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Sample records for 3h-thymidine incorporation assays

  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. 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.

  8. 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

  9. 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.

  10. 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).

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  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. 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

  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. 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.

  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. 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.

  3. 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.

  4. 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.

  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. Radiometric macrophage culture assay for rapid evaluation of antileprosy activity of rifampin

    SciTech Connect

    Mittal, A.; Seshadri, P.S.; Prasad, H.K.; Sathish, M.; Nath, I.

    1983-10-01

    The antileprosy effect of rifampin was evaluated by a newly developed rapid in vitro assay wherein 31 human-derived strains and 1 armadillo-derived strain of Mycobacterium leprae were maintained for 2 and 3 weeks, respectively, in murine and human macrophages in the presence of (3H)thymidine. Of these strains, 27 showed significant incorporation of the radiolabel in cultures of live bacilli as compared with control cultures of heat-killed bacilli of the same strain. Consistent and significant inhibition of (3H)thymidine uptake was observed in M. leprae resident cultures with 3 to 200 ng of rifampin per ml as compared with similar cultures without the drug. In general, an increase in percent inhibition was seen from 3 to 20 ng/ml, with marginal increases at 40, 50, and 100 ng/ml. M. leprae strains appear to be remarkably susceptible to this drug in the in vitro assay.

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. Evaluation of T and B lymphocyte function in clinical practice using a flow cytometry based proliferation assay.

    PubMed

    Marits, Per; Wikström, Ann-Charlotte; Popadic, Dusan; Winqvist, Ola; Thunberg, Sarah

    2014-08-01

    The golden standard for functional evaluation of immunodeficiencies is the incorporation of [(3)H]-thymidine in a proliferation assay stimulated with mitogens. Recently developed whole blood proliferation assays have the advantage of parallel lymphocyte lineage analysis and in addition provide a non-radioactive alternative. Here we evaluate the Flow-cytometric Assay for Specific Cell-mediated Immune-response in Activated whole blood (FASCIA) in a comparison with [(3)H]-thymidine incorporation in four patients with severe combined immunodeficiency. The threshold for the minimum number of lymphocytes required for reliable responses in FASCIA is determined together with reference values from 100 healthy donors when stimulated with mitogens as well as antigen specific stimuli. Finally, responses against PWM and SEA+SEB stimuli are conducted with clinically relevant immunomodulatory compounds. We conclude that FASCIA is a rapid, stable and sensitive functional whole blood assay that requires small amounts of whole blood that can be used for reliable assessment of lymphocyte reactivity in patients.

  12. An acid phosphatase assay for quantifying the growth of adherent and nonadherent cells.

    PubMed

    Yang, T T; Sinai, P; Kain, S R

    1996-10-01

    We describe an acid phosphatase assay for determination of cell growth based on quantification of cytosolic acid phosphatase activity. The assay is based on the hydrolysis of the p-nitrophenyl phosphate by intracellular acid phosphatases in viable cells to produce p-nitrophenol. For all cell types examined, absorbance of p-nitrophenol at 405 nm is directly proportional to the cell number in the range of 10(3)-10(5) cells. The assay can quantify as few as 1000 cells per well in 96-well microtiter plates. The acid phosphatase assay was used to count various adherent and nonadherent cells, including human tumors, L6, and HT-2 cells. We also demonstrate the utility of this assay for analysis of growth factor and cytokine bioactivity on mammalian cells in culture. In comparison to [3H]thymidine incorporation, the acid phosphatase assay has similar sensitivity but a wider linear response range. The method also shows higher sensitivity and reproducibility in comparison to cell proliferation assays based on the reduction of tetrazolium salts. Because of the ease of use, sensitivity, and low cost, the acid phosphatase method is especially suited to applications where a large number of samples are assayed.

  13. 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.

  14. Evaluation of adenosine deaminase assay for analyzing T-lymphocyte density in vitro.

    PubMed

    Kainthla, Rani Poonam; Kashyap, Rajpal Singh; Prasad, Sweta; Purohit, Hemant J; Taori, Giridhar M; Daginawala, Hatim F

    2006-01-01

    The proliferative capacity of T cells in response to various stimuli is commonly determined by radioactive assay based on incorporation of [3H]thymidine ([3H]TdR) into newly synthesized DNA. In order to assess techniques for application in laboratories where radioactive facilities are not present, an alternative method was tested. As an alternative, T-cell proliferation was measured by spectrophotometrically analyzing the presence of an enzyme adenosine deaminase in lymphocytes and also using a standard XTT assay. Jurkat (human) T-cell line (clone E6.1) was used for lymphocyte population. The Jurkat cell concentration was adjusted according to different cell densities and enzyme activity was determined. Cells were also seeded in complete medium up to 72 h and harvested for estimation of enzyme activity. A significant correlation between the standard cell-proliferation assay and adenosine deaminase assay was observed. The present study indicates that the assay of adenosine deaminase is a reliable and accurate method for measuring proliferation of T lymphocytes.

  15. 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.

  16. 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.

  17. Detection of mycobacterial DNA by a specific and simple lateral flow assay incorporating cadmium selenide quantum dots.

    PubMed

    Cimaglia, Fabio; Liandris, Emmanouil; Gazouli, Maria; Sechi, Leonardo; Chiesa, Maurizio; De Lorenzis, Enrico; Andreadou, Margarita; Taka, Styliani; Mataragka, Antonia; Ikonomopoulos, John

    2015-12-01

    Cadmium selenide quantum dots have been incorporated to a lateral flow assay for the specific and very simple detection of different mycobacterial DNA targets within only a few minutes, bypassing the complexity of conventional DNA hybridization assays. The method extends our previous work on protein detection using an identical procedure.

  18. Lack of DNA damage induction by okadaic acid, a marine toxin, in the CHO-Hprt and the in vitro UDS assays.

    PubMed

    Le Hégarat, Ludovic; Nesslany, Fabrice; Mourot, Annick; Marzin, Daniel; Fessard, Valérie

    2004-12-12

    Okadaic acid (OA) is a marine toxin produced by dinoflagellates and responsible for human intoxications. OA is a specific inhibitor of serine/threonine protein phosphatases PP1 and PP2A and a potent tumor promoter in mouse skin and rat glandular stomach. In a previous study, we demonstrated that OA induced aneuploidy in CHO-K1 cells using the cytokinesis-block micronucleus (CBMN) assay coupled to FISH and concluded that OA was not a direct mutagen. As some previous in vitro mutagenicity studies had given positive results with OA, we decided to perform two additional in vitro mutagenicity assays in accordance with the OECD guidelines: (i) the CHO/Hprt test, which provides end points about locus-specific gene mutation; (ii) the in vitro unscheduled DNA synthesis (UDS) assay in rat hepatocytes, which measures [(3)H]thymidine incorporation into DNA undergoing excision repair. In the CHO/Hprt assay, there was no significant increase in the number of mutants for doses ranging from 5 to 5000 nM in the presence or absence of rat liver S9 fraction. In the in vitro UDS assay, OA did not induce primary DNA damages in rat hepatocytes following 18 h exposure at concentrations between 1.32 and 100 nM. As OA could affect the DNA repair systems via the inