quadrupole Resonance Spectroscopy Data' of Landolt-Börnstein - Group III `Condensed Matter'. It contains an extract of Section `3.2 Data tables' of the Chapter `3 Nuclear quadrupole resonance data' providing the NQRS data for C3H12INO7 [C3H7NO2·HIO3·2(H2O)] (Subst. No. 0646)

  18. A designed equine herpes thymidine kinase (EHV4 TK) variant improves ganciclovir-induced cell-killing.

    PubMed

    McSorley, Theresa; Ort, Stephan; Monnerjahn, Christian; Konrad, Manfred

    2014-02-01

    The limitations of the ganciclovir (GCV)/herpes simplex virus thymidine kinase (HSV1 TK: EC 2.7.1.21) system as a suicide gene therapy approach have been extensively studied over the years. In our study, we focused on improving the cytotoxic profile of the GCV/equine herpes virus-4 thymidine kinase (EHV4 TK: EC 2.7.1.21) system. Our approach involved the structure-guided mutagenesis of EHV4 TK in order to switch its ability to preferentially phosphorylate the natural substrate deoxythymidine (dT) to that of GCV. We performed steady-state kinetic analysis, genetic complementation in a thymidine kinase-deficient Escherichia coli strain, isothermal titration calorimetry, and analysis of GCV-induced cell killing through generation of HEK 293 stable cell-lines expressing EHV4 TK mutants and wild-type EHV4 TK. We found that the EHV4 TK S144H-GFP mutant preferentially phosphorylates GCV and confers increased GCV-induced cytotoxicity compared to wild-type EHV4 TK.

  19. Characterization of (/sup 3/H)pirenzepine binding to muscarinic cholinergic receptors solubilized from rat brain

    SciTech Connect

    Luthin, G.R.; Wolfe, B.B.

    1985-07-01

    Membranes prepared from rat cerebral cortex were solubilized in buffer containing 1% digitonin. Material present in the supernatant after centrifugation at 147,000 X g was shown to contain binding sites for both (/sup 3/H)quinuclidinyl benzilate ((/sup 3/H)QNB) and (/sup 3/H)pirenzepine ((/sup 3/H)PZ). Recovery of binding sites was approximately 25% of the initial membrane-bound (/sup 3/H)QNB binding sites. The Kd values for (/sup 3/H)QNB and (/sup 3/H)PZ binding to solubilized receptors were 0.3 nM and 0.1 microM, respectively. As has been observed previously in membrane preparations, (/sup 3/H)PZ appeared to label fewer solubilized binding sites than did (/sup 3/H)QNB. Maximum binding values for (/sup 3/H)PZ and (/sup 3/H)QNB binding to solubilized receptors were approximately 400 and 950 fmol/mg of protein, respectively. Competition curves for PZ inhibiting the binding of (/sup 3/H)QNB, however, had Hill slopes of 1, with a Ki value of 0.24 microM. The k1 and k-1 for (/sup 3/H)PZ binding were 3.5 X 10(6) M-1 min-1 and 0.13 min-1, respectively. The muscarinic receptor antagonists atropine, scopolamine and PZ inhibited the binding of (/sup 3/H)QNB and (/sup 3/H)PZ to solubilized receptors with Hill slopes of 1, as did the muscarinic receptor agonist oxotremorine. The muscarinic receptor agonist carbachol competed for (/sup 3/H)QNB and (/sup 3/H)PZ binding with a Hill slope of less than 1 in cerebral cortex, but not in cerebellum. GTP did not alter the interactions of carbachol or oxotremorine with the solubilized receptor. Together, these data suggest that muscarinic receptor sites solubilized from rat brain retain their abilities to interact selectively with muscarinic receptor agonists and antagonists.

  20. Endocytic pathway for /sup 3/H-histamine (/sup 3/H-HIS) metabolism in cultured human vascular endothelial cells (HEC)

    SciTech Connect

    Haddock, R.C.; Mack, P.; Baenziger, N.L.

    1986-03-01

    Histamine metabolism by HEC utilizes enzymes from both endogenous and extracellular sources. HEC convert histamine to tele-methyl-histamine and accumulate this product. They exhibit a 6- to 10-fold enhancement of /sup 3/H-HIS uptake by exogenous amine oxidase activity from human placenta (P-AO) or fetal calf serum (FCS-AO) and accumulate methyl-imidazole-acetic acid (MIAA). /sup 3/H-HIS uptake is saturable with respect to /sup 3/H-HIS and to P-AO or FCS-AO, suggesting a limited number of interaction sites on HEC. Half maximal and saturated uptake occur at 1.6 ..mu..M and 4 ..mu..M for /sup 3/H-HIS, at 0.14 U/ml and 0.65 U/ml for P-AO, and 1.0- U/ml and 2.0 U/ml for FCS-AO (1 U/ml = 1 nmole putrescine degraded/hour). FCS-AO-enhanced uptake is inhibited 64% to 94% by 3-30 mM methylmaine and 98% by 75 ..mu..M chloroquine. Treatment of HEC with dextran sulfate removes bound FCS-AO activity, blocking subsequent /sup 3/H-HIS uptake by 84%. /sup 3/H-HIS-derived metabolites present in the plasma membrane fraction of HEC incubated in the absence or presence of FCS-AO activity, are tele-methyl-histamine or MIAA respectively. These results suggest the involvement of an endocytic-type process in /sup 3/H-HIS uptake and degradation, utilizing membrane-associated endogenous and exogenous enzymes.

  1. Modulation of [3H]diazepam binding in rat cortical membranes by GABAA agonists.

    PubMed

    Wong, E H; Iversen, L L

    1985-04-01

    GABAA receptor agonists modulate [3H]diazepam binding in rat cortical membranes with different efficacies. At 23 degrees C, the relative potencies for enhancement of [3H]diazepam binding by agonists parallel their potencies in inhibiting [3H]gamma-aminobutyric acid [( 3H]GABA) binding. The agonist concentrations needed for enhancement of [3H]diazepam binding are up to 35 times higher than for [3H]GABA binding and correspond closely to the concentrations required for displacement of [3H]bicuculline methochloride (BMC) binding. The maximum enhancement of [3H]diazepam varied among agonists: muscimol = GABA greater than isoguvacine greater than 3-aminopropane sulphonic acid (3APS) = imidazoleacetic acid (IAA) greater than 4,5,6,7-tetrahydroisoxazolo (4,5,6)-pyridin-3-ol (THIP) = taurine greater than piperidine 4-sulphonic acid (P4S). At 37 degrees C, the potencies of agonists remained unchanged, but isoguvacine, 3 APS, and THIP acquired efficacies similar to GABA, whereas IAA, taurine, and P4S maintained their partial agonist profiles. At both temperatures the agonist-induced enhancement of [3H]diazepam binding was reversible by bicuculline methobromide and by the steroid GABA antagonist RU 5135. These results stress the importance of studying receptor-receptor interaction under near-physiological conditions and offer an in vitro assay that may predict the agonist status of putative GABA receptor ligands.

  2. Absorption and metabolism of ( sup 3 H)arachidonic and ( sup 14 C)linoleic acid in essential fatty acid-deficient rats

    SciTech Connect

    Hjelte, L.; Melin, T.; Nilsson, A.; Strandvik, B. )

    1990-07-01

    ({sup 3}H)arachidonic acid (20:4) and ({sup 14}C)linoleic acid (18:2) were fed in a triolein emulsion to essential fatty acid-deficient (EFAD) rats and to age-matched controls. Tissues were analyzed for radioactivity of different lipid classes after 1, 2, and 4 h. As in earlier studies, control rats retained more ({sup 3}H)20:4 than ({sup 14}C)18:2 in all organs except adipose tissue. In EFAD rats, recovery of ({sup 14}C)18:2 was increased in small intestine, liver, heart, and kidneys. In comparison to controls, EFAD rats retained much more ({sup 14}C)18:2 in phospholipids of these organs. The increase in the incorporation of both {sup 3}H and {sup 14}C into phosphatidylethanolamine was particularly pronounced. Another striking feature was the drastic increase in the retention after 4 h of {sup 14}C in cardiolipin, which is specifically located in the inner mitochondrial membrane. In contrast, incorporation of both {sup 3}H and {sup 14}C into phosphatidylinositol was decreased or unchanged in EFAD rats. Although fecal fat excretion was increased there was no evidence for a malabsorption or an increased retention in intestinal triacyglycerol of the radioactive fatty acids in EFAD rats. The proportion of ({sup 14}C)18:2 that had been converted to ({sup 14}C)20:4 was generally low but increased significantly with time in the liver and intestine of EFAD rats.

  3. Possible role of thymidine phosphorylase in gynecological tumors as an individualized treatment strategy

    PubMed Central

    Shida, Masako; Yasuda, Masanori; Fujita, Mariko; Miyazawa, Masaki; Kajiwara, Hiroshi; Hirasawa, Takeshi; Ikeda, Masae; Matsui, Naruaki; Muramatsu, Toshinari; Mikami, Mikio

    2016-01-01

    Thymidine phosphorylase (TP) is structurally similar to platelet-derived endothelial cell growth factor, and it activates 5-fluorouracil (5-FU) prodrugs and also promotes angiogenesis. In the present study, the possibility of using TP expression as a biomarker for 5-FU prodrugs, and the significance of TP as an angiogenic factor, were investigated in patients with gynecological tumors. The subjects enrolled in the study were 188 patients with gynecological tumors who provided informed consent and underwent tumor resection at the Department of Obstetrics and Gynecology of Tokai University Hospital between February 2002 and January 2010. Measurement of the enzymatic activity of TP and dihydropyrimidine dehydrogenase (DPD) was performed by enzyme-linked immunosorbent assay. In addition, immunohistochemistry (IHC) analysis of microvessels by monochrome imaging, western blotting and reverse transcription-polymerase chain reaction were performed. The mean TP activity and the TP/DPD ratio were increased in squamous cell carcinoma of the cervix (306.9 and 2.2 U/mg protein, respectively) and adenosquamous carcinoma (317.6 and 1.4 U/mg protein, respectively) compared with benign tumors and other malignancies, including endometrial (uterine) carcinoma, ovarian serous adenocarcinoma and ovarian mucinous adenocarcinoma. However, these parameters were also elevated in other histological types of cancer such as clear cell adenocarcinoma of the ovary (115.2 and 2.1 U/mg protein, respectively), in which the microvessel area was the largest of all the histological types analyzed. Since high TP expression and a high TP/DPD ratio were identified in other tumors besides cervical cancer, it is possible that patients for whom 5-FU prodrugs are indicated could be selected appropriately if their TP activity is determined and their TP expression is analyzed by IHC prior to initiation of the treatment. PMID:27899985

  4. [18F]-fluoro-l-thymidine PET and advanced MRI for preoperative grading of gliomas

    PubMed Central

    Collet, S.; Valable, S.; Constans, J.M.; Lechapt-Zalcman, E.; Roussel, S.; Delcroix, N.; Abbas, A.; Ibazizene, M.; Bernaudin, M.; Barré, L.; Derlon, J.M.; Guillamo, J.S.

    2015-01-01

    Purpose Conventional MRI based on contrast enhancement is often not sufficient in differentiating grade II from grade III and grade III from grade IV diffuse gliomas. We assessed advanced MRI, MR spectroscopy and [18F]-fluoro-l-thymidine ([18F]-FLT) PET as tools to overcome these limitations. Methods In this prospective study, thirty-nine patients with diffuse gliomas of grades II, III or IV underwent conventional MRI, perfusion, diffusion, proton MR spectroscopy (1H-MRS) and [18F]-FLT-PET imaging before surgery. Relative cerebral blood volume (rCBV), apparent diffusion coefficient (ADC), Cho/Cr, NAA/Cr, Cho/NAA and FLT-SUV were compared between grades. Results Cho/Cr showed significant differences between grade II and grade III gliomas (p = 0.03). To discriminate grade II from grade IV and grade III from grade IV gliomas, the most relevant parameter was the maximum value of [18F]-FLT uptake FLTmax (respectively, p < 0.001 and p < 0.0001). The parameter showing the best correlation with the grade was the mean value of [18F]-FLT uptake FLTmean (R2 = 0.36, p < 0.0001) and FLTmax (R2 = 0.5, p < 0.0001). Conclusion Whereas advanced MRI parameters give indications for the grading of gliomas, the addition of [18F]-FLT-PET could be of interest for the accurate preoperative classification of diffuse gliomas, particularly for identification of doubtful grade III and IV gliomas. PMID:26106569

  5. The Structural Interface between HIV-1 Vif and Human APOBEC3H.

    PubMed

    Ooms, Marcel; Letko, Michael; Simon, Viviana

    2017-03-01

    Human APOBEC3H (A3H) is a cytidine deaminase that inhibits HIV-1 replication. To evade this restriction, the HIV-1 Vif protein binds A3H and mediates its proteasomal degradation. To date, little information on the Vif-A3H interface has been available. To decipher how both proteins interact, we first mapped the Vif-binding site on A3H by functionally testing a large set of A3H mutants in single-cycle infectivity and replication assays. Our data show that the two A3H α-helixes α3 and α4 represent the Vif-binding site of A3H. We next used viral adaptation and a set of Vif mutants to identify novel, reciprocal Vif variants that rescued viral infectivity in the presence of two Vif-resistant A3H mutants. These A3H-Vif interaction points were used to generate the first A3H-Vif structure model, which revealed that the A3H helixes α3 and α4 interact with the Vif β-sheet (β2-β5). This model is in good agreement with previously reported Vif and A3H amino acids important for interaction. Based on the predicted A3H-Vif interface, we tested additional points of contact, which validated our model. Moreover, these experiments showed that the A3H and A3G binding sites on HIV-1 Vif are largely distinct, with both host proteins interacting with Vif β-strand 2. Taken together, this virus-host interface model explains previously reported data and will help to identify novel drug targets to combat HIV-1 infection.IMPORTANCE HIV-1 needs to overcome several intracellular restriction factors in order to replicate efficiently. The human APOBEC3 locus encodes seven proteins, of which A3D, A3F, A3G, and A3H restrict HIV-1. HIV encodes the Vif protein, which binds to the APOBEC3 proteins and leads to their proteasomal degradation. No HIV-1 Vif-APOBEC3 costructure exists to date despite extensive research. We and others previously generated HIV-1 Vif costructure models with A3G and A3F by mapping specific contact points between both proteins. Here, we applied a similar approach to HIV

  6. Mapping Region of Human Restriction Factor APOBEC3H Critical for Interaction with HIV-1 Vif.

    PubMed

    Nakashima, Masaaki; Tsuzuki, Shinya; Awazu, Hiroaki; Hamano, Akiko; Okada, Ayaka; Ode, Hirotaka; Maejima, Masami; Hachiya, Atsuko; Yokomaku, Yoshiyuki; Watanabe, Nobuhisa; Akari, Hirofumi; Iwatani, Yasumasa

    2017-03-21

    The APOBEC3 (A3) family of cellular cytidine deaminases comprises seven members (A, B, C, D, F, G, and H) that potently inhibit retroviral replication. Human immunodeficiency virus type 1 (HIV-1) Vif is a small pleiotropic protein that specifically inactivates these enzymes, targeting them for ubiquitin-mediated proteasomal degradation. A3 Vif-interaction sites are presumed to fall into three distinct types: A3C/D/F, A3G, and A3H. To date, two types of A3G and A3C/D/F sites have been well characterized, whereas the A3H Vif-binding site remains poorly defined. Here, we explore the residues critical for the A3H-type Vif interaction. To avoid technical difficulties in performing experiments with human A3H haplotype II (hapII), which is relatively resistant to HIV-1 Vif, we employed its ortholog chimpanzee A3H (cA3H), which displays high Vif sensitivity, for a comparison of sensitivity with that of A3H hapII. The Vif susceptibility of A3H hapII-cA3H chimeras and their substitution mutants revealed a single residue at position 97 as a major determinant for the difference in their Vif sensitivities. We further surveyed critical residues by structure-guided mutagenesis using an A3H structural model and thus identified eight additional residues important for Vif sensitivity, which mapped to the α3 and α4 helices of A3H. Interestingly, this area is located on a surface adjacent to the A3G and A3C/D/F interfaces and is composed of negatively charged and hydrophobic patches. These findings suggest that HIV-1 Vif has evolved to utilize three dispersed surfaces for recognizing three types of interfaces on A3 proteins under certain structural constraints.

  7. Significance of skeletal muscle digitalis receptors for [3H]ouabain distribution in the guinea pig.

    PubMed

    Kjeldsen, K; Nørgaard, A; Hansen, O; Clausen, T

    1985-09-01

    The importance of specific digitalis glycoside binding sites in skeletal muscle for the digitalis glycoside distribution in the guinea pig was evaluated using [3H]ouabain and [3H]digoxin binding assays. Measurements of [3H]ouabain binding capacity (EOmax) in gastrocnemius and heart muscles in vitro gave values of 474 +/- 15 and 1,092 +/- 39 pmol/g wet wt., respectively, in 4-week-old guinea pigs. Hence the total amount of [3H]ouabain binding sites in skeletal muscle and the heart was around 42,700 and 1,200 pmol, respectively. The apparent dissociation constants (Kd) for ouabain receptor interaction was 0.7 X 10(-7) and 1.5 X 10(-7) M for skeletal muscle and heart, respectively. Comparison of [3H]ouabain and [3H]digoxin binding revealed that these drugs are competitive. From birth to maturity the concentration of [3H]ouabain binding sites in guinea pigs decreased from 803 +/- 58 to 304 +/- 28 pmol/g wet wt. in gastrocnemius muscle and from 1,458 +/- 31 to 1,079 +/- 19 pmol/g wet wt. in the heart. After i.p. injection, measurements of the distribution of [3H]ouabain in plasma, skeletal muscle and the heart showed an almost equal relative specific occupancy of digitalis glycoside receptors in skeletal muscle and the heart: When 10% of the digitalis receptors in the heart were occupied by [3H]ouabain, 13% of those in the skeletal muscles were occupied. It was calculated that 1 hr after the i.p. administration of [3H]ouabain the amount of [3H]ouabain specifically bound to the skeletal muscles and the heart corresponded to 5 times and 1/10 the amount available in the extracellular pool, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. 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.

  9. Investigation of tritium incorporation of tritium incorporation by means of excreted metabolites.

    PubMed

    Biró, T; Szilágyi, M

    1978-01-01

    The commonly accepted urine analysis by liquid scintillation method was applied for whole body dose estimating. After the separation of metabolite fractions the organically bound tritium in urine could be measured. Urine samples from workers repeatedly exposed to tritium incorporation during the chemical processing of various labeled compounds have been collected and analyzed. The time dependence of tritium activity in certain metabolites was found to be characteristic, significantly differing from the 3H concentration curve of the native or treated urine sample.

  10. Effects of Flutamide on [Methyl-3H]-Choline Uptake in Human Prostate Cancer-3 Cells: A Pilot Study

    PubMed Central

    Al-Saeedi, Fatma

    2007-01-01

    Background: Positron emission tomography using [methyl-11C]-choline is effective in imaging many types of cancer, especially prostate cancer (PC). The antiandrogen flutamide is often used as part of the initial treatment of PC. Data on the effect of flutamide on and methylcholine incorporation into PC-3 cells are lacking in the experimental and literature work. Objectives: The aims of this study were to assess whether human PC-3 cells are susceptible to flutamide and whether the drug modulates the uptake of [methyl-3H]-choline into these cells. Methods: PC-3 cells were treated for 3 days with flutamide (≤100 nmol/L), inhibiting growth by 20% to 70% with control cells included. Two viability tests (cytotoxic analyses), the thiazole blue assay and the trypan blue exclusion method, were used to determine the median inhibitory concentration for flutamide (10 nmol/L). Control and flutamide-treated cells were incubated with [methyl-3H]-choline for 10 minutes and then in nonradioactive medium for 10 minutes to simulate the rapid blood clearance of [methyl-11C]-choline tracer that occurs within 5 to 20 minutes, and then extracted using organic and aqueous solvents to determine the intracellular distribution of the tracer. Protein assay and flow-cytometry analysis were used to determine protein content and DNA synthesis in both control and treated cells. The uptake of [methyl-3H]-choline was normalized to protein content and expressed as mean (SD) dpm/1Jg protein (n = 6). Results: PC-3 cell proliferation was inhibited with flutamide treatment. After treatment of PC-3 cells with flutamide 10 nmol/L for 3 days, cells accumulated DNA during the S phase. Mean (SD) [methyl-3H]-choline uptake was found to be significantly lower with flutamide 10-nmol/L-treated cells compared with control cells (65.95 [0.72] vs 114.21 [0.57] dpm/1Jg protein; P < 0.001); the difference between the 5-nmol/L-treated cells and controls was nonsignificant. Conclusions: In this pilot study, flutamide

  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. 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.

  13. 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.

  14. 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

  15. 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.

  16. Nuclear accident-derived (3)H in river water of Fukushima Prefecture during 2011-2014.

    PubMed

    Ueda, Shinji; Hasegawa, Hidenao; Kakiuchi, Hideki; Ochiai, Shinya; Akata, Naofumi; Hisamatsu, Shun'ichi

    2015-08-01

    During 2011-2014, we measured (3)H concentrations in river water samples collected during base flow conditions and during several flood events from two small rivers in a mountainous area in Fukushima Prefecture, which received deposition of (137)Cs from the Fukushima Dai-ichi Nuclear Power Plant accident. (3)H concentrations above background levels were found in water samples collected during both base flow conditions and flood events in 2011. The (3)H concentrations during flood events were generally higher than those during base flow conditions. The (3)H concentrations in both rivers during base flow conditions and flood events decreased with time after the accident and reached almost background levels in 2013. We also measured (3)H concentrations in freshwater samples from 16 other rivers and one dam in eastern Fukushima Prefecture from 2012 to 2014 during base flow conditions. The measured (3)H concentrations were higher than the background level in 2012 and decreased with time. The (137)Cs inventory in the catchment area at each sampling point was estimated from air-borne monitoring results in the literature and compared with the (3)H concentrations. We found surprisingly good correlations between (137)Cs inventories in the catchment areas and (3)H concentrations in the water samples. Further studies will be necessary to clarify the reason for the good correlation.

  17. Elevated ZC3H15 increases HCC growth and predicts poor survival after surgical resection

    PubMed Central

    Li, Li-mei; Liu, Hui; Fu, Si-yuan; Yang, Yuan; Zhang, Jin; Yuan, Shen-xian; Wang, Ruo-yu; Yang, Yun; Gu, Fang-ming; Dong, Li-wei; Pan, Ze-ya; Zhou, Wei-ping

    2016-01-01

    Zinc finger CCCH-type containing 15 (ZC3H15), also known as DRG family regulatory protein 1 (DFRP1), is a highly conserved eukaryotic protein that associates with active translation machinery. The aim of our study was to explore the clinical relevance and intrinsic functions of ZC3H15 in hepatocellular carcinoma (HCC). We constructed a cohort with 261 tumor and matched normal tissues from HCC patients. ZC3H15 protein and mRNA levels were determined using immunohistochemistry, western blot analysis, and quantitative polymerase chain reaction. ZC3H15 was highly expressed in the majority of HCC cases, and high ZC3H15 levels were significantly associated with high serum a-fetoprotein (AFP) levels (>20 ng/mL) and vascular invasion. Kaplan-Meier and Cox regression data indicated that elevated ZC3H15 was an independent predictor for HCC-specific disease-free survival (hazards ratio [HR], 1.789; 95% confidence interval [95% CI], 1.298-2.466 [P=0.0004]) and overall survival (HR, 1.613; 95% CI, 1.120-2.322 [P=0.0101]). Interaction of ZC3H15 with TRAF2 increased activation of NFκB signaling. These results suggest ZC3H15 is an independent prognostic marker in HCC patients that is clinicopathologically associated with tumor invasion and serum AFP levels. PMID:27191988

  18. 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).

  19. 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).

  20. 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).

  1. A combined crossed beam and theoretical investigation of O(3P)+C3H3→C3H2+OH

    NASA Astrophysics Data System (ADS)

    Lee, Hohjai; Joo, Sun-Kyu; Kwon, Lee-Kyoung; Choi, Jong-Ho

    2004-02-01

    The radical-radical reaction dynamics of ground-state atomic oxygen [O(3P)] with propargyl radicals (C3H3) has first been investigated in a crossed beam configuration. The radical reactants O(3P) and C3H3 were produced by the photodissociation of NO2 and the supersonic flash pyrolysis of precursor propargyl bromide, respectively. A new exothermic channel of O(3P)+C3H3→C3H2+OH was identified and the nascent distributions of the product OH in the ground vibrational state (X 2Π:ν″=0) showed bimodal rotational excitations composed of the low- and high-N″ components without spin-orbit propensities. The averaged ratios of Π(A')/Π(A″) were determined to be 0.60±0.28. With the aid of ab initio theory it is predicted that on the lowest doublet potential energy surface, the reaction proceeds via the addition complexes formed through the barrierless addition of O(3P) to C3H3. The common direct abstraction pathway through a collinear geometry does not occur due to the high entrance barrier in our low collision energy regime. In addition, the major reaction channel is calculated to be the formation of propynal (CHCCHO)+H, and the counterpart C3H2 of the probed OH product in the title reaction is cyclopropenylidene (1c-C3H2) after considering the factors of barrier height, reaction enthalpy and structural features of the intermediates formed along the reaction coordinate. On the basis of the statistical prior and rotational surprisal analyses, the ratio of population partitioning for the low- and high-N″ is found to be about 1:2, and the reaction is described in terms of two competing addition-complex mechanisms: a major short-lived dynamic complex and a minor long-lived statistical complex. The observed unusual reaction mechanism stands in sharp contrast with the reaction of O(3P) with allyl radical (C3H5), a second significant conjugated hydrocarbon radical, which shows totally dynamic processes [J. Chem. Phys. 117, 2017 (2002)], and should be understood based

  2. Concentration of (3)H in plants around Fukushima Dai-ichi Nuclear Power Station.

    PubMed

    Kakiuchi, Hideki; Akata, Naofumi; Hasegawa, Hidenao; Ueda, Shinji; Tokonami, Shinji; Yamada, Masatoshi; Hosoda, Masahiro; Sorimachi, Atsuyuki; Tazoe, Hirofumi; Noda, Kaori; Hisamatsu, Shun'ichi

    2012-01-01

    A large amount of radionuclides was released from the Fukushima Dai-ichi Nuclear Power Station (FDNPS) following the damage caused by the tsunami due to the Great East Japan Earthquake on 11 March 2011. Although many radionuclides in various environmental samples around the FDNPS have been measured, (3)H in the terrestrial environment has not yet been reported. We present here the first survey results of (3)H concentrations in plant samples collected around the FDNPS in 2011 from shortly after the accident. The free-water (3)H concentrations in herbaceous plant shoots and evergreen tree leaves were considerably higher than the previous background concentration, and diminished with distance from the FDNPS. Although reconstruction of atmospheric (3)H concentrations after the accident is difficult, a rough estimate of the radiation dose due to (3)H inhalation about 20 km from the FDNPS is on the order of a few microsieverts (μSv).

  3. Photoaffinity labeling of the dopamine reuptake carrier protein with 3-azido sup 3 H GBR-12935

    SciTech Connect

    Berger, S.P.; Martenson, R.E.; Laing, P.; Thurkauf, A.; Decosta, B.; Rice, K.C.; Paul, S.M. )

    1991-04-01

    A high affinity tritiated azido-diphenylpiperazine derivative, 3-azido {sup 3}H GBR-12935, was synthesized as a potential photoaffinity probe of the dopamine transporter. Initially, the reversible binding of 3-azido {sup 3}H GBR-12935 to crude synaptosomal membranes from the rat striatum was characterized. Specific binding was sodium dependent and inhibited by a variety of drugs that are known to potently inhibit dopamine uptake. Other neurotransmitter uptake inhibitors, as well as cis-flupenthixol, a potent inhibitor of {sup 3}H GBR-12935 binding to piperazine binding sites, failed to inhibit specific binding at concentrations of less than or equal to 10 microM. A good correlation was observed between the relative potencies of these drugs in inhibiting dopamine uptake into synaptosomes and in inhibiting specific 3-azido {sup 3}H GBR-12935 binding to rat striatal membranes. These data suggest that 3-azido {sup 3}H GBR-12935, like other diphenylpiperazines such as {sup 3}H GBR-12935 and {sup 3}H GBR-12909, binds primarily to the dopamine transporter under defined assay conditions. After UV photolysis of crude synaptosomal membranes preincubated with 3-azido {sup 3}H GBR-12935 (1-2 nM), a single radiolabeled polypeptide with an apparent molecular mass of 80 kDa was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Photoincorporation of 3-azido {sup 3}H GBR-12935 into this polypeptide was inhibited selectively by compounds that inhibit the uptake of dopamine and was completely dependent on the presence of Na+. No photolabeled proteins were observed when cerebellar membranes were substituted for striatal membranes. Essentially complete adsorption of the radiolabeled 80-kDa polypeptide to wheat germ agglutinin and elution with N-acetyl-D-glucosamine strongly suggest that the dopamine transporter polypeptide photolabeled by 3-azido {sup 3}H GBR-12935 is glycosylated.

  4. Effect of xid on autoimmune C3H-gld/gld mice.

    PubMed

    Seldin, M F; Reeves, J P; Scribner, C L; Roths, J B; Davidson, W F; Morse, H C; Steinberg, A D

    1987-06-01

    The xid gene was introduced into C3H-gld/gld mice to determine its effects on the development of autoimmune disease. C3H-gld/gld.xid mice were compared with C3H-gld/gld mice for the development of lymphadenopathy, surface phenotype of lymph node (LN) cells, c-myb oncogene RNA production, serum immunoglobulin (Ig) levels, and autoantibody production. In addition, C3H-gld/gld and C3H-lpr/lpr mice were examined for serum Ig and autoantibody levels. The results showed that the xid gene had no effect on either the development of the severe lymphadenopathy characteristic of C3H-gld/gld mice or the phenotype of the Ly-2-, L3T4-, Ly-5(B220)+ T-cell subset that is expanded in the LN and spleens of these mice. Similarly, xid did not affect the high levels of c-myb oncogene RNA expression by C3H-gld/gld LN and spleen cells. By contrast, the xid gene caused a significant reduction in serum IgM but not IgA levels and almost completely ablated the generation of both IgM and IgG anti-ssDNA antibodies and anti-dsDNA antibodies. These data suggest that the xid gene can dramatically decrease the B-cell manifestations of autoimmunity in gld homozygotes without affecting their abnormal T-cell expansion. Comparisons of age-matched C3H-gld/gld and C3H-lpr/lpr mice showed that they had similarly elevated serum IgM and IgA levels and anti-ssDNA and anti-dsDNA antibody levels providing further evidence that gld and lpr produce parallel defects in C3H mice.

  5. 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.

  6. Establishment of conditions for the detection of bovine herpesvirus-1 by polymerase chain reaction using primers in the thymidine kinase region.

    PubMed Central

    Yason, C V; Harris, L M; McKenna, P K; Wadowska, D; Kibenge, F S

    1995-01-01

    Polymerase chain reaction (PCR) for detection of bovine herpesvirus-1 (BHV-1) was developed and optimized using 22 bp sense and 20 bp antisense primers in the thymidine kinase (TK) coding region. The amplification product is 183 bp long. The PCR optimization was done using BHV-1 tissue culture supernate (BHV-1TCS), concentrated BHV-1 tissue culture supernate (cBHV-1TCS) and sucrose gradient purified BHV-1 (pBHV-1). The sensitivity of four methods of sample preparation which are standard DNA extraction, modified proteinase K (PK) digestion, GeneReleaserTM + 34 cycles or + 44 cycles, and boiling were compared with virus isolation (VI) using BHV-1TCS. The incorporation of 10% glycerol in the reaction mixture, the incubation in PK for 18 hours and predenaturation of samples and cooling in ice prior to PCR were essential for the amplification of BHV-1 DNA for samples prepared by standard DNA extraction and modified PK digestion. The preparation of samples by Gene-ReleaserTM, a proprietary nucleic acid releasing cocktail, showed 10 to 1,000-fold increase in sensitivity compared to standard DNA extraction and modified PK digestion. No amplification was observed in samples prepared by boiling. The sample preparation of BHV-1 LA strain by GeneReleaserTM showed sensitivity equivalent to virus isolation. The BHV-1 TK PCR using GeneReleaserTM has a detection limit of 1 picogram and 10 fentograms of purified BHV-1 DNA using ethidium bromide stained gel and Southern blot hybridization, respectively. It could detect viral DNA in 1,000 infected cells in a total suspension of 10,000 cells using either ethidium bromide stained gel or Southern blot hybridization. Images Fig. 1. Fig. 2. Fig. 3. PMID:7648533

  7. Combination of thymidine phosphorylase gene transfer and deoxyinosine treatment greatly enhances 5-fluorouracil antitumor activity in vitro and in vivo.

    PubMed

    Ciccolini, J; Cuq, P; Evrard, A; Giacometti, S; Pelegrin, A; Aubert, C; Cano, J P; Iliadis, A

    2001-12-01

    We reported previously that 5-fluorouracil (FUra) efficacy could be enhanced by increasing tumoral thymidine phosphorylase (TP) activity. Potentiated TP yield was achieved by either transfecting cells with human TP gene (A. Evrard et al., Br. J. Cancer, 80: 1726-1733, 1999) or associating FUra with 2'-deoxyinosine (d-Ino), a modulator providing the tumors with TP cofactor deoxyribose 1-phosphate (J. Ciccolini et al., Clin. Cancer Res., 6: 1529-1535, 2000). The purpose of the present work was to study the effects of a combined modulation (TP gene transfer + use of d-Ino) on the sensitivity to FUra of the LS174T human colorectal cell line. Results showed a near 4000 times increase of cell sensitivity in vitro after double (genetic + biochemical) modulation. This potentiation of tumor response was accompanied by a total change in the FUra anabolic pathway with a 5000% increase of cytosolic fluorodeoxyuridine monophosphate, a stronger and longer inhibition of thymidylate synthase, and 300% augmentation of DNA damage. Besides, whereas thymidine failed to inhibit FUra cytotoxicity in LS174T wild-type cells, the potentiation of the antitumor activity observed in the modulating regimen was partly reversed by thymidine, indicative of thymidylate synthase as the main drug target. The impact of this double modulation was next investigated in xenograft-bearing nude mice. Results showed that whereas FUra alone was completely ineffective on wild-type tumor growth, the size of TP-transfected tumors in animals treated with the FUra/d-Ino combination was reduced by 80% (P < 0.05). Our results suggest that FUra exhibits stronger antiproliferative activity when activated via TP through the DNA pathway and that high tumoral TP activity therefore leads to enhanced sensitivity to fluoropyrimidines.

  8. Anomalous absorption in c-C_3H and c-C_3D radicals

    NASA Astrophysics Data System (ADS)

    Chandra, S.; Shinde, S. V.; Kegel, W. H.; Sedlmayr, E.

    Yamamoto et al. (1987) reported the first detection of the c-C_3H radical in TMC-1 through its transition 2_1 2 rightarrow 1_1 1 at 91.5 GHz. The column density of c-C_3H in TMC-1 was estimated to be 6 times 10^12 cm^-2, which is about one order of magnitude lower than that of the c-C_3H_2 which is ubiquitous in galactic objects. Mangum & Wootten (1990) detected c-C_3H through the transition 1_1 0 rightarrow 1_1 1 at 14.8 GHz in 12 additional galactic objects. The most probable production mechanism of both the c-C_3H and c-C_3H_2 in dark clouds is a common dissociation reaction of the C_3H_3^+ ion (Adams & Smith 1987). Although the c-C_3H is 0.8 eV less stable than its isomer l-C_3H, finding of comparable column densities of both the isomers in TMC-1 suggests that the formation rate for both, c-C_3H and l-C_3H, are of about the same order in the cosmic objects. The existence of a metastable isomer under interstellar conditions is a well known phenomenon in astronomy. The aim of this investigation is a quantitative estimate of relative line intensities under NLTE conditions. For wide ranges of physical parameters, where these molecules may be found, we have solved a set of statistical equilibrium equations coupled with the equations of radiative transfer in an on-the-spot approximation. For c-C_3H, we accounted for 51 energy levels connected by 207 radiative transitions and for c-C_3D, we accounted for 51 energy levels connected by 205 radiative transitions. Our results show that the 3_3 1 rightarrow 3_3 0 transition of c-C_3H and c-C_3D may be found in absorption against the cosmic microwave background (CMB). Furthermore, we found population inversion for the 1_1 0 rightarrow 1_1 1 transition. These findings may be useful in identifying these molecules in other cosmic objects, as well as for the determination of physical parameters in these objects.

  9. Accumulation of radioactivity after repeated infusion of 3H-adrenaline and 3H-noradrenaline in the rat as a model animal.

    PubMed

    Lepschy, M; Filip, T; Palme, R G

    2014-10-01

    Besides enzymatic inactivation, catecholamines bind non-enzymatically and irreversible to proteins. The physiological impact of these catecholamine adducts is still unclear. We therefore collected basic data about the distribution of catecholamine adducts in the rat after repeated intravenous administration of (3)H-adrenaline and (3)H-noradrenaline. In all animals radioactivity in blood increased until the last injection on Day 7 and decreased then slowly close to background values (plasma) or remained higher (erythrocytes). In all sampled tissues radioactivity could be found, but only in hair high amounts remained present even after 3 weeks. Half-life of rat serum albumin loaded with (3)H-adrenaline or (3)H-noradrenaline was not altered. This study provides basic knowledge about the distribution of catecholamines or their adducts, but physiological effects could not be demonstrated. However, for the first time deposition and accumulation of catecholamines (adducts) in the hair could be proven, suggesting that hair might be used for evaluating long term stress.

  10. 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.

  11. A comparison of growth and development of three major agricultural insect pests infected with Heliothis virescens ascovirus 3h (HvAV-3h).

    PubMed

    Li, Shun-Ji; Wang, Xing; Zhou, Zhong-Shi; Zhu, Jie; Hu, Jue; Zhao, Yi-Pei; Zhou, Gui-Wei; Huang, Guo-Hua

    2013-01-01

    Ascoviruses are double-stranded DNA viruses that are pathogenic to lepidopteran hosts, particularly noctuid larvae. Infection of a larva is characterized by retarded growth, reduced feeding and yellowish body color. In this paper, we reported the growth and development of three major agricultural noctuid insect pests, Helicoverpa armigera (Hübner), Spodoptera exigua (Hübner) and Spodoptera litura (Fabricius), infected with Heliothis virescens ascovirus 3h (HvAV-3h). Using 10-fold serial dilutions (0 to 7) of HvAV-3h-containing hemolymph to infect S. litura larvae, we found no significant difference in larval mortalities from 0 to 10(3)-fold dilutions; however, significant differences were observed at 10(4)-fold dilution and above. Using a 10-fold dilution of HvAV-3h-containing hemolymph to infect H. armigera, S. exigua and S. litura larvae, we found that the growth and development were significantly affected. All infected larvae could not pupate; the survival times of treated H. armigera, S. litura and S. exigua larvae were significantly longer than untreated control larvae. Body weight showed significant difference between treated and untreated control group from day 1 after inoculation in H. armigera and S. exigua, but day 2 in S. litura. Additionally, food intake also showed significant difference between treated and untreated control group from day 2 after inoculation in H. armigera and S. litura, but day 3 in S. exigua.

  12. Is thymidine glycol containing DNA a substrate of E. coli DNA mismatch repair system?

    PubMed

    Perevozchikova, Svetlana A; Trikin, Roman M; Heinze, Roger J; Romanova, Elena A; Oretskaya, Tatiana S; Friedhoff, Peter; Kubareva, Elena A

    2014-01-01

    The DNA mismatch repair (MMR) system plays a crucial role in the prevention of replication errors and in the correction of some oxidative damages of DNA bases. In the present work the most abundant oxidized pyrimidine lesion, 5,6-dihydro-5,6-dihydroxythymidine (thymidine glycol, Tg) was tested for being recognized and processed by the E. coli MMR system, namely complex of MutS, MutL and MutH proteins. In a partially reconstituted MMR system with MutS-MutL-MutH proteins, G/Tg and A/Tg containing plasmids failed to provoke the incision of DNA. Tg residue in the 30-mer DNA duplex destabilized double helix due to stacking disruption with neighboring bases. However, such local structural changes are not important for E. coli MMR system to recognize this lesion. A lack of repair of Tg containing DNA could be due to a failure of MutS (a first acting protein of MMR system) to interact with modified DNA in a proper way. It was shown that Tg in DNA does not affect on ATPase activity of MutS. On the other hand, MutS binding affinities to DNA containing Tg in G/Tg and A/Tg pairs are lower than to DNA with a G/T mismatch and similar to canonical DNA. Peculiarities of MutS interaction with DNA was monitored by Förster resonance energy transfer (FRET) and fluorescence anisotropy. Binding of MutS to Tg containing DNAs did not result in the formation of characteristic DNA kink. Nevertheless, MutS homodimer orientation on Tg-DNA is similar to that in the case of G/T-DNA. In contrast to G/T-DNA, neither G/Tg- nor A/Tg-DNA was able to stimulate ADP release from MutS better than canonical DNA. Thus, Tg residue in DNA is unlikely to be recognized or processed by the E. coli MMR system. Probably, the MutS transformation to active "sliding clamp" conformation on Tg-DNA is problematic.

  13. Interactions of dopamine and the release of [3H]-taurine and [3H]-glycine from the isolated retina of the rat.

    PubMed

    Pycock, C J; Smith, L F

    1983-02-01

    1 The dose-related, calcium-dependent, potassium-stimulated release of preloaded [(3)H]-dopamine from the superfused rat retina has been demonstrated.2 A high-affinity uptake system for dopamine exists in rat retina in vitro; K(m) value was calculated as 1.89 muM, V(max) value as 1.4 nmol g(-1) tissue h(-1).3 Dopamine (0.8 and 4 mM) inhibited the spontaneous release of [(3)H]-glycine from retina, and in the case of 0.8 mM dopamine this inhibitory effect was antagonized by 10 muM (+)-butaclamol but not by 10 muM (-)-butaclamol.4 The potassium-evoked (25 mM) release of [(3)H]-glycine from rat retina was similarly inhibited by dopamine (0.4-4 mM) in a dose-related manner when added to the superfusate with the potassium. The effect of 0.8 mM dopamine was antagonized by 10 muM (+)-butaclamol but not by 10 muM (-)-butaclamol.5 Dopamine (4 mM) significantly reduced the spontaneous release of [(3)H]-taurine from rat retina.6 The potassium-stimulated (25 mM) release of [(3)H]-taurine occurred after the cessation of the depolarizing stimulus. This delayed release of [(3)H]-taurine was unaffected if dopamine was applied to the superfusate at the same time as the potassium, but it was significantly reduced if dopamine (0.8 and 4 mM) was applied after the depolarizing stimulus had been removed and during the actual amino acid release phase.7 The inhibition of K(+)-stimulated (25 mM) delayed release of [(3)H]-taurine by applying dopamine (0.8 mM) after the depolarizing stimulus was blocked by 10 muM (+)-butaclamol but not by 10 muM (-)-butaclamol.8 The results are discussed with respect to the possible neurotransmitter role for dopamine within the rat retina, and its possible interaction with glycine and taurine.

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. Binding of [3H]SCH23390 to a non-dopaminergic site in bovine kidney.

    PubMed

    Hollis, C M; Turner, J D; Strange, P G

    1992-05-08

    Binding of the D1 dopamine receptor antagonist [3H]SCH23390 to bovine renal cortical membranes has been studied. Specific binding of [3H]SCH23390 was saturable and reversible and stereoisomers of SCH23390 competed stereoselectively. In contrast, competition with the isomers of butaclamol was not stereoselective and dopamine failed to compete for the [3H]SCH23390 binding site. The site is therefore not a D1 dopamine receptor. Competition studies with a very wide range of compounds failed to define the nature of the [3H]SCH23390 binding sites in renal cortex whereas in parallel studies the characteristics of [3H]SCH23390 binding in caudate nucleus were entirely consistent with those of D1 dopamine receptors. The nature of [3H]SCH23390 binding in preparations of tubular and glomerular membranes was found to be virtually identical to those of crude renal cortical membranes indicating lack of compartmentation of these sites. Autoradiographic studies of [3H]SCH23390 binding in bovine kidney showed significantly higher levels of binding sites in renal cortex compared with renal medulla and this was confirmed by direct ligand binding studies.

  19. 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.

  20. 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.

  1. A New Sandwich ELISA for Quantification of Thymidine Kinase 1 Protein Levels in Sera from Dogs with Different Malignancies Can Aid in Disease Management.

    PubMed

    Jagarlamudi, Kiran Kumar; Moreau, Laura; Westberg, Sara; Rönnberg, Henrik; Eriksson, Staffan

    2015-01-01

    Thymidine kinase 1 (TK1) is a DNA precursor enzyme whose expression is closely correlated with cell proliferation and cell turnover. Sensitive serum TK1 activity assays have been used for monitoring and prognosis of hematological malignancies in both humans and dogs. Here we describe the development of a specific sandwich TK1-ELISA for the quantification of TK1 protein levels in sera from dogs with different malignancies. A combination of rabbit polyclonal anti-dog TK1 antibody and a mouse monoclonal anti-human TK1 antibody was used. Different concentrations of recombinant canine TK1 was used as standard. Clinical evaluation of the ELISA was done by using sera from 42 healthy dogs, 43 dogs with hematological tumors and 55 with solid tumors. An established [3H]-dThd phosphorylation assay was used to determine the TK1 activity levels in the same sera. The mean TK1 activities in dogs with hematological tumors were significantly higher than those found in healthy dogs. In agreement with earlier studies, no significant difference was observed in serum TK1 activities between healthy dogs and dogs with solid tumors. However, the mean TK1 protein levels determined by new TK1-ELISA were significantly higher not only in hematological tumors but also in solid tumors compared to healthy dogs (mean ± SD = 1.30 ± 1.16, 0.67 ± 0.55 and 0.27± 0.10 ng/mL, respectively). Moreover, TK1-ELISA had significantly higher ability to distinguish lymphoma cases from healthy based on receiver operating characteristic analyses (area under the curve, AUC, of 0.96) to that of the activity assay (AUC, 0.84). Furthermore, fluctuations in TK1 protein levels during the course of chemotherapy in dogs with lymphoma closely associated with clinical outcome. Overall, the TK1-ELISA showed significant linear correlation with the TK1 activity assay (rs = 0.6, p<0.0001). Thus, the new TK1-ELISA has sufficient sensitivity and specificity for routine clinical use in veterinary oncology.

  2. The (--)(/sup 3/H)dihydroalprenolol binding to rat adipocyte membranes: an explanation of curvilinear Scatchard plots and implications for quantitation of beta-adrenergic sites

    SciTech Connect

    Dax, E.M.; Partilla, J.S.; Gregerman, R.I.

    1982-09-01

    In rat adipocyte membranes, both beta-adrenergic agonists and beta-adrenergic antagonists competed with (--)(/sup 3/H)dihydroalprenolol for high affinity (KD 2-4 nM) and low capacity binding sites. The antagonists but not the agonists competed with (--)(/sup 3/H)dihydroalprenolol for lower affinity and higher capacity sites. The present studies were performed in order to characterize the adipocyte beta-adrenergic receptor and distinguish it from low affinity, higher capacity sites which were heat-labile and not stereoselective. When isoproterenol was used to define the nonspecific binding, saturation studies showed a single binding site with a capacity of approximately 100 fmol/mg membrane protein (corresponding to approximately 50,000 sites/adipocyte). Binding was saturated by 10 nM (--)(/sup 3/H)dihydroalprenolol. Approximate KD's of 204 nM were observed. Kinetic analysis of (--)(/sup 3/H)dihydroalprenolol binding provided an independent measurement of KD between 0.75 and 1.1 nM. This binding site had the characteristics of a beta 1-adrenergic receptor with the potency of isoproterenol greater than norepinephrine greater than or equal to epinephrine as competitors of binding. Furthermore, the KD of inhibition of (--)(/sup 3/H)dihydroalprenolol binding correlated with the Ki of inhibition by antagonists or Ka of activation by agonists of glycerol release in isolated adipocytes (r . 0.968, P less than 0.001). These results suggest that beta-adrenergic agonists compete with (--)(/sup 3/H)dihydroalprenolol for the high affinity binding site which represents the physiological site. Furthermore, the use of antagonists (propranolol, alprenolol) to define specific beta-binding includes nonspecific site(s) as well as the beta-adrenergic site. Previous characterization and quantitation of beta receptors in rat fat cell membranes may have been in error by incorporating both types of binding in their measurement.

  3. Thymidine decomposition induced by low-energy electrons and soft X rays under N2 and O2 atmospheres.

    PubMed

    Alizadeh, Elahe; Sanz, Ana G; Madugundu, Guru S; García, Gustavo; Wagner, J Richard; Sanche, Léon

    2014-06-01

    A novel technique has been employed to investigate the simultaneous damage to DNA components induced by soft X rays (1.5 keV) and low-energy electrons (0-30 eV) in thin films of thymidine deposited on glass and tantalum substrates and irradiated under atmospheric pressure and temperature. The films were surrounded by either an N2 or O2 environment. The formation of four radiation-induced products is reported in this article: base release, 5-hydroxymethyl-2'-deoxyuridine (5-HMdUrd), 5-formyl-2'-deoxyuridine (5-FordUrd) and 5,6-dihydrothymidine (5,6-DHThd). Analysis with LC-MS/MS shows larger damage yields in the samples deposited on tantalum than in those deposited on glass, which is attributed to the interaction of the additional low-energy electrons that are photoemitted from the metal surface. From a comparison of the results obtained from N2 and O2 environment, we report a dramatic effect from 6 O2: an approximately threefold increase in the yield of products, attributed to the reaction of O2 with initial carbon-centered thymidine radicals generated in the film during irradiation.

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

    PubMed

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

    2015-07-15

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

  5. Single-molecule study of thymidine glycol and i-motif through the alpha-hemolysin ion channel

    NASA Astrophysics Data System (ADS)

    He, Lidong

    Nanopore-based devices have emerged as a single-molecule detection and analysis tool for a wide range of applications. Through electrophoretically driving DNA molecules across a nanosized pore, a lot of information can be received, including unfolding kinetics and DNA-protein interactions. This single-molecule method has the potential to sequence kilobase length DNA polymers without amplification or labeling, approaching "the third generation" genome sequencing for around $1000 within 24 hours. alpha-Hemolysin biological nanopores have the advantages of excellent stability, low-noise level, and precise site-directed mutagenesis for engineering this protein nanopore. The first work presented in this thesis established the current signal of the thymidine glycol lesion in DNA oligomers through an immobilization experiment. The thymidine glycol enantiomers were differentiated from each other by different current blockage levels. Also, the effect of bulky hydrophobic adducts to the current blockage was investigated. Secondly, the alpha-hemolysin nanopore was used to study the human telomere i-motif and RET oncogene i-motif at a single-molecule level. In Chapter 3, it was demonstrated that the alpha-hemolysin nanopore can differentiate an i-motif form and single-strand DNA form at different pH values based on the same sequence. In addition, it shows potential to differentiate the folding topologies generated from the same DNA sequence.

  6. Adoptive immunotherapy for leukemia: donor lymphocytes transduced with the herpes simplex thymidine kinase gene for remission induction. HGTRI 0103.

    PubMed

    Link, C J; Burt, R K; Traynor, A E; Drobyski, W R; Seregina, T; Levy, J P; Gordon, L; Rosen, S T; Burns, W H; Camitta, B; Casper, J; Horowitz, M; Juckett, M; Lawton, C; Margolis, D; Pietryga, D; Rowlings, P; Taylor, C; Furtado, M; Stefka, J; Gupta-Burt, S; Kaiser, H; Vesole, D H

    1998-01-01

    This study will evaluate the safety and efficacy of allogenic donor lymphocyte infusions in patients who have relapsed hematologic malignancies after allogeneic bone marrow transplantation (BMT). Donor lymphocyte transfusions have resulted in the cure of some patients with relapsed leukemia or lymphoproliferative disorder after allogeneic BMT, but has been complicated by the development of graft versus host disease (GvHD). We hypothesize that a retroviral vector containing the Herpes simplex thymidine kinase (HStk) gene will allow for retention of the anti-leukemia response of transfused donor lymphocytes while allowing for the adverse effects of GVHD to be mitigated. Patients with relapsed hematologic malignancies after allogeneic BMT will be infused with ex vivo gene modified donor lymphocytes. The Herpes Simplex thymidine kinase (HStk) gene will be transduced into the cells ex vivo using LTKOSN. 1 vector supernate. Insertion of the HStk gene into lymphocytes confers a sensitivity to the anti-herpes drug ganciclovir (GCV). This selective destruction of donor lymphocytes in situ will be used to abrogate the effect of graft versus host disease, if it develops.

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

    PubMed Central

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

    2015-01-01

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

  8. 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.

  9. Precise site-selective termination of DNA replication by caging the 3-position of thymidine and its application to polymerase chain reaction.

    PubMed

    Kuzuya, Akinori; Okada, Fuminori; Komiyama, Makoto

    2009-10-21

    A new caged thymidine, 3-N-(2-(2-nitrophenyl)propyloxymethyl)thymidine (T(NPPOM)) was synthesized and used as a site-selective terminator of DNA-polymerase reaction in light-assisted cohesive-ending PCR (LACE-PCR), which directly gives sticky-ended PCR products after brief UVA irradiation. Primer-extension experiments using a template involving T(NPPOM) have shown that this caged nucleotide efficiently and site-selectively blocks reactions of a variety of polymerases commonly used in PCR. Misincorporation of nucleobases, observed with the use of other previously reported caged thymidines, scarcely occurred. It has turned out that a slight structural difference of caging groups can significantly improve the termination yield of polymerase reactions. A LACE-PCR product coding GFP gene was prepared by using primers containing T(NPPOM) and was ligated with a vector fragment prepared using restriction enzymes. The resulting recombinant vector successfully transformed E. coli.

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

    NASA Astrophysics Data System (ADS)

    Panicali, Dennis; Paoletti, Enzo

    1982-08-01

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

  11. Increased Clearance and Degradation of [3H]Insulin in Streptozotocin Diabetic Rats

    PubMed Central

    Philippe, Jacques; Halban, Philippe A.; Gjinovci, Asllan; Duckworth, William C.; Estreicher, Jurek; Renold, Albert E.

    1981-01-01

    The role of the insulin-receptor compartment in the pharmacokinetics of intravenously injected insulin in rats was studied. Since streptozotocin-diabetes in rats results in increased insulin binding to tissues in vitro, insulin pharmacokinetics in streptozotocin-diabetic rats were compared to controls, using semisynthetic [3H]insulin as the tracer. The initial distribution volume for [3H]insulin was elevated by 60% in diabetic rats. By contrast, no difference in initial distribution volume for [14C]inulin was observed, and the absolute values were lower than those found for [3H]insulin. The metabolic clearance rate of [3H]insulin was elevated by 44% in diabetic rats. That these differences were the result of increased binding of insulin to a specific receptor compartment in diabetic rats was shown by three additional experiments. The first involved receptor saturation by injection of 10 U native insulin 2 min before the tracer injection, resulting in identical [3H]insulin disappearance rates in the two groups of rats. The second consisted of displacing [3H]insulin from receptors by injecting 10 U unlabeled insulin 6 min after the tracer injection. Displacement of intact [3H]insulin from receptors and subsequent reappearance in the circulation occurred in both control and diabetic animals; however, such displacement was 25% greater in the diabetic rats. Finally, treatment of diabetic rats with insulin for 8 d normalized [3H]insulin clearance even though the tracer was injected at a time when the animals were again hyperglycemic and hypoinsulinemic. This suggests that down-regulation of insulin receptors had occurred during insulin therapy. These results confirm that a specific compartment for insulin exists (the insulin-receptor compartment) and that this compartment plays an important role in insulin clearance. PMID:6451633

  12. 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.

  13. Low energy n-{sup 3}H scattering: A novel testground for nuclear interactions

    SciTech Connect

    Lazauskas, R.; Carbonell, J.; Fonseca, A.C.; Viviani, M.; Kievsky, A.; Rosati, S.

    2005-03-01

    Low energy n-{sup 3}H elastic cross sections near the resonance peak are calculated by solving the four-nucleon problem with realistic NN interactions. Three different methods - Alt, Grassberger, and Shandas; hyperspherical harmonics; and Faddeev-Yakubovsky - have been used and their respective results are compared. We conclude on a failure of the existing NN forces to reproduce the n-{sup 3}H total cross section.

  14. 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.

  15. 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.

  16. 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.

  17. Human retina contains polyamine sensitive [3H]-ifenprodil binding sites: implications for neuroprotection?

    PubMed Central

    Sharif, N; Xu, S

    1999-01-01

    AIMS—This study characterised the pharmacology of [3H]-ifenprodil binding to the polyamine binding sites (PBS) on the N-methyl-D-aspartate (NMDA) receptor channel complex on human retinas. These data were correlated with the known neuroprotective effects of ifenprodil and eliprodil.
METHODS—Specific binding of [3H]-ifenprodil (under sigma site blockade) was investigated using human retinal homogenates and radioligand binding techniques. Scatchard and competition analyses were utilised to define the pharmacology of the [3H]-ifenprodil binding sites.
RESULTS—Specific binding of [3H]-ifenprodil comprised 73% (SEM 3%) of total and reflected interaction with two affinity sites (Kds = 0.39 and 4.3 µM) of different densities (Bmax = 14.4 and 105 pmol/ mg protein) (n = 5). The rank order of affinity of compounds competing for [3H]-ifenprodil binding to the high affinity PBS was: ifenprodil > eliprodil > arcaine > spermine > diaminodecane > spermidine > putrescine >> MK-801 (n = 3-7). However, [3H]-ifenprodil binding was minimally inhibited by glutamate, NMDA, and kainate.
CONCLUSION—These studies have shown, for the first time, the presence of specific [3H]-ifenprodil binding sites in the human retina with pharmacological characteristics of PBS associated with the NMDA receptor ionophore complex. The neuroprotective effects of eliprodil and ifenprodil may, in part, be mediated via these [3H]-ifenprodil labelled sites.

 Keywords: retina; neuroprotection; eliprodil; NMDA receptors PMID:10396205

  18. Following macromolecular interactions and sugar metabolism using site specific /sup 3/H labelling and NMR spectroscopy

    SciTech Connect

    Williams, P.; Morimoto, Hiromi; Gehring, K.B.; Nikaido, Hiroshi; Carson, P.; Un, Sun; Klein, M.; Wemmer, D.E.

    1988-06-01

    In this paper we discuss the application of /sup 3/H NMR to biological problems. Two specific examples will be described; first, analysis of the binding of maltose to its transport protein from E. coli, called MBP; and second, following the glycolytic metabolism of glucose in erythrocytes. In both of these cases the unique properties of /sup 3/H for magnetic resonance make possible observations which are difficult with other methods. 4 refs., 2 figs.

  19. First Detection of c-C3H2 in a Circumstellar Disk

    NASA Astrophysics Data System (ADS)

    Qi, Chunhua; Öberg, Karin I.; Wilner, David J.; Rosenfeld, Katherine A.

    2013-03-01

    We report the first detection of c-C3H2 in a circumstellar disk. The c-C3H2 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-C3H2 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-C3H2 6-5 emission and find that c-C3H2 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 1012-1013 cm-2. The clear detection and intriguing ring structure suggest that c-C3H2 has the potential to become a useful probe of radiation penetration in disks.

  20. 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.

  1. Inhibition of brain [(3)H]cimetidine binding by improgan-like antinociceptive drugs.

    PubMed

    Stadel, Rebecca; Carpenter, Amanda B; Nalwalk, Julia W; de Esch, Iwan J P; Janssen, Elwin; Hough, Lindsay B

    2010-04-25

    [(3)H]cimetidine, a radiolabeled histamine H(2) receptor antagonist, binds with high affinity to an unknown hemoprotein in the brain which is not the histamine H(2) receptor. Improgan, a close chemical congener of cimetidine, is a highly effective pain-relieving drug following CNS administration, yet its mechanism of action remains unknown. To test the hypothesis that the [(3)H]cimetidine-binding site is the improgan antinociceptive target, improgan, cimetidine, and 8 other chemical congeners were studied as potential inhibitors of [(3)H]cimetidine binding in membrane fractions from the rat brain. All compounds produced a concentration-dependent inhibition of [(3)H]cimetidine binding over a 500-fold range of potencies (K(i) values were 14.5 to >8000nM). However, antinociceptive potencies in rats did not significantly correlate with [(3)H]cimetidine-binding affinities (r=0.018, p=0.97, n=10). These results suggest that the [(3)H]cimetidine-binding site is not the analgesic target for improgan-like drugs.

  2. 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.

  3. Calcium channel receptor sites for (+)-[3H]PN 200-110 in coronary artery.

    PubMed

    Yamada, S; Kimura, R; Harada, Y; Nakayama, K

    1990-01-01

    The receptor sites for 1,4-dihydropyridine (DHP) Ca++ channel antagonists in porcine coronary artery were identified and characterized by a binding assay using (+)-[3H]PN 200-110 as a radioligand. Specific (+)-[3H]PN 200-110 binding in porcine coronary artery was saturable, reversible and of high affinity (Kd = 0.24 nM) and it showed a pharmacological specificity as well as stereoselectivity which characterized the receptor sites for DHP Ca++ channel antagonists. DHP antagonists competed for the (+)-[3H]PN 200-110 binding in order: PN 200-110 greater than mepirodipine greater than nisoldipine greater than nicardipine greater than nitrendipine greater than nimodipine greater than nifedipine greater than (-)-PN 200-110. (+)-PN 200-110 was approximately 140 times as potent as the (-)-isomer. The potencies (PKi) of these eight DHP Ca++ channel antagonists in competing for (+)-[3H]PN 200-110 binding sites in porcine coronary artery correlated well with their pharmacological potencies. Specific (+)-[3H]PN 200-110 binding in the coronary artery was enhanced by d-cis-diltiazem and was inhibited incompletely by verapamil and D-600. In EDTA-pretreated coronary artery, the maximal number of binding sites for specific (+)-[3H]PN 200-110 binding was reduced (80%) markedly, and it was restored to the untreated level by the addition of Ca++ and Mg++.(ABSTRACT TRUNCATED AT 250 WORDS)

  4. 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

  5. Synthesis and spectroscopic properties of two classes of 5,6-dihydrothymidine derivatives. Action on the Ehrlich's ascites cells thymidine kinase.

    PubMed Central

    Teoule, R; Fouque, B; Cadet, J

    1975-01-01

    Trans (+) and (-) 6-alkoxy-5-bromo-5,6 dihydrothymidine and trans (+) and (-) 6-alkoyloxy-5-bromo-5,6-dihydrothymidine compounds have been prepared. The synthesis of these substances (alkoxy : methoxy, ethoxy, butyloxy and isoamyloxy and alkoyloxy : acetoxy and bensovloxy) is described. Diastereoisomers of all products have been isolated by thin layer chromatography and their spectroscopic properties (IR, UV, NMR, mass spectrometry) studied. These compounds have been shown to be competitive inhibitors of Ehrlich's ascites cells thymidine kinase with respect ot thymidine. PMID:1169761

  6. Cloning and characterization of the Streptomyces peucetius dnmZUV genes encoding three enzymes required for biosynthesis of the daunorubicin precursor thymidine diphospho-L-daunosamine.

    PubMed Central

    Otten, S L; Gallo, M A; Madduri, K; Liu, X; Hutchinson, C R

    1997-01-01

    Characterization of the dnmZ, dnmU, and dnmV genes from the daunorubicin-producer Streptomyces peucetius by DNA sequence analysis indicated that these genes encode a protein of unknown function plus a putative thymidine diphospho-4-keto-6-deoxyglucose-3(5)-epimerase and thymidine diphospho-4-ketodeoxyhexulose reductase, respectively. Inactivation of each of the three genes by gene disruption and replacement in the wild-type strain demonstrated that all of them are required for daunosamine biosynthesis. PMID:9209071

  7. Characterization of [3H]thiocolchicoside binding sites in rat spinal cord and cerebral cortex.

    PubMed

    Balduini, W; Cimino, M; Depoortere, H; Cattabeni, F

    1999-07-02

    Thiocolchicoside, a semi-synthetic derivative of the naturally occurring compound colchicoside with a relaxant effect on skeletal muscle, has been found to displace both [3H]gamma-aminobutyric acid ([3H]GABA) and [3H]strychnine binding, suggesting an interaction with both GABA and strychnine-sensitive glycine receptors. In order to gain further insight into the interaction of thiocolchicoside with these receptors, the binding of [3H]thiocolchicoside in rat spinal cord-brainstem and cortical synaptic membranes was characterized. [3H]Thiocolchicoside binding was saturable in both tissues examined. In spinal cord-brainstem membranes, we found a K(D) of 254 +/- 47 nM and a Bmax of 2.39 +/- 0.36 pmol/mg protein, whereas in cortical membranes, a K(D) of 176 nM and a Bmax of 4.20 pmol/mg protein was observed. A similar K(D) value was found in kinetic experiments performed in spinal cord-brainstem membranes. Heterologous displacement experiments showed that GABA and strychnine displaced the binding in a dose-dependent manner, whereas glycine was ineffective. [3H]Thiocolchicoside binding was also displaced by several GABA(A) receptor agonists and antagonists, but not by baclofen, flunitrazepam, guvacine, picrotoxin or by other drugs unrelated to GABA transmission. In spinal cord-brainstem, and to a lower extent, in cortical membranes, GABA and its analogs were not able to completely displace [3H]thiocolchicoside specific binding indicating that, besides GABA(A) receptors, thiocolchicoside can bind to another unidentified site. Unlabelled thiocolchicoside, however, completely displaced [3H]muscimol binding both in cortical and in spinal cord-brainstem synaptic membranes with an IC50 in the low microM range. Neurosteroids were found to modulate the binding in cortical but not in spinal cord-brainstem synaptic membranes. We conclude that [3H]thiocolchicoside binding shows a pharmacological profile indicating an interaction with the GABA(A) receptor. The different affinities

  8. Changes in [(3)H]-ouabain and [(3)H]-neurotensin binding to rat cerebral cortex membranes after administration of antipsychotic drugs haloperidol and clozapine.

    PubMed

    Rosin, Carina; López Ordieres, María Graciela; Rodríguez de Lores Arnaiz, Georgina

    2017-03-01

    Evidences indicate the relationship between neurotensinergic and dopaminergic systems. Neurotensin inhibits synaptosomal membrane Na(+), K(+)-ATPase activity, an effect blocked by SR 48692, antagonist for high affinity neurotensin receptor (NTS1) type. Assays of high affinity [(3)H]-ouabain binding (to analyze K(+) site of Na(+), K(+)-ATPase) show that in vitro addition of neurotensin decreases binding. Herein potential interaction between NTS1 receptor, dopaminergic D2 receptor and Na(+), K(+)-ATPase was studied. To test the involvement of dopaminergic D2 receptors in [(3)H]-ouabain binding inhibition by neurotensin, Wistar rats were administered i.p.with antipsychotic drugs haloperidol (2mg/kg) and clozapine (3, 10 and 30mg/kg). Animals were sacrificed 18h later, cerebral cortices harvested, membrane fractions prepared and high affinity [(3)H]-ouabain binding assayed in the absence or presence of neurotensin at a 10 micromolar concentration. No differences versus controls for basal binding or for binding inhibition by neurotensin were recorded, except after 10mg/kg clozapine. Rats were administered with neurotensin (3, 10y 30μg, i.c.v.) and 60min later, animals were sacrificed, cerebral cortices harvested and processed to obtain membrane fractions for high affinity [(3)H]-ouabain binding assays. Results showed a slight but statistically significant decrease in binding with the 30μg neurotensin dose. To analyze the interaction between dopaminergic D2 and NTS1 receptors, [(3)H]-neurotensin binding to cortical membranes from rats injected with haloperidol (2mg/kg, i.p.) or clozapine (10mg/kg) was assayed. Saturation curves and Scatchard transformation showed that the only statistically significant change occurred in Bmax after haloperidol administration. Hill number was close to the unit in all cases. Results indicated that typical and atypical antipsychotic drugs differentially modulate the interaction between neurotensin and Na(+), K(+)-ATPase. At the same time

  9. 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.

  10. Metabolism of D, L-/sup 3/H-norepinephrine in essential hypertension

    SciTech Connect

    Gitlow, S.; Dziedzic, S.; Dziedzic, L.; Roubein, I.

    1981-01-01

    Defective control of the cardiovascular system by the sympathetic nerves continues to be incriminated as the potential primary physiologic defect in essential hypertension (EH). The need to measure sympathetic tone has progressed from physiologic mensuration by assessment of reflex and pharmacological responses to the recent assay of norepinephrine (NE) and its congeners in both urine and plasma. The way in which the body handles D,L-B-/sup 3/H-NE represents yet another technique by which to evaluate sympathetic function. Previous studies of EH by this method demonstrated more rapid plasma disappearance of /sup 3/H-NE as well as elevated 24 hour tritium accumulation in the urine following D,L-B-/sup 3/H-NE injection. The present study of 7 normotensive subjects and 7 patients with EH was designed to depict more precisely these abnormalities in /sup 3/H-NE-metabolism. Following a one minute injection of 8 micrograms D,L-B-/sup 3/H-NE, (200 microCi) intravenously, the excretion of unlabeled endogenous metabolites and their labeled congeners was assayed. By these means one could estimate catecholamine synthesis, turnover of the labeled pools, and by comparison of relative specific activities of the metabolites, gain some insight into the distribution of the injected material. Alternative catabolic pathways were evaluated by measurement of the excretion of /sup 3/H/sub 2/O. Patients with EH excreted more label per 24 hours, revealed a more rapid decline of /sup 3/H2O excretion and lower specific activity of normetanephrine (NM). These findings are compatible with changes in pool dynamics and distribution of administered label which gave additional support to the concept of adrenergic dysfunction in association with essential hypertension.

  11. Autoradiographic localization of [3H]thiocolchicoside binding sites in the rat brain and spinal cord.

    PubMed

    Balduini, W; De Angelis, V; Mazzoni, E; Depoortere, H; Cattabeni, F; Cimino, M

    2001-06-01

    Thiocolchicoside is used in humans as a myorelaxant drug with anti-inflammatory and analgesic activity. Recently we established the experimental conditions that allowed the identification of [3H]thiocolchicoside binding sites in synaptic membranes of rat spinal cord and cerebral cortex. The pharmacological characterization of these sites indicated that GABA and several of its agonists and antagonists, as well as strychnine, were able to interact with [3H]thiocolchicoside binding in a dose-dependent manner and with different affinities. In order to gain more insight into the nature and the anatomical distribution of the binding sites labeled by [3H]thiocolchicoside, in the present study we examined the localization of these sites on parasagittal and coronal sections of the rat brain and spinal cord, respectively, using receptor autoradiography. In the spinal cord an intense signal was observed in the gray matter, with the highest density occurring in the superficial layers of the dorsal horns. Strychnine completely displaced [3H]thiocolchicoside binding, whereas GABA only partially removed the radioligand from its binding sites. In the brain, specific binding occurred in several areas and was displaced by both GABA and strychnine. The distribution of [3H]thiocolchicoside binding sites in brain sections, however, did not match that found for [3H]muscimol. Furthermore, cold thiocolchicoside was not able to completely displace [3H]muscimol binding, and showed a different efficacy in the various areas labeled by the radioligand. We conclude that thiocolchicoside may interact with a subpopulation of GABA(A) receptors having low-affinity binding sites for GABA. Furthermore, the observed sensitivity to strychnine in the spinal cord indicates an interaction also with strychnine-sensitive glycine receptors, suggesting that the pharmacological effects of thiocolchicoside may be the result of its interaction with different receptor populations.

  12. 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.

  13. 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.

  14. Binding of (/sup 3/H)forskolin to platelet membranes and solubilized proteins from bovine brain

    SciTech Connect

    Nelson, C.A.; Seamon, K.B.

    1986-05-01

    (/sup 3/H)Forskolin ((/sup 3/H)FSK) bound to platelet membranes with a Kd of 20 nM and a Bmax of 125 fmol/mg protein. The Bmax was increased to 400 fmol/mg protein in the presence of GppNHp (or NaF) and MgCl/sub 2/ with no change in Kd. PGE/sub 1/ decreased the EC50 of GppNHp to increase the Bmax for (/sup 3/H)FSK binding from 600 nM to 35 nM. In contrast, PGE/sub 1/ had no effect on the EC50 of NaF to increase (/sup 3/H)FSK binding. (/sup 3/H)FSK binding increased slowly over 60 min when forskolin and GppNHp were added to membranes simultaneously at 20/sup 0/C. Preincubation of membranes with GppNHp at 20/sup 5/C also caused a linear increase in adenylate cyclase specific activity over 60 minutes. (/sup 3/H)FSK bound to solubilized protein from bovine brain membrane with a Kd of 22 nM. GppNHp increased the number of binding sites in solubilized proteins only if membranes were not preincubated with GppNHp prior to solubilization. In conclusion the number of binding sites for (/sup 3/H)FSK is increased by agents that activate adenylate cyclase through the Ns protein. These sites appear to be associated with an activated complex of the Ns protein and adenylate cyclase.

  15. 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.

  16. Separate [3H]-nitrendipine binding sites in mitochondria and plasma membranes of bovine adrenal medulla.

    PubMed Central

    Ballesta, J. J.; Garcia, A. G.; Gutierrez, L. M.; Hidalgo, M. J.; Palmero, M.; Reig, J. A.; Viniegra, S.

    1990-01-01

    1. Two binding sites for the 1,4-dihydropyridine (DHP) derivative [3H]-nitrendipine have been found in the bovine adrenal medulla. The high-affinity site (Kd = 0.48 nM and Bmax = 128 fmol mg-1 protein) was specifically located in purified plasma membranes. The low-affinity site (Kd = 252 nM and Bmax = 169 pmol mg-1 protein) was located only in mitochondria. Chromaffin granule membranes lacked specific binding sites for [3H]-nitrendipine. 2. Kinetic analysis of the rates of association and dissociation of [3H]-nitrendipine, saturation isotherms and displacement experiments with unlabelled nitrendipine and PN200-110 revealed single, homogeneous populations of high- and low-affinity sites in plasma and mitochondrial membranes, respectively. 3. The high affinity site was sensitive to Ca2+ deprivation and heating; it was practically unaffected by changes in ionic strength of the medium and its optimal pH was slightly alkaline. This site exhibited a strong DHP stereoselectivity; diltiazem increased and verapamil decreased the affinity of [3H]-nitrendipine. 4. In contrast, binding of [3H]-nitrendipine to the low affinity site was more heat resistant and less affected by Ca2+ removal. Its optimal pH was slightly acid and the increase in ionic strength enhanced the number of available sites. The site had no DHP stereoselectivity. Verapamil decreased the dissociation constant of [3H]-nitrendipine acting in a non-competitive manner; diltiazem did not affect equilibrium binding parameters of [3H]-nitrendipine. 5. These results suggest that both biding sites reflect different receptor entities. The high-affinity binding site corresponds to the dihydropyridine receptor associated with the L-type calcium channel. The function of the mitochondrial, low-affinity binding site is, at present, unknown. PMID:1704272

  17. Metabolism studies of unformulated internally [3H]-labeled short interfering RNAs in mice.

    PubMed

    Christensen, Jesper; Litherland, Karine; Faller, Thomas; van de Kerkhof, Esther; Natt, François; Hunziker, Jürg; Krauser, Joel; Swart, Piet

    2013-06-01

    Absorption, distribution, metabolism, and excretion properties of two unformulated model short interfering RNA (siRNAs) were determined using a single internal [(3)H]-radiolabeling procedure, in which the full-length oligonucleotides were radiolabeled by Br/(3)H -exchange. Tissue distribution, excretion, and mass balance of radioactivity were investigated in male CD-1 mice after a single intravenous administration of the [(3)H]siRNAs, at a target dose level of 5 mg/kg. Quantitative whole-body autoradiography and liquid scintillation counting techniques were used to determine tissue distribution. Radiochromatogram profiles were determined in plasma, tissue extracts, and urine. Metabolites were separated by liquid chromatography and identified by radiodetection and high-resolution accurate mass spectrometry. In general, there was little difference in the distribution of total radiolabeled components after administration of the two unformulated [(3)H]siRNAs. The radioactivity was rapidly and widely distributed throughout the body and remained detectable in all tissues investigated at later time points (24 and 48 hours for [(3)H]MRP4 (multidrug resistance protein isoform 4) and [(3)H]SSB (Sjögren Syndrome antigen B) siRNA, respectively). After an initial rapid decrease, concentrations of total radiolabeled components in dried blood decreased at a much slower rate. A nearly complete mass balance was obtained for the [(3)H]SSB siRNA, and renal excretion was the main route of elimination (38%). The metabolism of the two model siRNAs was rapid and extensive. Five minutes after administration, no parent compound could be detected in plasma. Instead, radiolabeled nucleosides resulting from nuclease hydrolysis were observed. In the metabolism profiles obtained from various tissues, only radiolabeled nucleosides were found, suggesting that siRNAs are rapidly metabolized and that the distribution pattern of total radiolabeled components can be ascribed to small molecular

  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. 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.

  20. Effect of peroxides on [3H]D-aspartate release from bovine isolated retinae.

    PubMed

    LeDay, Angela M; Awe, Sunday O; Kulkarni, Kaustubh; Harris, Lydia C; Opere, Catherine; Dash, Alekha; Ohia, Sunny E

    2004-04-01

    In the present study, we investigated the effect of naturally occurring and synthetic peroxides on K+-depolarization-evoked release of [3H]D-aspartate from bovine isolated retinae. Furthermore, effect of peroxides on endogenous glutamate concentrations were measured by HPLC in bovine neural retinae and vitreous humor of eyes treated with hydrogen peroxide (H2O2) ex vivo. Both naturally occurring H2O2 (1-100 microM) and synthetic (cumene hydroperoxide, cuOOH; 1-100 microM) peroxides caused a concentration-dependent inhibition of K+-evoked [3H]D-aspartate release without affecting basal tritium efflux. The antioxidant, trolox (2 mM) prevented the inhibition of evoked [3H]D-aspartate overflow elicited by both H2O2 (30 microM) and cuOOH (10 microM). Inhibition of catalase by 3-amino-triazole (3- AT 100 mM) enhanced an inhibitory effect of a low concentration of H2O2 (1 microM) but antagonized the effect of H2O2 (30 microM) on K+-induced [3H]D-aspartate release. In ex vivo experiments, exogenously applied H2O2 (1-100 microM) also caused a concentration-related decrease in glutamate levels in the bovine retina. We conclude that peroxides can inhibit K+-evoked release of [3H]D-aspartate and also decrease endogenous glutamate concentrations in the bovine retina.

  1. [[sup 3]H]QNB displays in vivo selectivity for the m2 subtype

    SciTech Connect

    Gitler, M.S.; De La Cruz, R.; Zeeberg, B.R. ); Reba, R.C. Univ. of Chicago Hospital, Chicago, IL )

    1994-01-01

    Alzheimer's disease (AD) involves selective loss of muscarinic m2, but not m1, subtype neuroreceptors in the posterior parietal cortex of the human brain. Emission tomographic study of the loss of m2 receptors in AD is limited by the fact that there is currently no available m2-selective radioligand which can penetrate the blood-brain barrier. [[sup 3]H](R)-3-quinuclidinylbenzilate ([[sup 3]H]QNB) is commonly used for performing in vitro studies of the muscarinic acetylcholine receptor (mAChR), either with membrane homogenates or with autoradiographic slices, in which [[sup 3]H]QNB is nonsubtype-selective. We report here the results of in vivo studies, using both carrier-free and low specific activity [[sup 3]H]QNB, which show that [[sup 3]H]QNB exhibits a substantial in vivo m2-selectivity. Previously reported in vivo (R)-3-quinuclidinyl (R)-4-iodobenzilate ((R,R)-[[sup 125]I]lQNB) binding appears to be nonsubtype-selective. Apparently the bulky iodine substitution in the 4 position reduces the subtype selectivity of QNB. It is possible that a less bulky fluorine substitution might permit retention of the selectivity exhibited by QNB itself. We conclude that a suitably radiolabeled derivative of QNB, possibly labeled with [sup 18]F, may be of potential use in positron emission tomographic (PET) study of the loss of m2 receptors in AD. 39 refs., 8 figs., 2 tab.

  2. Immunogenicity and protective efficacy of DMT liposome-adjuvanted tuberculosis subunit CTT3H vaccine.

    PubMed

    Teng, Xindong; Tian, Maopeng; Li, Jianrong; Tan, Songwei; Yuan, Xuefeng; Yu, Qi; Jing, Yukai; Zhang, Zhiping; Yue, Tingting; Zhou, Lei; Fan, Xionglin

    2015-01-01

    Different strategies have been proposed for the development of protein subunit vaccine candidates for tuberculosis (TB), which shows better safety than other types of candidates and the currently used Bacillus Calmette-Guérin (BCG) vaccine. In order to develop more effective protein subunits depending on the mechanism of cell-mediated immunity against TB, a polyprotein CTT3H, based on 5 immunodominant antigens (CFP10, TB10.4, TB8.4, Rv3615c, and HBHA) with CD8(+) epitopes of Mycobacterium tuberculosis, was constructed in this study. We vaccinated C57BL/6 mice with a TB subunit CTT3H protein in an adjuvant of dimethyldioctadecylammonium/monophosphoryl lipid A/trehalose 6,6'-dibehenate (DDA/MPL/TDB, DMT) liposome to investigate the immunogenicity and protective efficacy of this novel vaccine. Our results demonstrated that DMT liposome-adjuvanted CTT3H vaccine not only induced an antigen-specific CD4(+) Th1 response, but also raised the number of PPD- and CTT3H-specific IFN-γ(+) CD8(+) T cells and elicited strong CTL responses against TB10.4, which provided more effective protection against a 60 CFU M. tuberculosis aerosol challenge than PBS control and DMT adjuvant alone. Our findings indicate that DMT-liposome is an effective adjuvant to stimulate CD8(+) T cell responses and the DMT-adjuvanted subunit CTT3H vaccine is a promising candidate for the next generation of TB vaccine.

  3. In vivo benzodiazepine receptor binding and imaging using (/sup 3/H)-flunitrazepam

    SciTech Connect

    Ciliax, B.J.

    1987-01-01

    The use of (/sup 3/H)-flunitrazepam as a ligand to image and measure alterations in benzodiazepine receptors in vivo in rat was investigated. Animals were injected with (/sup 3/H)-flunitrazepam intravenously, arterial samples of (/sup 3/H)-flunitrazepam were obtained and later the animals were sacrificed to assay brain binding. (/sup 3/H)-Flunitrazepam entered brain rapidly and bound to benzodiazepine receptors. A series of rats were lesioned unilaterally with kainic acid in the caudate-putamen several months prior to the infusion of (/sup 3/H)-flunitrazepam. In vivo autoradiography in lesioned rats showed that benzodiazepine binding in the lesioned striatum was significantly decreased compared to the control side and that benzodiazepine binding in globus pallidus and substantia nigra on the side of the lesion was significantly increased as compared to the intact side. The observed changes in benzodiazepine binding were similar to those observed previously in lesioned rats using in vitro techniques. Thus, benzodiazepine receptor regulation could be imaged quantitatively using in vivo binding techniques.

  4. 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.

  5. In vivo (/sup 3/H)flunitrazepam binding: imaging of receptor regulation

    SciTech Connect

    Ciliax, B.J.; Penney, J.B. Jr.; Young, A.B.

    1986-08-01

    The use of (/sup 3/H)flunitrazepam as a ligand to measure alterations in benzodiazepine receptors in vivo in rats was investigated. Animals were injected with (/sup 3/H)flunitrazepam i.v., arterial samples of (/sup 3/H)flunitrazepam were obtained and, later, the animals were sacrificed to assay brain binding. (/sup 3/H)flunitrazepam enters the brain rapidly and binds to benzodiazepine receptors. About two-thirds of this binding is blocked by predosing the animals with 5 mg/kg of clonazepam. The amount of remaining (nonspecific) binding correlates very well (r = 0.88) with the amount of radioactivity found in plasma at the time of death. A series of rats were lesioned unilaterally with kainic acid in the caudate-putamen several months before the infusion of (/sup 3/H)flunitrazepam. In vivo autoradiography in lesioned rats showed that benzodiazepine binding in globus pallidus and substantia nigra on the side of the lesion was increased significantly as compared to the intact side. The observed changes in benzodiazepine binding were similar to those observed previously in lesioned rats using in vitro techniques. Thus, benzodiazepine receptor regulation can be imaged quantitatively using in vivo binding techniques.

  6. Pharmacokinetics and autoradiography of [3H] or [14C]stepholidine.

    PubMed

    Zhang, Z D; Zhou, C M; Jin, G Z; Zhang, X; Yang, L

    1990-07-01

    After iv [3H]stepholidine (SPD) 12 MBq/kg, the concentration-time curve in rats was found to be a two-compartment open model. The distribution phase T1/2 alpha = 3.6 min, the elimination phase T1/2 beta = 168 min. The absorbed radioactivities of [3H] SPD in 15-30 min were 80-87%. The amounts of [3H]SPD bound to plasma protein, liver and kidney homogenates were estimated to be 37, 31, and 30%, respectively. During 3 d after ip [3H]SPD 50 MBq/kg, 56% of the radioactivity was excreted in urine and 5% in feces, thus, it suggested that [3H]SPD was mainly excreted by kidneys. After iv a single dose of [14C]SPD in mice, the whole-body autoradiography showed that [14C]SPD was rapidly distributed among various tissues. High radioactivities were found in kidneys, liver, brain, salivary glands, Harder's glands, heart blood and muscle at 2 min and intensively localized in kidneys and stomach mucosa at 30 min. The radioactivities in these tissues disappeared 4 and 8 h later, while that in intestine could not be detected 24 h later.

  7. 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.

  8. 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.

  9. /sup 3/H-imipramine binding in aged mouse brain: regulation by ions and serotonin

    SciTech Connect

    Severson, J.A.

    1986-03-01

    The density of binding sites (Bmax) for /sup 3/H-imipramine was elevated in cerebral cortical, hypothalamic and hippocampal membranes from 24 month old male C57BL/6J mice. Cerebellar binding was constant with increasing age. There were no changes in the equilibrium dissociation constant (Kd) for /sup 3/H-imipramine in any brain region. The increase in the binding of /sup 3/H-imipramine induced by sodium and chloride ions in vitro was diminished in cerebral cortical homogenates from aged mice; both the sodium-sensitive and chloride-sensitive components of binding were about 50% less in aged mice. Dose-response curves indicated that the effectiveness with which chloride enhanced binding was similar with age, even though the absolute increase in binding was less. The rate of dissociation of /sup 3/H-imipramine from cerebral cortical homogenates was similar with age and serotonin slowed the rate of dissociation equally at all ages. Possible mechanisms for the age-related increase in brain /sup 3/H-imipramine binding are discussed. Ion-sensitive binding is discussed in relationship to the current controversy surrounding desipramine-sensitive versus ion-sensitive binding.

  10. 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.

  11. Preservation of peripheral benzodiazepine receptors: differential effects of freezing on (/sup 3/H)Ro 5-4864 and (3H)PK 11195 binding

    SciTech Connect

    Basile, A.S.; Ostrowski, N.L.; Skolnick, P.

    1987-04-01

    A statistically significant decrease in the density of peripheral benzodiazepine receptors was observed in renal membranes of rats beginning 2 weeks after adrenalectomy when compared with sham-operated controls. This decrease in peripheral benzodiazepine receptor density was manifest as a decrease in the Bmax of two ligands (/sup 3/H)Ro 5-4864 and (/sup 3/H)PK 11195, without accompanying changes in their apparent affinity (Kd) for this site. Similar changes were not seen in another aldosterone-sensitive organ, the submandibular salivary gland. The decrease in peripheral benzodiazepine receptor density in observed in adrenalectomized rat renal membranes was restored to control levels after 1 week of aldosterone administration using a dose (12.5 micrograms/kg/day) that had no effect on peripheral benzodiazepine receptor density in sham-operated animals. In contrast, dexamethasone administration (50 micrograms/kg/day, 1 week) had no effect on renal peripheral benzodiazepine receptor density when administered to either adrenalectomized or sham-operated rats. Further, adrenal demedullation had no effect on renal peripheral benzodiazepine receptor density or affinity. The decrease in peripheral benzodiazepine receptor density was localized to the renal cortex and the outer stripe of the medulla by gross dissection of renal slices and renal tissue section autoradiography. The specific effect of adrenalectomy on renal peripheral benzodiazepine receptor density, the lack of direct effect of aldosterone on (/sup 3/H)Ro 5-4864 binding, and the localization of the change in peripheral benzodiazepine receptor density to the renal cortex and outer stripe suggests that these changes may reflect an adaptation of the renal nephron (possibly the distal convoluted tubule, intermediate tubule and/or the collecting duct) to the loss of mineralocorticoid hormones.

  12. Effect of malathion on nucleic acid synthesis in phytohemagglutinin-stimulated human lymphocytes.

    PubMed

    Czajkowska, A; Walter, Z

    1980-01-01

    The effect of malathion, an organophosphorus insecticide, on DNA and RNA synthesis was investigated by measuring the rate of incorporation of 3H thymidine and 3H uridine, respectively, into human lymphocytes stimulated by phytohemagglutinin (PHA). Increasing concentrations of malathion, from 10 to 70 micrograms/ml, were added to human lymphocyte cultures at different times in relation to PHA introduction. The lowest applied dose of malathion (10 micrograms/ml) in most cases led to increased incorporation of both 3H thymidine and 3H uridine. Higher concentrations of malathion (30, 50, 70 micrograms/ml) caused a time- and dose-dependent decrease of radioisotope incorporation.

  13. Aminosilanes derived from 1H-benzimidazole-2(3H)-thione.

    PubMed

    Palomo-Molina, Juliana; García-Báez, Efrén V; Contreras, Rosalinda; Pineda-Urbina, Kayim; Ramos-Organillo, Angel

    2015-09-01

    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 R2(2)(8) rings via N-H···S interactions, along with parallel π-π interactions between imidazole and benzene rings.

  14. 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.

  15. 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

  16. Interaction of thiocolchicoside with [3H]strychnine binding sites in rat spinal cord and brainstem.

    PubMed

    Cimino, M; Marini, P; Cattabeni, F

    1996-12-27

    Radioreceptor binding assays and receptor autoradiography were used to investigate the activity of thiocolchicoside on strychnine-sensitive binding sites in rat brain and spinal cord using [3H]strychnine as a ligand. Thiocolchicoside displaced the binding of [3H]strychnine with an affinity similar to that of unlabeled glycine, and showed a Hill coefficient and proportionality parameter (P) less than unity. The activity of thiocolchicoside toward [3H]strychnine binding sites was confirmed in autoradiographic studies. The results suggest that thiocolchicoside behaves as an allosteric compound acting on the strychnine-sensitive glycine receptor in rat brainstem and spinal cord, and that this may provide a possible mechanism for the myorelaxant activity of this colchicoside derivative, the first clinically useful drug acting on this receptor.

  17. (/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.

  18. 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.

  19. Selective retrograde labeling of cholinergic neurons with (/sup 3/H)choline

    SciTech Connect

    Bagnoli, P.; Beaudet, A.; Stella, M.; Cuenod, M.

    1981-07-01

    Evidence is presented which is consistent with a specific retrograde labeling of cholinergic neurons following (/sup 3/H)choline application in their zone of termination. (/sup 3/H)Choline injection in the rat hippocampus leads to perikaryal retrograde labeling in the ipsilateral medial septal nuclease and nucleus of the diagonal band, thus delineating an established cholinergic pathway, while only diffuse presumably anterograde labeling was observed in the lateral septum, the entorhinal cortex, and the opposite hippocampus. After (/sup 3/H)choline injection in the pigeon visual Wulst, only the ipsilateral thalamic relay, of all inputs, showed similar perikaryal retrograde labeling, an observation supporting the suggestion that at least some thalamo-Wulst neurons are cholinergic.

  20. Metabolism of [3H]pentosan polysulfate sodium (PPS) in healthy human volunteers.

    PubMed

    Simon, M; McClanahan, R H; Shah, J F; Repko, T; Modi, N B

    2005-08-01

    Pentosan polysulfate sodium (PPS) is the active ingredient in ELMIRON, a drug approved for the relief of bladder pain associated with interstitial cystitis. The study objective was to characterize the pharmacokinetic and metabolic profiles of PPS following oral dosing of [3H]PPS. As specific assays for PPS do not exist, metabolic profiling was accomplished through multiple fraction collections and radiochromatographic techniques. Two groups of eight healthy female subjects sequentially received a single oral dose of 200 microCi [3H]PPS supplemented with 300 mg unlabelled PPS or 300 microCi [3H]PPS supplemented with 450 mg unlabelled PPS. Most of the administered dose (84%) was excreted in faeces as intact PPS, and a smaller percentage (6%) was excreted in urine. In summary, orally administered PPS was very poorly absorbed, with the majority of the drug being excreted in faeces as intact PPS and in urine as low molecular weight and desulfated PPS.

  1. 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.

  2. Dynamic uptake of radioactive substance in rat salivary gland following /sup 3/H-melatonin administration

    SciTech Connect

    Withyachumnarnkul, B.; Wongprapairot, P.; Trakulrungsi, W.

    1987-01-01

    Dynamics of radioactive accumulation in rat greater salivary gland following systemic administration of /sup 3/H-melatonin was studied to determine a possible action of the hormone in the gland. Progressive decline of /sup 3/H-melatonin concentrations was found in the serum, lung, skeletal muscle, liver, kidney, and salivary gland during 60 min following the administration. On the contrary, there was a progressive accumulation of radioactive substance other than /sup 3/H-melatonin in the salivary gland but not in other tissues mentioned. The radioactivity was also progressively and preferentially localized in the nuclear fraction of the gland cells. These results suggest a possible direct action of melatonin derivative in rat salivary gland.

  3. 3H-GABA uptake selectively labels identifiable neurons in the leech central nervous system

    SciTech Connect

    Cline, H.T.

    1983-04-10

    Segmental ganglia of the leech ventral nerve cord synthesize the neurotransmitter gamma-aminobutyric acid (GABA) when incubated in the presence of the precursor glutamate, suggesting that there may be GABA-ergic neurons in the leech nerve cord. GABA-accumulating neurons of the two taxonomically distant leech species, Haementeria ghilianii and Hirudo medicinalis, have been labeled by taking advantage of their high-affinity uptake system for the neurotransmitter. Autoradiography of sectioned segmental ganglia previously exposed to 3H-GABA reveals a reproducible pattern of about thirty 3H-GABA-labeled neuronal cell bodies per ganglion. The majority of 3H-GABA-labeled neuronal cell bodies are bilaterally paired, although some apparently unpaired cell bodies also accumulate label. Neuronal processes were reproducibly labeled by GABA uptake and could be traced in the neuropil through commissures and fiber tracts into the segmental nerve roots and interganglionic connectives, respectively.

  4. Excited-state lifetime of propadienylidene, l-C3H2.

    PubMed

    Noller, Bastian; Margraf, Markus; Schröter, Christian; Schultz, Thomas; Fischer, Ingo

    2009-07-14

    The excited-state dynamics of the singlet carbene propadienylidene, l-C(3)H(2), were investigated by femtosecond time-resolved photoionisation. The carbene was excited into the C (1)A(1) state with 250 nm pulses and the subsequent excited state dynamics were probed by multiphoton ionization with 800 nm pulses. The lifetime of the C (1)A(1) state was determined to be 70 fs. In agreement with recent nanosecond experiments, we assume that the carbene deactivates to the electronic ground state where it subsequently dissociates. Since propadienylidene was generated from 3-bromo-1-iodopropyne, two further radical intermediates were studied, IC(3)H(2) and C(3)H(2)Br. For both species, an ultrafast excited state decay was observed with an upper limit of 40 fs for the respective lifetimes.

  5. Low-lying states of Li 3H: Is there an ion-pair minimum?

    NASA Astrophysics Data System (ADS)

    Talbi, Dahbia; Saxon, Roberta P.

    1989-05-01

    The 1 1A', 1 1A″, 1 3A', and 1 3A″ states of Li 3H have been investigated at the MCSCF/SOCI level. The global minimum is a planar conformation of 1A' symmetry. A local minimum on the same potential surface at a C 3v pyramidal geometry, of mixed ion-pair and covalent character, is found at a relative energy of 20.30 kcal/mol. The barrier height for isomerization is predicted to be 1.3 kcal/mol. The correlation diagram linking states of Li 3H to those of Li 3 + H, LiH + Li 2 and Li 2H + Li is presented.

  6. Selective removal of undifferentiated embryonic stem cells from differentiation cultures through HSV1 thymidine kinase and ganciclovir treatment.

    PubMed

    Naujok, Ortwin; Kaldrack, Joanna; Taivankhuu, Terbish; Jörns, Anne; Lenzen, Sigurd

    2010-09-01

    Pluripotent cell lines such as embryonic stem cells are an attractive source for a potential cell replacement therapy. However, transplantation of differentiated cells harbors the risk of teratoma formation, presenting a serious health risk. To overcome this obstacle, a negative selection system was established that permits selective removal of undifferentiated cells during in vitro differentiation. Use of the HSV1 thymidine kinase and eGFP under the control of the Oct4 promoter allowed the destruction of undifferentiated ES cells by ganciclovir treatment; differentiated cells were unharmed. Clonal ES cells remained pluripotent and showed positive staining for a wide range of embryonic markers. Thus, treatment with ganciclovir during in vitro differentiation effectively removed the population of undifferentiated cells and provided a pure population of completely differentiated cells. This approach may pave the way for a safe application of ES cells in regenerative medicine in the future.

  7. [Cytotoxicity of cytosine deaminase and herpes simplex virus thymidine kinase genes in melanoma cells is independent on promoter strength].

    PubMed

    Alekseenko, I V; Kuz'min, D V; Pleshkan, V V; Zinov'eva, M V; Sverdlov, E D

    2013-01-01

    In preparation of the therapeutic genetic constructs aimed to the gene-programmed enzymatic transformation of the non-toxic prodrug into toxin within cancer cells the right choice of regulatory elements (promoters and enhancers) is essential. This is widely accepted that the efficiency of the gene therapy constructions is dependent, in particular, on the strength of promoters driving the expression of the therapeutic genes. In this work we demonstrated, using the melanoma-specific promoters and enhancers of human melanoma inhibitory activity and mouse tyrosinase gene, that for the development of cytotoxic effect the promoter strength is not of primary importance. In the case of HSVtk, coding for the herpes simplex virus thymidine kinase, and FCU1, coding for cytosine deaminase/uracil phosphoribosyltransferase hybrid protein genes, their cytotoxic activity was determined by the quantity of the added prodrug.

  8. Far-UV photochemistry and photosensitization of 2'-deoxycytidylyl-(3'-5')-thymidine: isolation and characterization of the main photoproducts.

    PubMed

    Douki, T; Cadet, J

    1992-08-31

    Far-UV irradiation of 2'-deoxycytidylyl-(3'-5')-thymidine (dCpT) gave rise to the pyrimidine (6-4) pyrimidone adduct and its Dewar valence isomer as the main photoproducts. The absolute configuration of the former adduct was determined and its photoisomerization studied. A comparison of the alkali lability of both compounds showed that hydrolysis of the phosphodiester bond occurs for the Dewar valence isomer but not for its (6-4) precursor. In addition, the trans-syn and cis-syn cyclobutane dimers of dCpT were obtained by acetophenone photosensitization and characterized. Finally, the deamination rate constants for this series of compounds were shown to be dramatically influenced by the nature and the configuration of the photoproducts.

  9. The alkylation of 2′-deoxyguanosine and of thymidine with diazoalkanes. Some observations on O-alkylation

    PubMed Central

    Farmer, P. B.; Foster, A. B.; Jarman, M.; Tisdale, M. J.

    1973-01-01

    The reaction in ether–methanol between 2′-deoxyguanosine and diazomethane or its ethyl or n-butyl homologue gives 1-, O6- and 7-alkyl-2′-deoxyguanosine. N2,O6-Dimethyl-2′-deoxyguanosine was also detected. The hydrolysis of the methyl and the ethyl derivatives gives the corresponding alkylguanines: the O6-alkyl-2′-deoxyguanosines were sequentially hydrolysed, first to 2-amino-6-alkoxypurines, subsequently to guanine. The mass spectra of O6-alkyl-2′-deoxyguanosines (methyl and ethyl) and of the corresponding 2-amino-6-alkoxypurines were determined. The reaction of diazomethane with thymidine afforded O4-methylthymidine, in addition to the previously detected 3-methylthymidine. PMID:4776868

  10. Prediction of the binding mode of N2-phenylguanine derivative inhibitors to herpes simplex virus type 1 thymidine kinase

    NASA Astrophysics Data System (ADS)

    Gaudio, Anderson Coser; Takahata, Yuji; Richards, William Graham

    1998-01-01

    The probable binding mode of the herpes simplex virus thymidine kinase (HSV1 TK) N2-[substituted]-phenylguanine inhibitors is proposed. A computational experiment was designed to check some qualitative binding parameters and to calculate the interaction binding energies of alternative binding modes of N2-phenylguanines. The known binding modes of the HSV1 TK natural substrate deoxythymidine and one of its competitive inhibitors ganciclovir were used as templates. Both the qualitative and quantitative parts of the computational experiment indicated that the N2-phenylguanine derivatives bind to the HSV1 TK active site in the deoxythymidine-like binding mode. An experimental observation that N2-phenylguanosine derivatives are not phosphorylated during the interaction with the HSV1 TK gives support to the proposed binding mode.

  11. Improved synthesis of the two-photon caging group 3-nitro-2-ethyldibenzofuran and its application to a caged thymidine phosphoramidite

    PubMed Central

    Lusic, Hrvoje; Uprety, Rajendra; Deiters, Alexander

    2010-01-01

    A new and efficient route to the recently reported 3-nitro-2-ethyldibenzofuran caging group was developed. Furthermore, its installation on a thymidine phosphoramidite is described. This caging group is efficiently removed through light-irradiation at 365 nm. PMID:20112966

  12. Role of ribose in the initial excited state structural dynamics of thymidine in water solution: a resonance Raman and density functional theory investigation.

    PubMed

    Zhu, Xin-Ming; Wang, Hui-gang; Zheng, Xuming; Phillips, David Lee

    2008-12-11

    Resonance Raman spectra were obtained for thymidine and thymine with excitation wavelengths in resonance with the approximately 260 nm band absorption spectrum. The spectra indicate that the Franck-Condon (FC) region photodissociation dynamics of thymidine have multidimensional character with motion predominantly along the nominal C5=C6 stretch + C6-H bend nu17 (delta = 0.75, lambda = 468 cm(-1)), the nominal thymine ring stretch + C6-H bend + N1-C1, stretch nu29 (delta = 0.73, lambda = 363 cm(-1)), the nominal thymine ring stretch + C5-CH3/ N1-C1, stretch nu37 (delta = 0.69, lambda = 292 cm(-1)), and accompanied by the moderate and minor changes in the nu40, nu20 and nu23, nu55, nu60, nu61, nu63 modes. A preliminary resonance Raman intensity analysis was done, and these results for thymidine and thymine were compared to each other. The roles of ribose in the FC structure dynamics of thymidine were explored and the results were used to correlate to its lifetime constants tau1 and tau2 for two nonradiative decay channels. Spi/Sn conical intersection versus a distorted structure of Spi,min in the FC region was briefly discussed.

  13. 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.

  14. Dopamine transporter; solubilization and characterization of ( sup 3 H) GBR-12935 binding in canine caudate

    SciTech Connect

    Sallee, F.R.

    1988-01-01

    The dopamine (DA) transporter protein, as indexed by ({sup 3}H)GBR-12935 binding, was solubilized from canine striatal membranes with the detergent digitonin. This solubilized protein retained the same pharmacological characteristics as membrane attached uptake sites. The binding of ({sup 3}H)GBR-12935 to solubilized preparations was specific, saturable and reversible with an equilibrium dissociation constant of approximately 3 nM and a maximum ligand binding (B{sub max}) of 3.4 pmol/mg protein. ({sup 3}H)GBR-12935 also bound to solubilized sites in a sodium-independent manner with a K{sub D} of approximately 6 nM and a B{sub max} of 1.2 {plus minus} 0.2 pmol/mg protein. Dopamine uptake inhibitors and substrates of DA uptake inhibited ({sup 3}H)GBR-12935 binding in a stereoselective and concentration dependent manner. For these compounds rank order of potency for inhibition of ({sup 3}H)GBR-12935 binding correlated with their potency for inhibition of dopamine uptake. K{sub D} values for DA uptake inhibitors in solubilized preparations correlated with those obtained on ({sup 3}H)GBR-12935 binding in the native state. The dopamine transporter appears to be a transmembrane glycoprotein by virtue of its absorption and specific elution from wheat germ agglutinin (WGA)-lectin column. Solubilization of the putative dopamine transporter with full retention of binding activity now allows for the purification and biochemical characterization of this important membrane protein.

  15. 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.

  16. 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.

  17. Neurotensin decreases high affinity [3H]-ouabain binding to cerebral cortex membranes.

    PubMed

    Rosin, Carina; Ordieres, María Graciela López; Arnaiz, Georgina Rodríguez de Lores

    2011-12-10

    Previous work from this laboratory showed the ability of neurotensin to inhibit synaptosomal membrane Na(+), K(+)-ATPase activity, the effect being blocked by SR 48692, a non-peptidic antagonist for high affinity neurotensin receptor (NTS1) [López Ordieres and Rodríguez de Lores Arnaiz 2000; 2001]. To further study neurotensin interaction with Na(+), K(+)-ATPase, peptide effect on high affinity [(3)H]-ouabain binding was studied in cerebral cortex membranes. It was observed that neurotensin modified binding in a dose-dependent manner, leading to 80% decrease with 1 × 10(-4)M concentration. On the other hand, the single addition of 1 × 10(-6)M, 1 × 10(-5)M and 1 × 10(-4)M SR 48692 (Sanofi-Aventis, U.S., Inc.) decreased [(3)H]-ouabain binding (in %) to 87 ± 16; 74 ± 16 and 34 ± 17, respectively. Simultaneous addition of neurotensin and SR 48692 led to additive or synergic effects. Partial NTS2 agonist levocabastine inhibited [(3)H]-ouabain binding likewise. Saturation assays followed by Scatchard analyses showed that neurotensin increased K(d) value whereas failed to modify B(max) value, indicating a competitive type interaction of the peptide at Na(+), K(+)-ATPase ouabain site. At variance, SR 48692 decreased B(max) value whereas it did not modify K(d) value. [(3)H]-ouabain binding was also studied in cerebral cortex membranes obtained from rats injected i. p. 30 min earlier with 100 μg and 250 μg/kg SR 48692. It was observed that the 250 μg/kg SR 48692 dose led to 19% decrease in basal [(3)H]-ouabain binding. After SR 48692 treatments, addition of 1 × 10(-6)M led to additive or synergic effect. Results suggested that [(3)H]-ouabain binding inhibition by neurotensin hardly involves NTS1 receptor.

  18. [3H]-verapamil binding to rat cardiac sarcolemmal membrane fragments; an effect of ischaemia.

    PubMed Central

    Dillon, J. S.; Nayler, W. G.

    1987-01-01

    The [3H]-verapamil binding activity of rat cardiac sarcolemmal fragments was studied, using membranes harvested from non-perfused, aerobically-perfused and ischaemic hearts. Glass-fibre filters were found to contain specific, high affinity--(KD 38 +/- 3.1 nM) [3H]-verapamil binding sites--making them unsuitable for use in [3H]-verapamil binding studies. Incubation of membranes from non-perfused hearts in a medium containing 150 mM NaCl, 1 mM CaCl2 and 50 mM Tris revealed two populations of [3H]-verapamil binding sites. When centrifugation instead of filtration was used to separate bound and free [3H]-verapamil, high affinity sites with a KD of 0.57 +/- 0.19 microM and a Bmax of 38 +/- 5.2 pmol mg-1 protein, and low affinity sites with a KD of 78 +/- 27.5 microM and a Bmax of 2.9 +/- 1.3 nmol mg-1 protein were detected. However, only low affinity binding sites could be detected in membranes which had been incubated in a cation-free medium containing 50 mM Tris. [3H]-verapamil binding to the low and high affinity sites was saturable, reversible, stereospecific and displaceable by D600 greater than diltiazem greater than Ca2+ but not by nifedipine, nitrendipine, nisoldipine or prazosin. The two populations of binding sites survived aerobic perfusion and 60 min ischaemia at 37 degrees C. Ischaemia reduced the Bmax and KD but selectivity was maintained. PMID:3028561

  19. 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.

  20. Modafinil evokes striatal [(3)H]dopamine release and alters the subjective properties of stimulants.

    PubMed

    Dopheide, Marsha M; Morgan, Russell E; Rodvelt, Kelli R; Schachtman, Todd R; Miller, Dennis K

    2007-07-30

    Modafinil is a mild psychostimulant used for the treatment of sleep and arousal-related disorders, and has been considered a pharmacotherapy for cocaine and amphetamine dependence; however, modafinil's mechanism of action is largely unclear. The present study investigated modafinil using drug discrimination and slice superfusion techniques. Rats were trained to discriminate cocaine (1.6 or 5 mg/kg) or amphetamine (0.3 mg/kg) from saline injection for food reinforcement. Modafinil (64-128 mg/kg) substituted partially for both cocaine doses and amphetamine. Pretreatment with a lower modafinil dose (32 mg/kg) augmented the discriminative stimulus properties of cocaine (1.6 mg/kg dose group) and amphetamine. In neurochemical experiments, modafinil (100-300 microM) evoked [(3)H]overflow from rat striatal slices preloaded with [(3)H]dopamine in a concentration-dependent manner; however, modafinil was less potent and efficacious than amphetamine and nicotine. The dopamine transporter inhibitor nomifensine (10 microM) blocked modafinil-evoked [(3)H]overflow, and concentrations of modafinil (<100 microM) that did not have intrinsic activity attenuated amphetamine (1 and 3 microM)-evoked [(3)H]overflow. Modafinil-evoked [(3)H]overflow was not altered by the nicotinic acetylcholine receptor antagonist mecamylamine, and modafinil did not alter nicotine-evoked [(3)H]overflow, indicating that nicotinic acetylcholine receptors likely are not important for modafinil's mechanism of action. The present results indicate that modafinil evokes dopamine release from striatal neurons and is a psychostimulant that is pharmacologically similar to, but much less potent and efficacious than, amphetamine.

  1. Liposomal delivery of the herpes simplex virus thymidine kinase gene in glioma: improvement of cell sensitization to ganciclovir.

    PubMed

    Zerrouqi, A; Rixe, O; Ghoumari, A M; Yarovoi, S V; Mouawad, R; Khayat, D; Soubrane, C

    1996-01-01

    In this study, we investigated whether the regulation and the copy number of the herpes simplex virus thymidine kinase (HSVtk) gene increased the sensitization to ganciclovir (GCV) of glioma cell lines (Rat C6 and human U118-MG) using liposome-mediated gene transfer. Three recombinant plasmids carrying the HSVtk gene driven by the thymidine kinase promoter in single (pAGo) and double copy (pYED) or by the human cytomegalovirus promoter (pCMVtk) were used for the transfection. The DNA delivery was optimized by screening a panel of cationic liposomes using Lac-Z and luciferase as reporter genes. The efficiency of transfection reached 33% to 36% in vitro but only 18.6% in vivo after an intratumoral injection of DNA-liposome complexes. Moreover, after transfection of the three plasmids, the cell-killing effect of GCV was evaluated. A significant enhancement (four- to fivefold) of the cell sensitivity to GCV was shown in pCMVtk and pYED as compared with pAGo-transfected cells in both cell lines. According to the plasmid, the effect of the HSVtk/GCV system was confirmed by in vivo experiments and was objectified by a higher tumor weight reduction with pCMVtk (49%) than pAGo (27%). From these results, we conclude that (1) the gene transfer can be achieved by cationic liposomes both in vitro and in vivo and that (2) using this type of vector, the antitumor effect of the HSVtk/GCV system could be potentiated by the up-regulation of HSVtk gene duplication.

  2. 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.

  3. 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.

  4. Inhibition of platelet (/sup 3/H)- imipramine binding by human plasma protein fractions

    SciTech Connect

    Strijewski, A.; Chudzik, J.; Tang, S.W.

    1988-01-01

    Inhibition of high-affinity (/sup 3/H)-imipramine binding to platelet membranes by human plasma fractions and isolated plasma proteins was investigated. Several plasma proteins were found to contribute to the observed apparent inhibition and this contribution was assessed in terms of inhibitor units. Alpha/sub 1/ acid glycoprotein, high density and low density lipoprotein, IgG and ..cap alpha../sub 1/-antitrypsin were identified as effective non-specific inhibitors. Alpha-1-acid glycoprotein was confirmed to be the most potent plasma protein inhibitor. Cohn fractions were evaluated for the presence of the postulated endocoid of (/sup 3/H)-imipramine binding site.

  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. 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.

  7. Human kidney thiopurine methyltransferase (TPMT): Photoaffinity labeling with ( sup 3 H-methyl)- S-adenosyl-L-methionine ( sup 3 H-ado-met)

    SciTech Connect

    Van Loon, J.A.; Weinshilboum, R.M. )

    1990-02-26

    TPMT (EC 2.1.1.67) catalyzes the S-methylation of 6-mercaptopurine (6-MP) and other heterocyclic and aromatic thiol drugs. TPMT activity and immunoreactive protein in humans are regulated by a common genetic polymorphism. Human kidney contains two isozymes of TPMT which can be separated by ion exchange chromatography. Partially purified isozymes of human kidney TPMT were exposed to UV light in the presence of {sup 3}H-Ado-Met. After photolysis, these preparations were subjected to SDS-polyacrylamide gel electrophoresis. Autoradiography of gels revealed that a 34 kDa protein was the predominant species that was radioactively labeled for both isozymes. Photoaffinity labeling of TPMT was inhibited in a concentration-dependent fashion by the methyl acceptor substrate, 6-MP, and by three TPMT inhibitors, 3,4-dimethoxy-5-hydroxybenzoic acid, 6-methylmercaptopurine and S-adenosyl-L-homocysteine. Of three wavelengths tested, 254, 300, and 350 nm, labeling was the greatest at 254 nm, near the absorption maximum for Ado-Met. Photoaffinity labeling of TPMT with {sup 3}H-Ado-Met should make it possible to determine the amino acid sequence of the active site and may make it possible to define the molecular basis of the genetic polymorphism for this important drug-metabolizing enzyme.

  8. Crystal structures of [NEt3H]5[XCoIIW11O39]·3H2O (X = P or As)

    USGS Publications Warehouse

    Evans, H.T.; 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.

  9. Distribution of 1-(3H)-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (3H-MPTP) in the frog: uptake in neuromelanin.

    PubMed

    Sokolowski, A L; Larsson, B S; Lindquist, N G

    1990-04-01

    The nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes selective destruction of pigmented monoaminergic neurons of the brain, mainly in the substantia nigra. Primates and amphibians, whose nerve cells contain melanin, have shown a higher sensitivity for the toxic effects of MPTP than species which are lacking neuromelanin, e.g. rodents. In the present study the distribution after intraperitoneal injection of 3H-MPTP in frogs (Rana temporaria) was studied by whole-body autoradiography. Histochemical staining methods for melanin were used in order to identify the pigment in various tissues. Melanin-containing nerve cells were present bilaterally in the ventral motor parts of the frog brain. Melanin was also found in the meninges, around the cerebral ventricles and the aqueducts, and in the eyes, skin and liver. The results from the autoradiographic study of 3H-MPTP revealed a high accumulation and retention in all melanin-containing structures up to 15 days after administration (the longest survival time). The pigmented tissues showed the highest concentration of radioactivity in the body at all survival times. The MPTP-induced destruction of pigmented nerve cells may be related to the binding and storage of MPTP and/or its metabolites in neuromelanin, causing toxic cytoplasmic concentrations through the continuous release of substance from the melanin depot.

  10. Effect of fluvoxamine on platelet 5-HT2A receptors as studied by [3H]lysergic acid diethylamide ([3H]LSD) binding in healthy volunteers.

    PubMed

    Spigset, O; Mjörndal, T

    1997-09-01

    Alterations in platelet 5-HT2A receptor characteristics have been reported in major depression as well as in other psychiatric diseases, and some effort has been made to utilize platelet 5-HT2A receptor status as a biological correlate to antidepressant drug response. In order to investigate whether treatment with a selective serotonin reuptake inhibitor affects platelet 5-HT2A receptors, we have studied platelet [3H]lysergic acid diethylamide ([3H]LSD) binding in healthy subjects treated with fluvoxamine in increasing dosage once weekly for 4 weeks. After 1 week of fluvoxamine treatment (25 mg/day), both Bmax and Kd were significantly lower than before the start of the treatment (19.9 versus 25.5 fmol/mg protein, P = 0.005 for Bmax; 0.45 versus 0.93 nM, P = 0.006 for Kd). Bmax returned to baseline during week 2, whereas Kd was lower than the baseline value throughout the treatment period. After discontinuation of fluvoxamine treatment, there was a significant increase in Kd (0.50 nM before discontinuation vs. 1.14 nM after discontinuation; P = 0.001), but not in Bmax. The study demonstrates that fluvoxamine affects platelet 5-HT2A receptor status irrespective of underlying psychiatric disease, and that this effect is evident already after 1 week at a subtherapeutic fluvoxamine dose.

  11. ( sup 3 H)PN200-110 and ( sup 3 H)ryanodine binding and reconstitution of ion channel activity with skeletal muscle membranes

    SciTech Connect

    Hamilton, S.L.; Alvarez, R.M.; Fill, M.; Hawkes, M.J.; Brush, K.L.; Schilling, W.P.; Stefani, E. )

    1989-11-15

    Skeletal muscle membranes derived either from the tubular (T) network or from the sarcoplasmic reticulum (SR) were characterized with respect to the binding of the dihydropyridine, ({sup 3}H)PN200-110, and the alkaloid, ({sup 3}H)ryanodine; polypeptide composition; and ion channel activity. Conditions for optimizing the binding of these radioligands are discussed. A bilayer pulsing technique is described and is used to examine the channels present in these membranes. Fusion of T-tubule membranes into bilayers revealed the presence of chloride channels and dihydropyridine-sensitive calcium channels with three distinct conductances. The dihydropyridine-sensitive channels were further characterized with respect to their voltage dependence. Pulsing experiments indicated that two different populations of dihydropyridine-sensitive channels existed. Fusion of heavy SR vesicles revealed three different ion channels; the putative calcium release channel, a potassium channel, and a chloride channel. Thus, this fractionation procedure provides T-tubules and SR membranes which, with radioligand binding and single channel recording techniques, provide a useful tool to study the characteristics of skeletal muscle ion channels and their possible role in excitation-contraction coupling.

  12. EdU Incorporation for FACS and Microscopy Analysis of DNA Replication in Budding Yeast.

    PubMed

    Talarek, Nicolas; Petit, Julie; Gueydon, Elisabeth; Schwob, Etienne

    2015-01-01

    DNA replication is a key determinant of chromosome segregation and stability in eukaryotes. The yeast Saccharomyces cerevisiae has been extensively used for cell cycle studies, yet simple but key parameters such as the fraction of cells in S phase in a population or the subnuclear localization of DNA synthesis have been difficult to gather for this organism. 5-ethynyl-2'-deoxyuridine (EdU) is a thymidine analogue that can be incorporated in vivo and later detected using copper-catalyzed azide alkyne cycloaddition (Click reaction) without prior DNA denaturation. This chapter describes a budding yeast strain and conditions that allow rapid EdU incorporation at moderate extracellular concentrations, followed by its efficient detection for the analysis of DNA replication in single cells by flow cytometry and fluorescence microscopy.

  13. Changes in labeling of soluble and insoluble rat brain proteins using [3H]-tyrosine as precursor during a learning experiment.

    PubMed

    Popov, N; Schulzeck, S; Schmidt, S; Matthies, H

    1975-01-01

    The incorporation of intraventricularly injected L-[3H]-tyrosine into proteins from three rat brain regions was studied during the acquisition of a shock-motivated brightness discrimination. Separating the proteins by means of polyacrylamide gel electrophoresis revealed an increased labeling confined to only a few of the resolved slow-moving bands of soluble and, especially insoluble hippocampus proteins. The labeling of visual cortex proteins showed only minute changes, while the auditory cortex proteins exhibited no differences between trained and nontrained animals.

  14. Heterogeneity of basal keratinocytes: nonrandom distribution of thymidine-labeled basal cells in confluent cultures is not a technical artifact

    SciTech Connect

    Milstone, L.M.; LaVigne, J.F.

    1985-06-01

    Basal surface autoradiography of (/sup 3/H)dThd-labeled, confluent, keratinocyte cultures reveals that proliferating cells have a nonrandom, patterned distribution. Unlabeled cells, likewise, appear nonrandomly in clusters. The authors show here that failure to detect DNA synthesis in some basal cells in culture is not an artifact caused either by physical separation of the labeled nuclei from the radiographic emulsion or by a diffusion barrier that would prevent (/sup 3/H)dThd from reaching basal cells.

  15. 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.

  16. Precise /sup 3/H-/sup 3/He mass difference for neutrino mass determination

    SciTech Connect

    Lippmaa, E.; Pikver, R.; Suurmaa, E.; Past, J.; Puskar, J.; Koppel, I.; Tammik, A.

    1985-01-28

    The precise /sup 3/H-/sup 3/He atomic mass difference has been measured by high-resolution (10/sup -8/) ion cyclotron resonance in a 4.7-T magnetic field. The result of 18 599 +- 2 eV favors a nonzero electron antineutrino mass.

  17. Theoretical microwave spectral constants for C2N, C2N/+/, and C3H

    NASA Technical Reports Server (NTRS)

    Green, S.

    1980-01-01

    Theoretical microwave spectral constants have been computed for C2N, C3H, and C2N(+). For C2N these are compared with values obtained from optical data. Calculated hyperfine constants are also presented for HNC, DNC, and HCNH(+). The possibility of observing these species in dense interstellar clouds is discussed.

  18. 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.

  19. Regulation of (/sup 3/H)GABA release from strips of guinea pig urinary bladder

    SciTech Connect

    Shirakawa, J.; Taniyama, K.; Iwai, S.; Tanaka, C.

    1988-12-01

    The presence of receptors that regulate the release of gamma-aminobutyric acid (GABA) was studied in strips of the guinea pig urinary bladder. GABA (10(-8)-10(-5) M) and muscimol (10(-8)-10(-5) M), but not baclofen (10(-5) M), reduced the Ca2+-dependent, tetrodotoxin-resistant release of (/sup 3/H)GABA evoked by high K+ from the urinary bladder strips preloaded with (/sup 3/H)GABA. The inhibitory effect of muscimol was antagonized by bicuculline and potentiated by diazepam, clonazepam, and pentobarbital sodium. The potentiating effect of clonazepam was antagonized by Ro 15-1788. Acetylcholine (ACh) inhibited the high K+-evoked release of (/sup 3/H)GABA. The inhibitory effect of ACh was antagonized by atropine sulfate and pirenzepine but not by hexamethonium. Norepinephrine (NE) inhibited the evoked release of (/sup 3/H)GABA. The inhibitory effect of NE was mimicked by clonidine, but not by phenylephrine, and was antagonized by yohimbine but not by prazosin. These results provide evidence that the release of GABA from strips of guinea pig urinary bladder is regulated via the bicuculline-sensitive GABAA receptor, M1-muscarinic, and alpha 2-adrenergic receptors.

  20. 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

    2015-10-05

    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.

  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.

  2. Anionic ordering and thermal properties of FeF3·3H2O

    DOE PAGES

    Burbano, Mario; Duttine, Mathieu; Borkiewicz, Olaf; ...

    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

  3. Autoradiographic Localization of [3H]Gentamicin in the Proximal Renal Tubules of Mice

    PubMed Central

    Kuhar, Michael J.; Mak, Linda L.; Lietman, Paul S.

    1979-01-01

    The site of localization of [3H]gentamicin within mouse kidney is shown to be the proximal renal tubule by coincidence of the radioactivity, as visualized by autoradiography, and the mucopolysaccharide-rich microvilli characteristic of proximal convoluted tubules, as visualized by histochemical staining. Images PMID:426500

  4. 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.

  5. Ascorbic acid reduces accumulation of (/sup 3/H)spiperone in mouse striatum in vivo

    SciTech Connect

    Dorris, R.L.

    1987-10-01

    (/sup 3/H)Spiperone was administered to mice. In agreement with other published reports, 2 hr later the accumulation of tritium was three to four times greater in the corpus striatum than in the cerebellum. Ascorbic acid (100, 1000, 2000 mg/kg, ip, 30 min) reduced the 2-hr accumulation in the corpus striatum 16, 42, and 63%, respectively, with only the highest does producing any significant reduction in the cerebellum. The effect was still evident in striatum 18 hr after a single dose of 1000 mg/kg. Striatal minces taken from mice treated 1 or 2 hr earlier with ascorbic acid showed no reduction in (/sup 3/H)spiperone binding. However, preincubation of striatal minces for 2 hr with ascorbic acid (10/sup -3/ M) produced an 82% reduction in specific binding while not having any effect on nonspecific binding. While it cannot be certain that the reduction of striatal (/sup 3/H) spiperone concentrations after ascorbic acid in vivo was not a result of some nonspecific alteration in the pharmacokinetics of (/sup 3/H)spiperone, the in vitro observation strongly suggests that it resulted from an alteration of binding characteristics at the receptor level.

  6. Point mutation of H3/H4 histones affects acetic acid tolerance in Saccharomyces cerevisiae.

    PubMed

    Liu, Xiangyong; Zhang, Xiaohua; Zhang, Zhaojie

    2014-10-10

    The molecular mechanism of acetic acid tolerance in yeast remains unclear despite of its importance for efficient cellulosic ethanol production. In this study, we examined the effects of histone H3/H4 point mutations on yeast acetic acid tolerance by comprehensively screening a histone H3/H4 mutant library. A total of 24 histone H3/H4 mutants (six acetic acid resistant and 18 sensitive) were identified. Compared to the wild-type strain, the histone acetic acid-resistant mutants exhibited improved ethanol fermentation performance under acetic acid stress. Genome-wide transcriptome analysis revealed that changes in the gene expression in the acetic acid-resistant mutants H3 K37A and H4 K16Q were mainly related to energy production, antioxidative stress. Our results provide novel insights into yeast acetic acid tolerance on the basis of histone, and suggest a novel approach to improve ethanol production by altering the histone H3/H4 sequences.

  7. Bearings Incorporating Deadband Rollers

    NASA Technical Reports Server (NTRS)

    Gualtieri, Guy V.

    1996-01-01

    Bearings in high-pressure turbopump redesigned to incorporate rollers allowing limited axial motion within small deadband. Does not permit radial deadband motion. Axial deadband motion used for rotor-thrust-balance control. Design eliminates some nonlinearities in dynamics of pump rotor and assists in suppressing vibrations at harmonics of frequency of rotation.

  8. 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.

  9. DNA methylation in 5-aza-2'-deoxycytidine-resistant variants of C3H 10T1/2 C18 cells.

    PubMed Central

    Flatau, E; Gonzales, F A; Michalowsky, L A; Jones, P A

    1984-01-01

    A cell line (T17) was derived from C3H 10T1/2 C18 cells after 17 treatments with increasing concentrations of 5-aza-2'-deoxycytidine. The T17 cell line was very resistant to the cytotoxic effects of 5-aza-2'-deoxycytidine, and the 50% lethal dose for 5-aza-2'-deoxycytidine was ca. 3 microM, which was 30-fold greater than that of the parental C3H 10T1/2 C18 cells. Increased drug resistance was not due to a failure of the T17 cell line to incorporate 5-aza-2'-deoxycytidine into DNA. The cells were also slightly cross-resistant to 5-azacytidine. The percentage of cytosines modified to 5-methylcytosine in T17 cells was 0.7%, a 78% decrease from the level of 3.22% in C3H 10T1/2 C18 cells. The DNA cytosine methylation levels in several clones isolated from the treated lines were on the order of 0.7%, and clones with methylation levels lower than 0.45% were not obtained even after further drug treatments. These highly decreased methylation levels appeared to be unstable, and DNA modification increased as the cells divided in the absence of further drug treatment. The results suggest that it may not be possible to derive mouse cells with vanishingly low levels of 5-methylcytosine and that considerable de novo methylation can occur in cultured lines. PMID:6209556

  10. 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.

  11. 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.

  12. 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.

  13. Endomorphin synthesis in rat brain from intracerebroventricularly injected [3H]-Tyr-Pro: a possible biosynthetic route for endomorphins.

    PubMed

    Rónai, András Z; Szemenyei, Erzsébet; Kató, Erzsébet; Kocsis, László; Orosz, György; Al-Khrasani, Mahmoud; Tóth, Géza

    2006-03-15

    In spite of concentrated efforts, the biosynthetic route of mu-opioid receptor agonist brain tetrapeptide endomorphins (Tyr-Pro-Trp-Phe-NH2 and Tyr-Pro-Phe-Phe-NH2), discovered in 1997, is still obscure. We report presently that 30 min after intracerebroventricular injection of 20 or 200 microCi [3H]Tyr-Pro (49.9 Ci mmol(-1)) the incorporated radioactivity was found in endomorphin-related tetra- and tripeptides in rat brain extracts. As detected by the combination of HPLC with radiodetection, a peak corresponding to endomorphin-2-OH could be identified in two of four extracts of "20 microCi" series. Radioactive peaks in position of Tyr, Tyr-Pro, Tyr-Pro-Phe or Tyr-Pro-Trp appeared regularly in both series and also in the "tetrapeptide cluster" constituted by endomorphins and their free carboxylic forms. In one of the four extracts in the "200 microCi" series a robust active peak in the position of endomorphin 2 could be detected. Intracerebroventricularly injected 100 nmol, but not 10 or 1000 nmol cold Tyr-Pro (devoid of opioid activity in vitro), caused a naloxone-reversible prolongation of tail-flick latency in rats, peaking between 15 and 30 min. We suggest that Tyr-Pro may serve as a biosynthetic precursor to endomorphin synthesis.

  14. Zinc Incorporation Into Hydroxylapatite

    SciTech Connect

    Tang, Y.; Chappell, H; Dove, M; Reeder, R; Lee, Y

    2009-01-01

    By theoretical modeling and X-ray absorption spectroscopy, the local coordination structure of Zn incorporated into hydroxylapatite was examined. Density function theory (DFT) calculations show that Zn favors the Ca2 site over the Ca1 site, and favors tetrahedral coordination. X-ray absorption near edge structure (XANES) spectroscopy results suggest one dominant coordination environment for the incorporated Zn, and no evidence was observed for other Zn-containing phases. Extended X-ray absorption fine structure (EXAFS) fitting of the synthetic samples confirms that Zn occurs in tetrahedral coordination, with two P shells at 2.85-3.07 {angstrom}, and two higher Ca shells at 3.71-4.02 {angstrom}. These fit results are consistent with the most favored DFT model for Zn substitution in the Ca2 site.

  15. Metabolism of /sup 3/H-dopamine by human chorioamnion in vitro

    SciTech Connect

    Phillippe, M.; Niloff, J.M.

    1982-08-01

    Previous investigation has demonstrated biologically significant concentrations of catecholamines in amniotic fluid, which increase with gestation. The half life, metabolic clearance rate, and metabolic fate of these hormones in the amniotic compartment are yet to be established. This study was undertaken to demonstrate the ability of human chorioamnion to metabolize dopamine in vitro. Incubation experiments demonstrated that /sup 3/H-dopamine is rapidly metabolized to dihydroxyphenylacetic acid, 3-methoxy, 4-hydroxyphenylacetic acid, and 3-methoxy, 4-hydroxyphenylethanol-all products of monoamine oxidase. No significant 3-methoxytyramine, a catechol-o-methyltransferase product, was observed. Incubation experiments with pargyline, a monoamine oxidase inhibitor, resulted in significant reduction in /sup 3/H-dopamine metabolism. Catecholamines and their interaction with prostaglandin synthesis have been theorized to be a fetal signal for the initiation of parturition. The ability of chorioamnion to metabolize catecholamine could, therefore, provide another control mechanism by which fetal catecholamines are modulated.

  16. Increased platelet membrane [3H]-LSD binding in patients on chronic neuroleptic treatment.

    PubMed Central

    Schächter, M; Geaney, D P; Grahame-Smith, D G; Cowen, P J; Elliott, J M

    1985-01-01

    Using a [3H]-lysergic acid diethylamide [( 3H]-LSD) binding technique, platelet 5-hydroxytryptamine (5-HT) receptor number and affinity were compared in schizophrenics treated with depot thioxanthenes and phenothiazines and controls. There was an approximately 30% increase in platelet receptor number (Bmax) in the patient group. There was a decrease in affinity (increase in Kd) of about 30% in the patient group. This was probably due to the persistence of the neuroleptic in the platelet membrane preparation. There was a weak positive correlation between receptor number and total neuroleptic dosage. The increased number of 5-HT receptors is consistent with the previously reported enhancement of 5-HT-induced platelet aggregation in patients treated with long-term phenothiazines and thioxanthenes. Our findings are compatible with 5-HT up-regulation in human platelets produced by depot neuroleptic therapy. It is not known whether parallel changes may be occurring in brain 5-HT receptors. PMID:2859873

  17. Expeditious diastereoselective synthesis of elaborated ketones via remote Csp3-H functionalization

    NASA Astrophysics Data System (ADS)

    Shu, Wei; Lorente, Adriana; Gómez-Bengoa, Enrique; Nevado, Cristina

    2017-01-01

    The quest for selective C-H functionalization reactions, able to provide new strategic opportunities for the rapid assembly of molecular complexity, represents a major focus of the chemical community. Examples of non-directed, remote Csp3-H activation to forge complex carbon frameworks remain scarce due to the kinetic stability and thus intrinsic challenge associated to the chemo-, regio- and stereoselective functionalization of aliphatic C-H bonds. Here we describe a radical-mediated, directing-group-free regioselective 1,5-hydrogen transfer of unactivated Csp3-H bonds followed by a second Csp2-H functionalization to produce, with exquisite stereoselectivity, a variety of elaborated fused ketones. This study demonstrates that aliphatic acids can be strategically harnessed as 1,2-diradical synthons and that secondary aliphatic C-H bonds can be engaged in stereoselective C-C bond-forming reactions, highlighting the potential of this protocol for target-oriented natural product and pharmaceutical synthesis.

  18. Synthesis of (3) H, (2) H4 and (14) C-SCH 417690 (Vicriviroc).

    PubMed

    Hesk, D; Borges, S; Hendershot, S; Koharski, D; McNamara, P; Ren, S; Saluja, S; Truong, V; Voronin, K

    2016-05-15

    Vicriviroc or SCH 417690 is a potent and selective antagonist of the CCR5 receptor. CCR5 receptor antagonists have the potential for the treatment of HIV infections. Four distinct isotopically labelled forms of SCH 417690 were synthesized. Low specific activity [(3) H]SCH 417690 was prepared for a preliminary absorption, distribution, metabolism and excretion evaluation of the compound and [(14) C]SCH 417690 for more definitive absorption, distribution, metabolism and excretion work, including an absorption, metabolism and excretion study in man. In addition, high specific activity [(3) H]SCH 417690 was prepared for CCR5 receptor binding work and [(2) H4 ]SCH 417690 was prepared as an internal standard for a liquid chromatography-mass spectrometry bioanalytical method. The paper discusses the synthesis of four isotopically labelled forms of SCH 417690.

  19. On the stability of (3)H and (63)Ni Ultima Gold liquid scintillation sources.

    PubMed

    Nedjadi, Youcef; Duc, Pierre-François; Bochud, François; Bailat, Claude

    2016-12-01

    The stability of (3)H and (63)Ni samples, made with Ultima Gold and Ultima Gold AB using eight aqueous fractions ranging between 0.1% and 10%, was monitored over a period of 32 weeks. Counting rate losses were measured in all cases, but were found to be particularly severe for samples with low aqueous fractions. Hydrodynamic diameter measurements of the reverse micelles of (3)H and (63)Ni in Ultima Gold and Ultima Gold AB samples were performed with dynamic light scattering. For Ultima Gold, the micelle diameters were measured to be about 2nm and slightly dependent of the aqueous fraction, while for Ultima Gold AB they were found to vary strongly with the aqueous fraction and range between 1 and 4nm. The hypothesis that the counting rate instabilities derive from changes in the sizes of micelles was tested by measuring them four times over the monitoring period. The measurement results do not support this claim.

  20. Synthesis of high specific activity (1- sup 3 H) farnesyl pyrophosphate

    SciTech Connect

    Saljoughian, M.; Morimoto, H.; Williams, P.G.

    1991-08-01

    The synthesis of tritiated farnesyl pyrophosphate with high specific activity is reported. trans-trans Farnesol was oxidized to the corresponding aldehyde followed by reduction with lithium aluminium tritide (5%-{sup 3}H) to give trans-trans (1-{sup 3}H)farnesol. The specific radioactivity of the alcohol was determined from its triphenylsilane derivative, prepared under very mild conditions. The tritiated alcohol was phosphorylated by initial conversion to an allylic halide, and subsequent treatment of the halide with tris-tetra-n-butylammonium hydrogen pyrophosphate. The hydride procedure followed in this work has advantages over existing methods for the synthesis of tritiated farnesyl pyrophosphate, with the possibility of higher specific activity and a much higher yield obtained. 10 refs., 3 figs.

  1. Effect of O3 and O3/H2O2 on algae harvesting using chitosan.

    PubMed

    Pranowo, R; Lee, D J; Liu, J C; Chang, J S

    2013-01-01

    We examined the effects of pre-oxidation using ozone (O3) and a combination of O3 and hydrogen peroxide (O3/H2O2) on algae suspensions and their harvesting. Inactivation of algae cells, release of intracellular organic matter (IOM), mineralization of extracellular organic matter (EOM), and changes in molecular weight distribution of EOM were found after pre-oxidation. Enhanced separation efficiency of turbidity, dissolved organic carbon (DOC), protein, and polysaccharide using chitosan and polyaluminum chloride (PACl) was found after pre-oxidation, especially when algae cells were subject to O3/H2O2. Chitosan showed higher efficiency than PACl. Judging from the remarkable increase in floc size, it was proposed that released IOM formed complexes with cationic chitosan and resulted in enhanced dual flocculation and facilitated algae separation.

  2. Quantitation of uveoscleral outflow in normotensive and glaucomatous Beagles by /sup 3/H-labeled dextran

    SciTech Connect

    Barrie, K.P.; Gum, G.G.; Samuelson, D.A.; Gelatt, K.N.

    1985-01-01

    In uveoscleral outflow, aqueous humor leaves the anterior chamber and passes caudally through the trabecular meshwork and the sclerociliary cleft to enter the supraciliary and suprachoroidal spaces. The fluid is then absorbed by choroidal and scleral circulations. Using /sup 3/H-labeled dextran, uveoscleral outflow was quantitated in normotensive and glaucomatous Beagles under general anesthesia. The intrascleral plexus was isolated and /sup 3/H-labeled dextran was injected into the anterior chamber. Intrascleral plexus contents were sampled every 5 minutes over a 30- to 60-minute period. The eyes were enucleated, sectioned, and prepared for scintillation counting. Uveoscleral outflow accounted for 15% and 3% of the total aqueous humor outflow in the normotensive dogs and in the advanced glaucomatous dogs, respectively. In the advanced glaucomatous Beagle, conventional and uveoscleral outflow pathways were reduced and contributed to the etiopathogenesis of glaucoma.

  3. 96-Well plate assays for measuring collagenase activity using (3)H-acetylated collagen.

    PubMed

    Koshy, P J; Rowan, A D; Life, P F; Cawston, T E

    1999-11-15

    We describe two alternative assays for measuring collagenolytic activity using (3)H-acetylated collagen. Both assays have been developed for the 96-well plate format and measure the amount of radiolabeled collagen fragments released into the supernatant from an insoluble (3)H-acetylated collagen fibril preparation. The first method separates digested solubilized fragments from the intact fibril by sedimentation of the undigested collagen by centrifugation. The second method achieves this separation by filtration of the supernatant through the membrane of a 96-well filtration plate which retains the undigested collagen fibril. Both methods give linear dose- and time-dependent responses of collagenase activity > or = 70% of total collagen lysis. In addition, both assays can be simply modified to measure tissue inhibitors of metalloproteinases (TIMPs) inhibitory activity, which is also linear between 20 and 75% of total collagen lysis with the amount of TIMP added.

  4. SO3H-functionalized ionic liquid: efficient catalyst for bagasse liquefaction.

    PubMed

    Long, Jinxing; Guo, Bin; Teng, Junjiang; Yu, Yinghao; Wang, Lefu; Li, Xuehui

    2011-11-01

    Liquefaction is a process for the production of biofuel or value-added biochemicals from non-food biomass. SO(3)H-, COOH-functionalized and HSO(4)-paired imidazolium ionic liquids were shown to be efficient catalysts for bagasse liquefaction in hot compressed water. Using SO(3)H-functionalized ionic liquid, 96.1% of bagasse was liquefied and 50.6% was selectively converted to low-boiling biochemicals at 543 K. The degree of liquefaction and selectivity for low-boiling products increased and the average molecular weight of the tetrahydrofuran soluble products decreased with increasing acidic strength of ionic liquids. Analysis of products and comparative characterization of raw materials and residues suggested that both catalytic liquefaction and hydrolysis processes contribute to the high conversion of bagasse. A possible liquefaction mechanism based on the generation of 3-cyclohexyl-1-propanol, one of the main products, is proposed.

  5. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography.

    PubMed

    Laskey, R A; Mills, A D

    1975-08-15

    Methods which use the scintillator PPO to record film images of 3H in chromatograms and polyacrylamide gels (fluorography) have been described elsewhere. This paper demonstrates that pre-exposure of the film to a brief flash of light greatly increases the sensitivity of fluorography. Pre-exposure also permits quantitative interpretation of the film image, because it corrects the non-linear relationship between radioactivity of the sample and absorbance of the film image. Therefore the distribution of radioactivity in the sample is accurately represented by microdensitometry of the image obtained on pre-exposed film. Using pre-exposed film 300 dis. 3H/min or 30 dis. 14C/min can be detected in a band in a gel in a 24-h exposure. The Appendix describes revisions and extensions of existing fluorographic procedures, including application to agarose gels and a rapid procedure for recovering PPO for re-use.

  6. Expeditious diastereoselective synthesis of elaborated ketones via remote Csp(3)-H functionalization.

    PubMed

    Shu, Wei; Lorente, Adriana; Gómez-Bengoa, Enrique; Nevado, Cristina

    2017-01-13

    The quest for selective C-H functionalization reactions, able to provide new strategic opportunities for the rapid assembly of molecular complexity, represents a major focus of the chemical community. Examples of non-directed, remote Csp(3)-H activation to forge complex carbon frameworks remain scarce due to the kinetic stability and thus intrinsic challenge associated to the chemo-, regio- and stereoselective functionalization of aliphatic C-H bonds. Here we describe a radical-mediated, directing-group-free regioselective 1,5-hydrogen transfer of unactivated Csp(3)-H bonds followed by a second Csp(2)-H functionalization to produce, with exquisite stereoselectivity, a variety of elaborated fused ketones. This study demonstrates that aliphatic acids can be strategically harnessed as 1,2-diradical synthons and that secondary aliphatic C-H bonds can be engaged in stereoselective C-C bond-forming reactions, highlighting the potential of this protocol for target-oriented natural product and pharmaceutical synthesis.

  7. Enhanced binding of /sup 3/H-arginine8-vasopressin in the Brattleboro rat

    SciTech Connect

    Shewey, L.M.; Dorsa, D.M.

    1986-07-01

    Specific binding sites for 3(H)-arginine8-vasopressin (AVP) were characterized using membrane preparations of liver, renal medulla and brain (septal) tissue of heterozygous (HE) and homozygous (HO) Brattleboro (BB) rats. Measurement of binding sites indicated that significantly greater numbers of AVP receptors are present in the liver and septum of HO-BB rats. Similar numbers of AVP receptors were present in renal medullary tissue from HO-BB and HE-BB rats. Higher equilibrium dissociation constants were measured in the HO-BB septal tissue indicating a lower affinity of the brain receptor for 3(H)-AVP than in heterozygotes. No significant differences in AVP receptor affinity were noted in liver or kidney tissue. It is concluded that up-regulation of AVP receptor number and, in the brain, alterations in AVP receptor affinity may occur in the absence of endogenous AVP.

  8. Evaluation of PM-3 Chemistry Data and Possible Interpretations of 3H Observations, Revision 0

    SciTech Connect

    Andrews, Robert; Marutzky, Sam J.

    2015-02-01

    This report summarizes the analyses of the groundwater results from sampling of PM-3-1 (deep) and PM-3-2 (shallow), with a particular focus of evaluating the groundwater geochemistry data in comparison to the geochemistry observed in other wells in the Thirsty Canyon area as well as to evaluate the potential source of 3H observed in these piezometers from previous sampling activities, which employed depth-discrete bailers or a Bennett submersible piston pump.

  9. Nickel-Catalyzed Stereoselective Alkenylation of C(sp(3))-H Bonds with Terminal Alkynes.

    PubMed

    Lin, Cong; Chen, Zhengkai; Liu, Zhanxiang; Zhang, Yuhong

    2017-02-17

    A nickel-catalyzed stereoselective alkenylation of an unactivated β-C(sp(3))-H bond in aliphatic amide with terminal alkynes using 8-aminoquinoline auxiliary is reported for the first time. This reaction displays excellent functional group tolerance with respect to both aliphatic amides and terminal alkynes and features a cheap nickel catalytic system. The 8-aminoquinolyl directing group could be smoothly removed, and the resultant β-styrylcarboxylic acid derivatives could serve as versatile building blocks for further transformation.

  10. Acetohydrazone: A Transient Directing Group for Arylation of Unactivated C(sp(3))-H Bonds.

    PubMed

    Ma, Fei; Lei, Min; Hu, Lihong

    2016-06-03

    A straightforward and efficient method has been developed for the synthesis of 2-benzylbenzaldehyde derivatives from 2-methylbenzaldehyde and iodobenzene via a C(sp(3))-H activation process. In the course of the activation reaction, acetohydrazone is formed between 2-benzylbenzaldehyde and acetohydrazine as a transient directing group. As a new kind of transient directing group, acetohydrazone exhibits a remarkable directing effect to give corresponding products in good to excellent yields.

  11. Characterization Results For The 2013 HTF 3H Evaporator Overhead Samples

    SciTech Connect

    Washington, A. L. II

    2013-12-04

    This report tabulates the radiochemical analysis of the 3H evaporator overhead sample for {sup 137}Cs, {sup 90}Sr, and {sup 129}I to meet the requirements in the Effluent Treatment Project (ETP) Waste Acceptance Criteria (WAC) (rev. 6). This report identifies the sample receipt date, preparation method, and analysis performed in the accumulation of the listed values. All data was found to be within the ETP WAC (rev. 6) specification for the Waste Water Collection Tanks (WWCT).

  12. Antihypertensive activity of 2[(2-hydroxyphenyl)amino]-4(3H)-quinazolinone.

    PubMed

    Hussain, M A; Chiu, A T; Price, W A; Timmermans, P B; Shefter, E

    1988-04-01

    Two 2-substituted 4(3H)-quinazolinones were synthesized through an easy one-step synthetic procedure. One of them (1), which was synthesized by the reaction of 1-(2-hydroxyphenyl)-2-thiourea and isatoic anhydride, contained a guanidino moiety and showed antihypertensive activity following cumulative intravenous administration to anesthetized spontaneously hypertensive rats. The other compound (2) does not contain the guanidino group and did not exhibit antihypertensive activity.

  13. UPTAKE OF [3H]-COLCHICINE INTO BRAIN AND LIVER OF MOUSE, RAT, AND CHICK

    SciTech Connect

    Bennett, Edward L.; Alberti, Marie Hebert; Flood, James F.

    1980-07-01

    The uptake of [ring A-4-{sup 3}H] colchicine and [ring C-methoxy-{sup 3}H]colchicine has been compared in mice from 1 to 24 hr after administration. Less radioactivity was found in brain after administration of ring-labeled colchicine than after administration of the methoxy-labeled colchicine. Three hr after administration of ring-labeled colchicine, 5% of the label was in liver and about 0.01% of the label was present in brain. Forty percent of the brain radioactivity was bound to tubulin as determined by vinblastine precipitation. After 3 hr, an average of 8% of the radioactivity from methoxy-labeled colchicine was found in the liver and 0.16% in brain. However, less than 5% of the activity in brain was precipitated by vinblastine, and the colchicine equivalent was comparable to that found after administration of the ring-labeled colchicine. The amount of colchicine entering mouse brain after subcutaneous injection is comparable to the minimum behaviorally effective dose when administered to the caudate. The metabolism of [ring C-methoxy-{sup 3}H] and [ring A-{sup 3}H]colchicine was also studied in rats. the general pattern was similar to mice; less radioactivity was found in brain after administration of the ring-labeled alkoloid than after administration of methoxy-labeled colchicine. Again, 40-50% of ring-labeled colchicine was precipitated by vinblastine. A much smaller percentage of the methoxy-labeled drug was precipitated by vinblastine than of the ring A-labeled colchicine. These experiments, together with behavioral experiments [7], support the hypotheses that structural alteration in synapses by recently synthesized proteins which are transported down the axons and dendrites may be an essential process for long-term memory formation.

  14. Characterization results for the October 2015-Tank for farm 3H evaporator overhead examples

    SciTech Connect

    Nicholson, J. C.

    2016-01-28

    This report contains the radioanalytical results of the 3H evaporator overhead sample received at SRNL on October 13, 2015. Specifically, concentrations of 137Cs, 90Sr, and 129I are reported and compared to the corresponding Waste Acceptance Criteria (WAC) limits of the Effluent Treatment Project (ETP) Waste Water Collection Tank (WWCT) (rev. 6). All of the radionuclide concentrations in the sample were found to be in compliance with the ETP WAC limits.

  15. Characteristics of (3H)2-Deoxyglucose Uptake by Slices of Rat Cerebral Cortex

    DTIC Science & Technology

    1983-05-17

    system because carrier sys- tems are generally stereoselective ( Lehninger , 1975). For these reasons, L -glucose is a valid control for estimating the...analogues to human erythrocytes in connection with inhibition of sugar transport. J. Biol. Chem. 234:3022-26. Lehninger , A . L . 1975. Biochemistry...3H]Glucose uptake by slices was a valid approximation of the space available for glucose diffusion because L -[^H]glucose is an inactive stereoisomer

  16. 1-aryl-6,7-methylenedioxy-3H-quinazolin-4-ones as anticonvulsant agents.

    PubMed

    Zappalà, Maria; Grasso, Silvana; Micale, Nicola; Zuccalà, Giuseppe; Menniti, Frank S; Ferreri, Guido; De Sarro, Giovambattista; De Micheli, Carlo

    2003-12-15

    A set of novel 1-aryl-6,7-methylenedioxy-3H-quinazolin-4-(thi)ones (3a-f) has been designed and screened as anticonvulsant agents in DBA/2 mice. The new compounds are provided with anticonvulsant properties comparable to those of GYKI 52466. To clarify the mode of action, their affinity for the quinazolinone/2,3-benzodiazepine site of the AMPA receptor complex has been assayed.

  17. Modulation of [3H]MK-801 binding to NMDA receptors in vivo and in vitro.

    PubMed

    Murray, F; Kennedy, J; Hutson, P H; Elliot, J; Huscroft, I; Mohnen, K; Russell, M G; Grimwood, S

    2000-06-02

    [3H]MK-801 binding in vivo was used to determine the occupancy of NMDA receptor ligands shown to allosterically modulate binding in vitro. ED(50) values (mg/kg) were obtained for the channel blockers (+)-5-methyl-10,11-dihydro-5,4-dibenzo[a,d]cyclohepten-5,10-imine maleate ((+)-MK-801, 0.2), 1-(1-phenylcyclohexyl)piperidine (phencyclidine, PCP, 1.7) and ketamine (4.4). Antagonists at the glutamate (DL-(2-carboxypiperazine-4-yl)propyl-1-phosphonate (DL-CPP, 5.7)) and glycine site (7-Chloro-4-hydroxy-3-(3-phenoxy)-phenyl-2(H)quinolinone (L-701,324, 14.1), 3R(+)cis-4-methyl-pyrrollid-2-one (L-687,414, 15.1)) inhibited [3H]MK-801 binding in vivo to varying maximum levels (69%, 103% and 45%, respectively). NR2B subunit-selective compounds acting at the ifenprodil site inhibited [3H]MK-801 in vivo by a maximum of 52-72% and gave ED(50) values (mg/kg) of: (+/-)-(1S*, 2S*)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol ((+/-)CP-101,606), 1.9; (+/-)-(3R, 4S)-3-[4-(4-fluorophenyl)-4-hydroxypiperidin-1-yl]chroman-4,7-diol ((+/-)CP-283,097), 1.8; (+/-)-(R*, S*)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperidine propanol ((+/-)Ro 25-6981), 1.0; ifenprodil, 6.0. The glycine site agonist D-serine stimulated binding to 151% of control with an ED(50) of 1.7 mg/kg. Results show that [3H]MK-801 binding in vivo may be used to measure receptor occupancy of ligands acting not only within the ion channel but also at modulatory sites on the NMDA receptor complex.

  18. Purification of neuropathy target esterase from avian brain after prelabelling with [3H]diisopropyl phosphorofluoridate.

    PubMed

    Rüffer-Turner, M E; Read, D J; Johnson, M K

    1992-01-01

    Neuropathy target esterase from hen brains was radiolabelled at the active site with [3H]diisopropyl phosphorofluoridate. The labelled protein was purified by differential centrifugation and Nonidet P40 solubilization, detergent phase partitioning, anion exchange, and preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The volatilizable counts assay and analytical SDS-PAGE were used to monitor the protein. The 150-kDa subunit polypeptide appears as a single band on analytical SDS-PAGE.

  19. Characterization of (/sup 3/H)nifedipine binding to intact vascular smooth muscle cells

    SciTech Connect

    Sumimoto, K.; Hirata, M.; Kuriyama, H.

    1988-01-01

    Specific binding of the dihydropyridine Ca2+ antagonist (/sup 3/H)nifedipine to dispersed smooth muscle cells of the porcine coronary artery was investigated and the findings were compared with the binding to microsomes of smooth muscles. Specific binding to intact cells was saturable and reversible. The dissociation constant was 1.93 +/- 0.42 nM and the maximal binding capacity was 59.6 +/- 12.4 fmol/10(6) cells, as assessed by Scatchard analysis of the equilibrium binding at 25 degrees C. The Kd value with intact cells was slightly higher than that observed with microsomes. Specific binding of (/sup 3/H)nifedipine to intact cells was completely displaced by unlabeled dihydropyridine derivatives. Among other Ca2+ antagonists, verapamil and d-cis-diltiazem partially and flunarizine completely inhibited the binding. In the case of microsomes, d-cis-diltiazem stimulated the binding of (/sup 3/H)nifedipine. These results suggest that there may be multiple binding sites for different subclasses of Ca2+ antagonists. Polyvalent cations had no effect on the binding to intact cells. In the case of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)-treated microsomes, the addition of CaCl/sub 2/ and BaCl/sub 2/ increased the Bmax, but the Kd value remained unchanged. MnCl/sub 2/ and CdCl/sub 2/ had stimulatory or inhibitory effects, depending on the concentrations, whereas LaCl3 had no effect. The effect of membrane depolarization on the binding was also examined. When the intact cells were incubated in high (K+)o solution for 60 min, the Kd was lowered to 1.4 nM from the control value of 2.0 nM, thereby indicating that (/sup 3/H)nifedipine binds to Ca2+ channels, with a higher affinity, at depolarized states.

  20. Endotoxin-induced early gene expression in C3H/HeJ (Lpsd) macrophages.

    PubMed

    Manthey, C L; Perera, P Y; Henricson, B E; Hamilton, T A; Qureshi, N; Vogel, S N

    1994-09-15

    C3H/HeJ (Lpsd) macrophages have been shown to respond to certain LPSs, especially from rough mutant bacteria. C3H/OuJ (Lpsn) macrophages are induced by wild-type LPS, rough LPS, or lipid A to express many genes, including TNF-alpha, TNFR-2, IL-1 beta, IP-10, D3, and D8. C3H/HeJ macrophages failed to induce any of these genes when cultured with wild-type LPS or synthetic lipid A, even when pretreated with IFN-gamma. However, rough mutant Salmonella minnesota Ra, Rc, and Rd LPS, and Escherichia coli D31 m3 Rd LPS induced Lpsd macrophages to express a subset of genes within the gene panel. Because bioactive preparations contained trace quantities of endotoxin protein(s), a deoxycholate-modified, phenol-water method was used to repurify rough LPS into an aqueous phase, and extract endotoxin proteins into a phenol phase. Repurified LPS failed to stimulate Lpsd macrophages; however, phenol fractions were approximately 10% as potent in Lpsd macrophages as crude rough LPS. Full potency was restored in C3H/HeJ macrophages when aqueous phase LPS and phenol-phase proteins were co-precipitated, suggesting that LPS and endotoxin proteins interact synergistically. Endotoxin proteins alone induced TNF-alpha, TNFR-2, and IL-1 beta, but not IP-10, D3, and D8 genes in both Lpsd and Lpsn macrophages. Tyrosine phosphorylation of three 41- to 47-kDa proteins was induced by endotoxin proteins, but not by LPS, in Lpsd macrophages. Thus, endotoxin proteins seem to activate a signaling pathway(s) that converges (distal to the Lps gene product) with a subset of LPS-signaling pathways.

  1. Quantitative tritium exchange of (/sup 3/H) aflatoxin B1 during penetration through isolated human skin

    SciTech Connect

    Riley, R.T.; Kemppainen, B.W.; Norred, W.P.

    1988-05-31

    Tritium labelled aflatoxin B1 ((G-/sup 3/H)AFB1) underwent an almost total tritium exchange with water during penetration through isolated human skin. The process was not enzymatic and the site of exchange appeared to be within the epidermis. The mechanism which mediated this extensive exchange was not determined. However, the tritium in (G-/sup 3/H)AFB1 was found to be very susceptible to chemical conditions which favored carbanion formation at the alpha-carbon of the cyclopentenone ring. The relative effectiveness of the various solvents in mediating the loss of the tritium label was 1 N/sub 3/NaOH much greater than methanol greater than 1 N HCl greater than water. This work serves as a warning that (G-/sup 3/H)AFB1 can easily undergo significant changes in specific activity in biological tissues and under relatively mild experimental conditions. It is possible that conditions within the skin favor carbanion formation.

  2. [3H]Methotrexate as a ligand for the folate receptor of Dictyostelium discoideum.

    PubMed Central

    Nandini-Kishore, S G; Frazier, W A

    1981-01-01

    Studies of the folate chemotactic receptor of vegetative Dictyostelium discoideum cells have been hampered by the presence of the degradative enzyme folate deaminase. The diaminopterin compounds aminopterin and methotrexate (MTX) are chemoattractants but are not attacked by the deaminase. [3',5',7,9-3H]methotrexate ([3H]MTX) is a nondegraded radioligand for the folate receptor. Binding to the receptor is rapid, reaching steady state in less than one min, and reversible in less than 15 s by an excess of unlabeled MTX. A single class of binding sites is found with a Kd of 2 x 10(-8) M, which correlates well with the concentration dependence of chemotaxis. Folate, aminopterin, and MTX all compete for [3H]MTX binding, whereas pterin, p-aminobenzoate, and nucleotides do not. Analysis of the receptor during differentiation indicates a decrease in site number by a factor of 3 with no change in affinity during the first 7 hr. During this time, the directional response (chemotaxis) to MTX and folate is lost, but a nondirectional stimulation of motility rate (chemokinesis) is retained. The response to cyclic AMP displays reciprocal behavior, first appearing as a chemokinetic response and then as a chemotactic response. PMID:6278468

  3. Immunopathology of experimental autoallergic sialadenitis in C3H/He mice.

    PubMed Central

    Hayashi, Y; Hirokawa, K

    1989-01-01

    We have shown that autoallergic sialadenitis develops in C3H/He (H-2k) mice thymectomized 3 days after birth and then immunized at 4 or 6 weeks of age with a homogenate of the submandibular salivary gland emulsified in Freund's complete adjuvant. Significant inflammatory changes did not develop in other inbred strains, such as BALB/c (H-2d), and C57BL/6 (H-2b) mice, examined by the same experimental protocol, or in the control groups, i.e. animals thymectomized at day 3 but not immunized, and animals not thymectomized but immunized. The cellular infiltrates observed in C3H/He mice with sialadenitis consisted of small and medium-sized lymophocytes stained with anti-Thy-1.2 antibody (the major proportion positive with anti-L3T4 and the lesser, with anti-Lyt 2). Anti-salivary duct antibodies were detected frequently in the sera of the C3H/He mice with sialadenitis. Images Fig. 1 PMID:2784749

  4. ( sup 3 H)QNB binding and contraction of rabbit colonic smooth muscle cells

    SciTech Connect

    Ringer, M.J.; Hyman, P.E.; Kao, H.W.; Hsu, C.T.; Tomomasa, T.; Snape, W.J. Jr. )

    1987-11-01

    The authors used radioligand binding and studies of cell contraction to characterize muscarinic receptors on dispersed smooth muscle cells from rabbit proximal and distal colon. Cells obtained after serial incubations in collagenase were used to measure binding of tritiated quinuclidinyl benzilate (({sup 3}H)QNB). At 37{degree}C, specific ({sup 3}H)QNB binding was saturable and linearly related to cell number. Nonlinear regression analysis was used to determine the affinity of ({sup 3}H)QNB for its receptor. The IC{sub 50} for the muscarinic agonists bethanechol and oxotremorine were 80 and 0.57 {mu}M, respectively. Hill coefficients were 0.67 for both, suggesting more complex interaction involving receptors of different affinities. In studies of cell contraction, bethanechol stimulated a dose-dependent decrease in cell length with half the maximal contraction occurring at 100 pM. These results suggest that (1) contraction is mediated by binding of bethanechol to M{sub 2}-muscarinic receptors and that (2) there are a large number of spare receptors in colonic smooth muscle.

  5. Partial purification of the mu opioid receptor irreversibly labeled with (/sup 3/H)b-funaltrexamine

    SciTech Connect

    Liu-Chen, L.Y.; Phillips, C.A.; Tam, S.W.

    1986-03-01

    The mu opioid receptor in bovine striatal membranes was specifically and irreversibly labeled by incubation with 5 nM (/sup 3/H)..beta..-funaltrexamine (approx.-FNA) at 37/sup 0/C for 90 min in the presence of 100 mM NaCl. The specific and irreversible binding of (/sup 3/H)..beta..-FNA as defined by that blocked by 1 /sup +/M naloxone was about 60% of total irreversible binding. The specific irreversible binding was saturable, stereospecific, time-, temperature, and tissue-dependent. Mu opioid ligands were much more potent than delta or kappa ligands in inhibiting the specific irreversible labeling. SDS polyacrylamide gel electrophoresis of solubilized membranes in the presence of 2-mercaptoethanol yielded a major radiolabeled broad band of MW 68-97K daltons, characteristic of a glycoprotein band. This band was not observed in membranes labeled in the presence of excess unlabeled naloxone. The glycoprotein nature of the (/sup 3/H)..beta..-FNA-labeled opioid receptor was confirmed by its binding to a wheat germ agglutinin-Sepharose column and its elution with N-acetylglucosamine.

  6. High affinity binding of (/sup 3/H)neurotensin of rat uterus

    SciTech Connect

    Pettibone, D.J.; Totaro, J.A.

    1987-11-01

    (/sup 3/H)Neurotensin (NT) was found to bind specifically and with high affinity to crude membranes prepared from rat uterus. Scatchard analysis of saturation binding studies indicated that (/sup 3/H)NT apparently binds to two sites (high affinity Kd 0.5 nM; low affinity Kd 9 nM) with the density of high affinity sites (41 fmoles/mg prot.) being about one-third that of the low affinity sites (100 fmoles/mg prot.). In competition studies, NT and various fragments inhibited (/sup 3/H)NT binding with the following potencies (approximately IC50): NT 8-13 (0.4 nM), NT 1-13 (4 nM), NT 9-13 (130 nM), NT 1-11, NT 1-8 (greater than 100 microM). Quantitatively similar results were obtained using brain tissue. These findings raise the possibility of a role for NT in uterine function.

  7. Absolute cross section for the reaction /sup 3/H(p,. gamma. /sub 0/)/sup 4/He and a review of /sup 4/He(. gamma. , p/sub 0/)/sup 3/H measurements

    SciTech Connect

    Calarco, J.R.; Hanna, S.S.; Chang, C.C.; Diener, E.M.; Kuhlmann, E.; Fisher, G.A.

    1983-08-01

    Accurate differential cross sections have been measured at 90/sup 0/ for the reaction /sup 3/H(p,..gamma..)/sup 4/He at E/sub p/ = 8.34 and 13.6 MeV. Previously published results for both /sup 3/H(p,..gamma..)/sup 4/He and /sup 4/He(..gamma..,p)/sup 3/H are reviewed and compared with the present data. The theoretical implications of the results are briefly discussed.

  8. Photochemical Modeling of the Distribution of C3H8 in the Atmosphere of Saturn

    NASA Astrophysics Data System (ADS)

    Edgington, S. G.; Simon-Miller, A.; Jennings, D.; Bjoraker, G.; Romani, P.; Achterberg, R.; Orton, G.; Flasar, M.; Cassini CIRS Team

    2005-08-01

    Cassini's Composite Infrared Spectrometer (CIRS) has measured the abundance of C2H2 and C3H8 (Propane) at several latitudes in the Southern hemisphere. An increase of radiance with latitude towards the pole has been observed, possibly implying a corresponding increase of C3H8. In an effort explain the observed distribution of both species, it is important to model the creation, destruction, and transport of these chemical species. Furthermore, since both molecules have overlapping absorption features in the same spectral region near 748 cm-1, such modeling will aid in refining derived abundances and separating temperature effects. The photochemistry model used in Edgington et al. (1998, 1999, 2000) to model simultaneously hydrocarbons, ammonia, and phosphine is updated and expanded to include paths relevant to the creation of C3H8. Destruction occurs through photolysis, while transport would tend to spread C3H8 from its source regions. With a series of exercises in 1- and 2- dimensions, we explore the extent to which photolysis, vertical, and/or meridional transport impacts the distribution of C2H2 and C3H8 with latitude. Thermal profiles derived from CIRS observations versus latitude are used as they have an impact on numerous reaction rates. We then compare these results with abundances derived from observations taken with the CIRS instrument. Edgington, S.G., West, R.A., Friedson, A.J., Atreya, S.K., 2000. A 2-D photochemical model with meridional circulation. Bull. American. Astron. Soc., 32, 1013. Edgington, S.G., S.K. Atreya, L.M. Trafton, J.J. Caldwell, R.F. Beebe, A.A. Simon, and R.A. West, 1999. Ammonia and eddy mixing variations in the southern hemisphere of Jupiter from HST Faint Object Spectrograph Observations. Icarus, 142, 342-357. Edgington, S.G., S.K. Atreya, L.M. Trafton, J.J. Caldwell, R.F. Beebe, A.A. Simon, R.A. West, and C. Barnet, 1998. On the latitude variation of ammonia, acetylene, and phosphine altitude profiles on Jupiter from HST Faint

  9. Characterization of high affinity (/sup 3/H)triazolam binding in rat brain

    SciTech Connect

    Earle, M.; Concas, A.; Yamamura, H.I.

    1986-03-01

    The hypnotic Triazolam (TZ), a triazolo (1,4)-benzodiazepine, displays a short physiological half life and has been used for the treatment of insomnia related to anxiety states. Specific binding properties of this recently tritiated TZ were characterized. The authors major objectives were the direct measurement of the temperature dependence and the GABA effect on (/sup 3/H)TZ binding. Saturation studies showed a shift to lower affinity at 37/sup 0/C (K/sub d/ = 0.25 +/- 0.01 nM at O/sup 0/C; K/sub d/ = 1.46 +/- 0.03 nM at 37/sup 0/C) while the B/sub max/ values remained unchanged (1003 +/- 37 fmoles/mg prot. at 0/sup 0/C and 1001 +/- 43 fmoles/mg prot. at 37/sup 0/C). Inhibition studies showed that (/sup 3/H)TZ binding displayed no GABA shift at 0/sup 0/C(K/sub i/ 0.37 +/- 0.03 nM/- GABA and K/sub i/ = 0.55 +/- 0.13 nM/+GABA) but a nearly two-fold shift was apparent at 37/sup 0/C (K/sub i/ = 2.92 +/- 0.2 nM/-GABA; K/sub i/ = 1.37 +/- 0.11 mM/+GABA). These results were also confirmed by saturation studies in the presence or absence of GABA showing a shift to higher affinity in the presence of GABA only at 37/sup 0/C. In Ro 15-1788/(/sup 3/H)TZ competition experiments the presence of GABA did not affect the inhibitory potency of Ro 15-1788 on (/sup 3/H)TZ binding at both temperatures. In conclusion (/sup 3/H)TZ binding showed an extremely high affinity for benzodiazepine receptors. In contrast to reported literature, the findings suggest that TZ interacts with benzodiazepine receptors similar to other benzodiazepine agonists.

  10. Differential effect of detergents on (/sup 3/H)Ro 5-4864 and (/sup 3/H)PK 11195 binding to peripheral-type benzodiazepine-binding sites

    SciTech Connect

    Awad, M.; Gavish, M.

    1988-01-01

    The present study demonstrates a differential effect of various detergent treatments on (/sup 3/H)Ro 5-4864 and (/sup 3/H)PK 11195 binding to peripheral benzodiazepine binding sites (PBS). Triton X-100 caused a decrease of about 70% in (/sup 3/H)Ro 5-4864 binding to membranes from various peripheral tissues of rat, but had only a negligible effect on (/sup 3/H)PK 11195 binding. A similar effect of Triton X-100 was observed on guinea pig and rabbit kidney membranes. The decrease in (/sup 3/H)Ro 5-4864 binding after treatment with Triton X-100 was apparently due to a decrease in the density of PBS, since the affinity remained unaltered. The detergents 3-((3-cholamidopropyl)-dimethylammonio)-1-propane sulfonate (CHAPS), Tween 20, deoxycholic acid, or digitonin (0.0125%) caused only a minor change in (/sup 3/H)Ro 5-4864 and (/sup 3/H)PK 11195 binding to rat kidney membranes; but when concentrations were substantially increased (0.1%), all detergents caused a decrease of at least 50% in (/sup 3/H)Ro 5-4864 binding, while (/sup 3/H)PK 11195 binding to rat kidney membranes remained unaffected by the first three detergents, with only a minor decrease (15%) after treatment with digitonin.

  11. Sequences contained within the promoter of the human thymidine kinase gene can direct cell-cycle regulation of heterologous fusion genes.

    PubMed Central

    Kim, Y K; Wells, S; Lau, Y F; Lee, A S

    1988-01-01

    Recent evidence on the transcriptional regulation of the human thymidine kinase (TK) gene raises the possibility that cell-cycle regulatory sequences may be localized within its promoter. A hybrid gene that combines the TK 5' flanking sequence and the coding region of the bacterial neomycin-resistance gene (neo) has been constructed. Upon transfection into a hamster fibroblast cell line K12, the hybrid gene exhibits cell-cycle-dependent expression. Deletion analysis reveals that the region important for cell-cycle regulation is within -441 to -63 nucleotides from the transcriptional initiation site. This region (-441 to -63) also confers cell-cycle regulation to the herpes simplex virus thymidine kinase (HSVtk) promoter, which is not expressed in a cell-cycle manner. We conclude that the -441 to -63 sequence within the human TK promoter is important for cell-cycle-dependent expression. Images PMID:3413063

  12. Sequences contained within the promoter of the human thymidine kinase gene can direct cell-cycle regulation of heterologous fusion genes

    SciTech Connect

    Kim, Yongkyu; Wells, S.; Lau, Yunfai Chris; Lee, A.S. )

    1988-08-01

    Recent evidence on the transcriptional regulation of the human thymidine kinase (TK) gene raises the possibility that cell-cycle regulatory sequences may be localized within its promoter. A hybrid gene that combines the TK 5{prime} flanking sequence and the coding region of the bacterial neomycin-resistance gene (neo) has been constructed. Upon transfection into a hamster fibroblast cell line K12, the hybrid gene exhibits cell-cycle-dependent expression. Deletion analysis reveals that the region important for cell-cycle regulation is within {minus}441 to {minus}63 nucleotides from the transcriptional initiation site. This region ({minus}441 to {minus}63) also confers cell-cycle regulation to the herpes simplex virus thymidine kinase (HSVtk) promoter, which is not expressed in a cell-cycle manner. The authors conclude that the {minus}441 to {minus}63 sequence within the human TK promoter is important for cell-cycle-dependent expression.

  13. PET imaging of oncolytic VSV expressing the mutant HSV-1 thymidine kinase transgene in a preclinical HCC rat model.

    PubMed

    Muñoz-Álvarez, Kim A; Altomonte, Jennifer; Laitinen, Iina; Ziegler, Sibylle; Steiger, Katja; Esposito, Irene; Schmid, Roland M; Ebert, Oliver

    2015-04-01

    Hepatocellular carcinoma (HCC) is the most predominant form of liver cancer and the third leading cause of cancer-related death worldwide. Due to the relative ineffectiveness of conventional HCC therapies, oncolytic viruses have emerged as novel alternative treatment agents. Our previous studies have demonstrated significant prolongation of survival in advanced HCC in rats after oncolytic vesicular stomatitis virus (VSV) treatment. In this study, we aimed to establish a reporter system to reliably and sensitively image VSV in a clinically relevant model of HCC for clinical translation. To this end, an orthotopic, unifocal HCC model in immune-competent Buffalo rats was employed to test a recombinant VSV vector encoding for an enhanced version of the herpes simplex virus 1 (HSV-1) thymidine kinase (sr39tk) reporter, which would allow the indirect detection of VSV via positron emission tomography (PET). The resulting data revealed specific tracer uptake in VSV-HSV1-sr39tk-treated tumors. Further characterization of the VSV-HSV1-sr39tk vector demonstrated its optimal detection time-point after application and its detection limit via PET. In conclusion, oncolytic VSV expressing the HSV1-sr39tk reporter gene allows for highly sensitive in vivo imaging via PET. Therefore, this imaging system may be directly translatable and beneficial in further clinical applications.

  14. Serum thymidine kinase 1 and C-reactive protein as biomarkers for screening clinically healthy dogs for occult disease.

    PubMed

    Selting, K A; Sharp, C R; Ringold, R; Knouse, J

    2015-12-01

    Thymidine kinase (TK1) is a biomarker that correlates well with diagnosis and prognosis in certain canine cancers. Canine C-reactive protein (cCRP) is a widely accepted marker of inflammation correlated with increased risk and severity of various diseases. We evaluated serum TK1 and cCRP concentrations in apparently healthy dogs (n = 360). All dogs were followed up for a minimum of 6 months by health questionnaire. All dogs with cancer were identified using a proprietary dual-biomarker algorithm [termed Neoplasia Index (NI)]. Specificity of positive NI is 0.91 and high positive is 0.98. All-cause mortality was 20% in dogs with elevated cCRP and 3% in dogs with low cCRP. The performance of serum TK1 and cCRP as tools for screening for occult cancer is improved when evaluated together. Serum TK1 and cCRP (unified in the NI) are useful in the screening of occult canine cancer. cCRP is useful in screening for other serious diseases.

  15. Thymidine kinase/ganciclovir and cytosine deaminase/5-fluorocytosine suicide gene therapy-induced cell apoptosis in breast cancer cells.

    PubMed

    Kong, H; Tao, L; Qi, K; Wang, Y; Li, Q; Du, J; Huang, Z

    2013-09-01

    The present study was conducted to explore the efficacy of suicide gene therapy with thymidine kinase (TK) in combination with cytosine deaminase (CD) for breast cancer. The expression of CD/TK was detected in the infected cells by RT-PCR. The killing effect on MCF-7 cells following treatment was analyzed by MTT assay. The morphological characteristics of the cells were observed by electron microscopy, and the distribution of the cell cycle was analyzed by flow cytometry. Caspase‑3 and -8 activities were detected by absorption spectrometry. Cytotoxic assays showed that cells transfected with CD/TK became more sensitive to the prodrugs. Morphological features characteristic of apoptosis were noted in the MCF‑7 cells via electron microscopy. The experimental data showed that the proportion of MCF-7 cells during the different phases of the cell cycle varied significantly following treatment with the prodrugs. The activity of caspase‑3 gradually increased following treatment with increasing concentrations of the prodrugs. We conclude that the TK/ganciclovir and CD/5-fluorocytosine suicide gene system used here induces apoptosis in breast cancer cells, and provides a promising treatment modality for breast cancer.

  16. The crystal structure of ribonuclease A in complex with thymidine-3'-monophosphate provides further insight into ligand binding.

    PubMed

    Doucet, Nicolas; Jayasundera, Thusitha B; Simonović, Miljan; Loria, J Patrick

    2010-08-15

    Thymidine-3'-monophosphate (3'-TMP) is a competitive inhibitor analogue of the 3'-CMP and 3'-UMP natural product inhibitors of bovine pancreatic ribonuclease A (RNase A). Isothermal titration calorimetry experiments show that 3'-TMP binds the enzyme with a dissociation constant (K(d)) of 15 microM making it one of the strongest binding members of the five natural bases found in nucleic acids (A, C, G, T, and U). To further investigate the molecular properties of this potent natural affinity, we have determined the crystal structure of bovine pancreatic RNase A in complex with 3'-TMP at 1.55 A resolution and we have performed NMR binding experiments with 3'-CMP and 3'-TMP. Our results show that binding of 3'-TMP is very similar to other natural and non-natural pyrimidine ligands, demonstrating that single nucleotide affinity is independent of the presence or absence of a 2'-hydroxyl on the ribose moiety of pyrimidines and suggesting that the pyrimidine binding subsite of RNase A is not a significant contributor of inhibitor discrimination. Accumulating evidence suggests that very subtle structural, chemical, and potentially motional variations contribute to ligand discrimination in this enzyme.

  17. In vivo measurement of carbon-11 thymidine uptake in non-Hodgkin's lymphoma using positron emission tomography

    SciTech Connect

    Martiat, P.; Ferrant, A.; Labar, D.; Cogneau, M.; Bol, A.; Michel, C.; Michaux, J.L.; Sokal, G.

    1988-10-01

    Carbon-11 thymidine (TdR) uptake using positron emission tomography (PET) has been measured in ten patients with non-Hodgkin's lymphoma (NHL). The rate of TdR uptake (mean +/- s.d.) was of 0.009 +/- 0.006 mumol.100 cc-1.min-1 in low-grade NHL. This rate was 0.063 +/- 0.049 mumol.100 cc-1.min-1 in intermediate-grade NHL and 0.159 mumol.100 cc-1.min-1 in a patient with high-grade NHL. Lymphoma radioactivity reached a plateau at 0.42 +/- 0.22%. 100 cc-1 of the injected dose from 10 min after injection. The highest /sup 11/C uptakes were observed in the kidneys and in the liver (3.30 +/- 1.30 and 2.10 +/- 0.05%. 100 cc-1 of the injected dose, respectively). The lymphoma-to-muscle ratio was of 11.8 +/- 1.7, whereas the lymphoma-to-intestine ratio was of 1.5 +/- 0.7. Accordingly, the measurement of (/sup 11/C)TdR uptake in the abdomen may need other imaging methods for adequate interpretation. The results suggest that (/sup 11/C)TdR uptake using PET might be a method for noninvasively measuring cell proliferation in vivo.

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

    PubMed

    Sanford, B; Holinka, L G; O'Donnell, V; Krug, P W; Carlson, J; Alfano, M; Carrillo, C; Wu, Ping; Lowe, Andre; Risatti, G R; Gladue, D P; Borca, M V

    2016-02-02

    African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal viral disease of domestic pigs. There are no vaccines to control Africa swine fever (ASF). Experimental vaccines have been developed using genetically modified live attenuated ASFVs obtained by specifically deleting virus genes involved in virulence, including the thymidine kinase (TK) gene. TK has been shown to be involved in the virulence of several viruses, including ASFV. Here we report the construction of a recombinant virus (ASFV-G/V-ΔTK) obtained by deleting the TK gene in a virulent strain of ASFV Georgia adapted to replicate in Vero cells (ASFV-G/VP30). ASFV-G/P-ΔTK demonstrated decreased replication both in primary swine macrophage cell cultures and in Vero cells compared with ASFV-G/VP30. In vivo, intramuscular administration of up to 10(6) TCID50 of ASFV-G/V-ΔTK does not result in ASF disease. However, these animals are not protected when challenged with the virulent parental Georgia strain.

  19. Monitoring of bystander effect of herpes simplex virus thymidine kinase/acyclovir system using fluorescence resonance energy transfer technique.

    PubMed

    Xiong, Tao; Li, Yongjun; Ni, Fenge; Zhang, Feng

    2012-02-01

    Cytotoxic gene therapy mediated by gene transfer of the herpes simplex virus thymidine kinase (HSV-tk) gene followed by acyclovir (ACV) treatment has been reported to inhibit malignant tumor growth in a variety of studies. The magnitude of "bystander effect" is an essential factor for this anti-tumor approach in vivo. However, the mechanism by which HSV-tk/ACV brings "bystander effect" is poorly understood. In this report, the plasmid CD3 (ECFP-CRS-DsRed) and TK-GFP were transferred to the human adenoid cystic carcinoma line ACC-M cell line. The CD3-expressing cells apoptosis was monitored using fluorescence resonance energy transfer (FRET) technique. First, CD3 and TK-GFP co-expressing ACC-M cells apoptosis was monitored using FRET technique. The apoptosis was induced by ACV and initiated by caspase3. The FRET efficient was remarkably decreased and then disappeared during cellular apoptosis, which indicated that the TK-GFP expressing ACC-M cells apoptosis, induced by ACV, was via a caspase3-dependent pathway. Secondly, CD3 and TK-GFP mixed expressing ACC-M cells apoptosis, induced by ACV, were monitored using FRET technique. The apoptotic phenomena appeared in the CD3-expressing ACC-M cells. The results show that HSV-tk/ACV system killed ACC-M cells using its bystander effect. These results confirm that HSV-tk/ACV system is potential for cancer gene therapy.

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

    NASA Astrophysics Data System (ADS)

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

    2016-11-01

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

  1. Natural Polymorphisms and Oligomerization of Human APOBEC3H Contribute to Single-stranded DNA Scanning Ability.

    PubMed

    Feng, Yuqing; Love, Robin P; Ara, Anjuman; Baig, Tayyba T; Adolph, Madison B; Chelico, Linda

    2015-11-06

    APOBEC3H is a deoxycytidine deaminase that can restrict the replication of HIV-1 in the absence of the viral protein Vif that induces APOBEC3H degradation in cells. APOBEC3H exists in humans as seven haplotypes (I-VII) with different cellular stabilities. Of the three stable APOBEC3H haplotypes (II, V, and VII), haplotypes II and V occur most frequently in the population. Despite APOBEC3H being a bona fide restriction factor, there has been no comparative biochemical characterization of APOBEC3H haplotypes. We characterized the ssDNA scanning mechanisms that haplotypes II and V use to search their ssDNA substrate for cytosine-containing deamination motifs. APOBEC3H haplotype II was able to processively deaminate multiple cytosines in a single enzyme-substrate encounter by using sliding, jumping, and intersegmental transfer movements. In contrast, APOBEC3H haplotype V exhibited diminished sliding and intersegmental transfer abilities but was able to jump along ssDNA. Due to an Asp or Glu at amino acid 178 differentiating these APOBEC3H haplotypes, the data indicated that this amino acid on helix 6 contributes to processivity. The diminished processivity of APOBEC3H haplotype V did not result in a reduced efficiency to restrict HIV-1 replication in single-cycle infectivity assays, suggesting a redundancy in the contributions of jumping and intersegmental transfer to mutagenic efficiency. Optimal processivity on ssDNA also required dimerization of APOBEC3H through the β2 strands. The findings support a model in which jumping can compensate for deficiencies in intersegmental transfer and suggest that APOBEC3H haplotypes II and V induce HIV-1 mutagenesis efficiently but by different mechanisms.

  2. Poplar PdC3H17 and PdC3H18 are direct targets of PdMYB3 and PdMYB21, and positively regulate secondary wall formation in Arabidopsis and poplar.

    PubMed

    Chai, Guohua; Qi, Guang; Cao, Yingping; Wang, Zengguang; Yu, Li; Tang, Xianfeng; Yu, Yanchong; Wang, Dian; Kong, Yingzhen; Zhou, Gongke

    2014-07-01

    Wood biomass is mainly made of secondary cell walls, whose formation is controlled by a multilevel network. The tandem CCCH zinc finger (TZF) proteins involved in plant secondary wall formation are poorly understood. Two TZF genes, PdC3H17 and PdC3H18, were isolated from Populus deltoides and functionally characterized in Escherichia coli, tobacco, Arabidopsis and poplar. PdC3H17 and PdC3H18 are predominantly expressed in cells of developing wood, and the proteins they encode are targeted to cytoplasmic foci. Transcriptional activation assays showed that PdMYB2/3/20/21 individually activated the PdC3H17 and PdC3H18 promoters, but PdMYB3/21 were most significant. Electrophoretic mobility shift assays revealed that PdMYB3/21 bound directly to the PdC3H17/18 promoters. Overexpression of PdC3H17/18 in poplar increased secondary xylem width and secondary wall thickening in stems, whereas dominant repressors of them had the opposite effects on these traits. Similar alteration in secondary wall thickening was observed in their transgenic Arabidopsis plants. qRT-PCR results showed that PdC3H17/18 regulated the expression of cellulose, xylan and lignin biosynthetic genes, and several wood-associated MYB genes. These results demonstrate that PdC3H17 and PdC3H18 are the targets of PdMYB3 and PdMYB21 and are an additional two components in the regulatory network of secondary xylem formation in poplar.

  3. (/sup 3/H)adrenaline release from hypothalamic synaptosomes and its modulation by clonidine: effects of chronic antidepressant drug regimens

    SciTech Connect

    McWilliam, J.R.; Campbell, I.C.

    1987-07-13

    (/sup 3/H)Adrenaline ((/sup 3/H)ADR, 40nM) was accumulated by rat hypothalamic synaptosomes (P/sub 2/) more rapidly and in significantly greater amounts than by similar preparations from cerebral cortex. There was no significant difference between these two tissues in the rate or amount of (/sup 3/H)noradrenaline ((/sup 3/H)NA, 40nM) accumulation. Talusupram (10..mu..M), maximally inhibited the uptake of (/sup 3/H)ADR into hypothalamic synaptosomes by 60%. Nomifensine further inhibited uptake by 14%. From these observations it was concluded that some (/sup 3/H)ADR was accumulated into non adrenergic neuronal terminals. The effects of desipramine (DMI, 10mg/kg/day and clorgyline (1mg/kg/day) administration for 28 days on K/sup +/-evoked release of (/sup 3/H)ADR was investigated using superfused hypothalamic synaptosomes. After both chronic antidepressant drug regimens, total (/sup 3/H)ADR release (spontaneous + evoked) was significantly reduced. Evoked release of (numberH)ADR (by KCl, 16mM) was significantly reduced after the DMI but not the clorgyline regimens. Presynaptic ..cap alpha../sub 2/-adrenoceptor function in the hypothalamus was assessed during superfusion by measuring the reduction in /sup +/-evoked release of (/sup 3/H)ADR caused by clonidine (1/sup +/M). 30 references, 3 figures, 1 table.

  4. Stereoselective L-(3H)quinuclidinyl benzilate-binding sites in nervous tissue of Aplysia californica: evidence for muscarinic receptors

    SciTech Connect

    Murray, T.F.; Mpitsos, G.J.; Siebenaller, J.F.; Barker, D.L.

    1985-12-01

    The muscarinic antagonist L-(/sup 3/H)quinuclidinyl benzilate (L-(/sup 3/H)QNB) binds with a high affinity (Kd = 0.77 nM) to a single population of specific sites (Bmax = 47 fmol/mg of protein) in nervous tissue of the gastropod mollusc, Aplysia. The specific L-(/sup 3/H)QNB binding is displaced stereoselectively by the enantiomers of benzetimide, dexetimide, and levetimide. The pharmacologically active enantiomer, dexetimide, is more potent than levetimide as an inhibitor of L-(/sup 3/H)QNB binding. Moreover, the muscarinic cholinergic ligands, scopolamine, atropine, oxotremorine, and pilocarpine are effective inhibitors of the specific L-(/sup 3/H)QNB binding, whereas nicotinic receptor antagonists, decamethonium and d-tubocurarine, are considerably less effective. These pharmacological characteristics of the L-(/sup 3/H)QNB-binding site provide evidence for classical muscarinic receptors in Aplysia nervous tissue. The physiological relevance of the dexetimide-displaceable L-(/sup 3/H)QNB-binding site was supported by the demonstration of the sensitivity of the specific binding to thermal denaturation. Specific binding of L-(/sup 3/H)QNB was also detected in nervous tissue of another marine gastropod, Pleurobranchaea californica. The characteristics of the Aplysia L-(/sup 3/H)QNB-binding site are in accordance with studies of numerous vertebrate and invertebrate tissues indicating that the muscarinic cholinergic receptor site has been highly conserved through evolution.

  5. The DNA cytosine deaminase APOBEC3H haplotype I likely contributes to breast and lung cancer mutagenesis.

    PubMed

    Starrett, Gabriel J; Luengas, Elizabeth M; McCann, Jennifer L; Ebrahimi, Diako; Temiz, Nuri A; Love, Robin P; Feng, Yuqing; Adolph, Madison B; Chelico, Linda; Law, Emily K; Carpenter, Michael A; Harris, Reuben S

    2016-09-21

    Cytosine mutations within TCA/T motifs are common in cancer. A likely cause is the DNA cytosine deaminase APOBEC3B (A3B). However, A3B-null breast tumours still have this mutational bias. Here we show that APOBEC3H haplotype I (A3H-I) provides a likely solution to this paradox. A3B-null tumours with this mutational bias have at least one copy of A3H-I despite little genetic linkage between these genes. Although deemed inactive previously, A3H-I has robust activity in biochemical and cellular assays, similar to A3H-II after compensation for lower protein expression levels. Gly105 in A3H-I (versus Arg105 in A3H-II) results in lower protein expression levels and increased nuclear localization, providing a mechanism for accessing genomic DNA. A3H-I also associates with clonal TCA/T-biased mutations in lung adenocarcinoma suggesting this enzyme makes broader contributions to cancer mutagenesis. These studies combine to suggest that A3B and A3H-I, together, explain the bulk of 'APOBEC signature' mutations in cancer.

  6. Preferential affinity of /sup 3/H-2-oxo-quazepam for type I benzodiazepine recognition sites in the human brain

    SciTech Connect

    Corda, M.G.; Giorgi, O.; Longoni, B.; Ongini, E.; Montaldo, S.; Biggio, G.

    1988-01-01

    The hypnotic drug quazepam and its active metabolite 2-oxo-quazepam (2-oxo-quaz) are two benzodiazepines (BZ) containing a trifluoroethyl moiety on the ring nitrogen at position 1, characterized by their preferential affinity for Type I BZ recognition sites. In the present study we characterized the binding of /sup 3/H-2-oxo-quaz in discrete areas of the human brain. Saturation analysis demonstrated specific and saturable binding of /sup 3/H-2-oxo-quaz to membrane preparations from human cerebellum. Hill plot analysis of displacement curves of /sup 3/H-flunitrazepam binding by 2-oxo-quaz yielded Hill coefficients of approximately 1 in the cerebellum and significantly less than 1 in the cerebral cortex, hippocampus, caudate nucleus, thalamus and pons. Self and cross displacement curves for /sup 3/H-FNT and /sup 3/H-2-oxo-quaz binding in these brain areas indicated that 2-oxo-quaz binds with different affinities to two populations of binding sites. High affinity binding sites were more abundant in the cerebellum, cerebral cortex, hippocampus and thalamus, whereas low affinity sites were predominant in the caudate nucleus and pons. Competition studies of /sup 3/H-2-oxo-quaz and /sup 3/H-FNT using unlabelled ligands indicated that compounds which preferentially bind to Type I sites are more potent at displacing /sup 3/H-2-oxo-quaz than /sup 3/H-FNT from cerebral cortex membrane preparations. 26 references, 2 figures, 3 tables.

  7. The DNA cytosine deaminase APOBEC3H haplotype I likely contributes to breast and lung cancer mutagenesis

    PubMed Central

    Starrett, Gabriel J.; Luengas, Elizabeth M.; McCann, Jennifer L.; Ebrahimi, Diako; Temiz, Nuri A.; Love, Robin P.; Feng, Yuqing; Adolph, Madison B.; Chelico, Linda; Law, Emily K.; Carpenter, Michael A.; Harris, Reuben S

    2016-01-01

    Cytosine mutations within TCA/T motifs are common in cancer. A likely cause is the DNA cytosine deaminase APOBEC3B (A3B). However, A3B-null breast tumours still have this mutational bias. Here we show that APOBEC3H haplotype I (A3H-I) provides a likely solution to this paradox. A3B-null tumours with this mutational bias have at least one copy of A3H-I despite little genetic linkage between these genes. Although deemed inactive previously, A3H-I has robust activity in biochemical and cellular assays, similar to A3H-II after compensation for lower protein expression levels. Gly105 in A3H-I (versus Arg105 in A3H-II) results in lower protein expression levels and increased nuclear localization, providing a mechanism for accessing genomic DNA. A3H-I also associates with clonal TCA/T-biased mutations in lung adenocarcinoma suggesting this enzyme makes broader contributions to cancer mutagenesis. These studies combine to suggest that A3B and A3H-I, together, explain the bulk of ‘APOBEC signature' mutations in cancer. PMID:27650891

  8. A rapid non-radioactive technique for measurement of repair synthesis in primary human fibroblasts by incorporation of ethynyl deoxyuridine (EdU).

    PubMed

    Limsirichaikul, Siripan; Niimi, Atsuko; Fawcett, Heather; Lehmann, Alan; Yamashita, Shunichi; Ogi, Tomoo

    2009-03-01

    Xeroderma pigmentosum (XP) is an autosomal recessive genetic disorder. Afflicted patients show extreme sun-sensitivity and skin cancer predisposition. XP is in most cases associated with deficient nucleotide excision repair (NER), which is the process responsible for removing photolesions from DNA. Measuring NER activity by nucleotide incorporation into repair patches, termed 'unscheduled DNA synthesis (UDS)', is one of the most commonly used assays for XP-diagnosis and NER research. We have established a rapid and accurate procedure for measuring UDS by replacement of thymidine with 5-ethynyl-2'-deoxyuridine (EdU). EdU incorporated into repair patches can be directly conjugated to fluorescent azide derivatives, thereby obviating the need for either radiolabeled thymidine or denaturation and antibody detection of incorporated bromodeoxyuridine (BrdU). We demonstrate that the EdU incorporation assay is compatible with conventional techniques such as immunofluorescent staining and labeling of cells with micro-latex beads. Importantly, we can complete the entire UDS assay within half a day from preparation of the assay coverslips; this technique may prove useful as a method for XP diagnosis.

  9. In vitro Characterization of Uptake Mechanism of L-[methyl-3H]-methionine in Hepatocellular Carcinoma

    PubMed Central

    Kuang, Yu; Wang, Fangjing; Corn, David J.; Tian, Haibin; Lee, Zhenghong

    2015-01-01

    MAT2A expression (the duration of SAM treatment). The incorporated 3H was mostly distributed in the protein phase and, to a lesser degree, in the lipid phase via PE methylation pathway in HCC cells with SAM treatment. The downregulated MAT2A expression led to the decreased uptake in protein and water-soluble phases. In addition, an increased uptake in the lipid phase was observed in WCH17 cells transfected with PEMT2 expression vector. Conclusions The amino acid transport processes may be responsible for the rapid accumulation of radiolabeled Met after the intravenous injection of tracers for the imaging of HCC. Upregulated MAT2A expression and impaired PEMT2 activities in HCC are associated with the specific metabolic pattern of L-[methyl-11C]-Met detected by PET. PMID:24488484

  10. Anomalous absorption in c-C3H and c-C3D radicals

    NASA Astrophysics Data System (ADS)

    Chandra, S.; Shinde, S. V.; Kegel, W. H.; Sedlmayr, E.

    2007-05-01

    Context: The c-C3H radical was first detected in TMC-1 by Yamamoto et al. (1987, ApJ, 322, L55), who observed the 2{12} → 1{11} transition at 91.5 GHz in emission. Mangum & Wootten (1990, A&A, 239, 319) observed the 1{10} → 1{11} transition at 14.8 GHz in emission in 12 additional galactic objects. Aims: The aim of this investigation is a quantitative estimate of relative line intensities under NLTE conditions. Methods: For wide ranges of physical parameters, where these molecules may be found, we have solved a set of statistical equilibrium equations coupled with the equations of radiative transfer in an on-the-spot approximation. For c-C3H, we accounted for 51 energy levels connected by 207 radiative transitions, and for c-C3D, we accounted for 51 energy levels connected by 205 radiative transitions. Results: Our results show that the 3{31} → 3{30} transition of c-C3H and c-C3D may be found in absorption against the cosmic microwave background (CMB). Furthermore, we found population inversion for the 1{10} → 1{11} transition. These findings may be useful in identifying these molecules in other cosmic objects, as well as for the determination of physical parameters in these objects. Tables 1-3 and Figs. 4, 5 are only available in electronic form at http://www.aanda.org

  11. Control by fibroblast growth factor of differentiation in the BC3H1 muscle cell line

    PubMed Central

    1985-01-01

    The regulation of creatine phosphokinase (CPK) expression by polypeptide growth factors has been examined in the clonal mouse muscle BC3H1 cell line. After arrest of cell growth by exposure to low concentrations of serum, BC3H1 cells accumulate high levels of muscle- specific proteins including CPK. The induction of this enzyme is reversible in the presence of high concentrations of fetal calf serum, which cause quiescent, differentiated cells to reenter the cell cycle. Under these conditions, the rate of CPK synthesis is drastically reduced. We show in the present communication that either pituitary- derived fibroblast growth factor (FGF) or brain-derived FGF are as effective as serum in repressing the synthesis of CPK when added to quiescent, differentiated cells. The decrease in the rate of synthesis of CPK occurs within 22 h after the addition of pituitary FGF to the cells. Pituitary FGF had very little effect, if any, on the rate CPK degradation. The overall rate of protein synthesis and the pattern of synthesis of the major polypeptides made by these cells was not altered by the addition of FGF. Although pituitary FGF was mitogenic for BC3H1 cells, the rate of cell growth was not absolutely correlated with the extent of repression of CPK. Brain-derived FGF fully repressed CPK induction under conditions where it showed no significant mitogenic activity. These results show that the expression of a muscle-specific protein, CPK, can be controlled by a single defined polypeptide growth factor in fully differentiated cultures, and that initiation of cell division is not required for their regulation to take place. PMID:3988800

  12. Matrix isolation ESR spectroscopy and magnetic anisotropy of D{sub 3h} symmetric septet trinitrenes

    SciTech Connect

    Misochko, Eugenii Ya.; Akimov, Alexander V.; Masitov, Artem A.; Korchagin, Denis V.; Aldoshin, Sergei M.; Chapyshev, Sergei V.

    2013-05-28

    The fine-structure (FS) parameters D of a series of D{sub 3h} symmetric septet trinitrenes were analyzed theoretically using density functional theory (DFT) calculations and compared with the experimental D values derived from ESR spectra. ESR studies show that D{sub 3h} symmetric septet 1,3,5-trichloro-2,4,6-trinitrenobenzene with D=-0.0957 cm{sup -1} and E= 0 cm{sup -1} is the major paramagnetic product of the photolysis of 1,3,5-triazido-2,4,6-trichlorobenzene in solid argon matrices at 15 K. Trinitrenes of this type display in the powder X-band ESR spectra intense Z{sub 1}-transition at very low magnetic fields, the position of which allows one to precisely calculate the parameter D of such molecules. Thus, our revision of the FS parameters of well-known 1,3,5-tricyano-2,4,6-trinitrenobenzene [E. Wasserman, K. Schueller, and W. A. Yager, Chem. Phys. Lett. 2, 259 (1968)] shows that this trinitrene has Double-Vertical-Line D Double-Vertical-Line = 0.092 cm{sup -1} and E= 0 cm{sup -1}. DFT calculations reveal that, unlike C{sub 2v} symmetric septet trinitrenes, D{sub 3h} symmetric trinitrenes have the same orientations of the spin-spin coupling tensor D-caret{sub SS} and the spin-orbit coupling tensor D-caret{sub SOC} and, as a result, have negative signs for both the D{sub SS} and D{sub SOC} values. The negative magnetic anisotropy of septet 2,4,6-trinitrenobenzenes is considerably strengthened on introduction of heavy atoms in the molecules, owing to an increase in contributions of various excitation states to the D{sub SOC} term.

  13. THE MAGNETIZED ENVIRONMENT OF THE W3(H{sub 2}O) PROTOSTARS

    SciTech Connect

    Chen, Huei-Ru; Rao, Ramprasad; Liu, Sheng-Yuan; Wilner, David J.

    2012-05-20

    We present the first interferometric polarization map of the W3(OH) massive star-forming region observed with the Submillimeter Array (SMA) at 878 {mu}m with an angular resolution of 1.''5 (about 3 Multiplication-Sign 10{sup 3} AU). Polarization is detected in the W3(H{sub 2}O) hot core, an extended emission structure in the northwest of W3(H{sub 2}O), and part of the W3(OH) ultracompact H II region. The W3(H{sub 2}O) hot core is known to be associated with a synchrotron jet along the east-west direction. In this core, the inferred magnetic field orientation is well aligned with the synchrotron jet and close to the plane of sky. Using the Chandrasekhar-Fermi method with the observed dispersion in polarization angle, we estimate a plane-of-sky magnetic field strength of 17.0 mG. Combined with water maser Zeeman measurements, the total magnetic field strength is estimated to be 17.1 mG, comparable to the field strength estimated from the synchrotron model. The magnetic field energy dominates over turbulence in this core. In addition, the depolarization effect is discerned in both SMA and James Clerk Maxwell Telescope measurements. Despite the great difference in angular resolutions and map extents, the polarization percentage shows a similar power-law dependence with the beam averaged column density. We suggest that the column density may be an important factor to consider when interpreting the depolarization effect.

  14. Mitotic Catastrophe in BC3H1 Cells following Yessotoxin Exposure

    PubMed Central

    Korsnes, Mónica Suárez; Korsnes, Reinert

    2017-01-01

    The marine toxin yessotoxin (YTX) can cause various cytotoxic effects depending on cell type and cell line. It is well known to trigger distinct mechanisms for programmed cell death which may overlap or cross-talk. The present contribution provides the first evidence that YTX can cause genotoxicity and induce mitotic catastrophe which can lead to different types of cell death. This work also demonstrates potential information gain from non-intrusive computer-based tracking of many individual cells during long time. Treatment of BC3H1 cells at their exponential growth phase causes atypical nuclear alterations and formation of giant cells with multiple nuclei. These are the most prominent morphological features of mitotic catastrophe. Giant cells undergo slow cell death in a necrosis-like manner. However, apoptotic-like cell death is also observed in these cells. Electron microscopy of treated BC3H1 cells reveal uncondensed chromatin and cells with double nuclei. Activation of p-p53, p-H2AX, p-Chk1, p-ATM, and p-ATR and down-regulation of p-Chk2 indicate DNA damage response and cell cycle deregulation. Micronuclei formation further support this evidence. Data from tracking single cells reveal that YTX treatment suppresses a second round of cell division in BC3H1 cells. These findings suggest that YTX can induce genomic alterations or imperfections in chromosomal segregation leading to permanent mitotic failure. This understanding extends the list of effects from YTX and which are of interest to control cancer and tumor progression.

  15. The 3 H and BMSEST Models for Spirituality in Multicultural Whole-Person Medicine

    PubMed Central

    Anandarajah, Gowri

    2008-01-01

    PURPOSE The explosion of evidence in the last decade supporting the role of spirituality in whole-person patient care has prompted proposals for a move to a biopsychosocial-spiritual model for health. Making this paradigm shift in today’s multicultural societies poses many challenges, however. This article presents 2 theoretical models that provide common ground for further exploration of the role of spirituality in medicine. METHODS The 3 H model (head, heart, hands) and the BMSEST models (body, mind, spirit, environment, social, transcendent) evolved from the author’s 12-year experience with curricula development regarding spirituality and medicine, 16-year experience as an attending family physician and educator, lived experience with both Hinduism and Christianity since childhood, and a lifetime study of the world’s great spiritual traditions. The models were developed, tested with learners, and refined. RESULTS The 3 H model offers a multidimensional definition of spirituality, applicable across cultures and belief systems, that provides opportunities for a common vocabulary for spirituality. Therapeutic options, from general spiritual care (compassion, presence, and the healing relationship), to specialized spiritual care (eg, by clinical chaplains), to spiritual self-care are discussed. The BMSEST model provides a conceptual framework for the role of spirituality in the larger health care context, useful for patient care, education, and research. Interactions among the 6 BMSEST components, with references to ongoing research, are proposed. CONCLUSIONS Including spirituality in whole-person care is a way of furthering our understanding of the complexities of human health and well-being. The 3 H and BMSEST models suggest a multidimensional and multidisciplinary approach based on universal concepts and a foundation in both the art and science of medicine. PMID:18779550

  16. Inhibition of (/sup 3/H)nitrendipine binding by phospholipase A/sub 2/

    SciTech Connect

    Goldman, M.E.; Pisano, J.J.

    1985-10-07

    Phospholipase A/sub 2/ from several sources inhibited (/sup 3/H)nitrendipine binding to membranes from brain, heart and ileal longitudinal muscle. The enzymes from bee venom and Russell's viper venom were most potent, having IC/sub 50/ values of approximately 5 and 14 ng/ml, respectively, in all three membrane preparations. Inhibition of binding by bee venom phospholipase A/sub 2/ was time- and dose-dependent. Mastoparan, a known facilitator of phospholipase A/sub 2/ enzymatic activity, shifted the bee venom phospholipase A/sub 2/ dose-response curve to the left. Pretreatment of brain membranes w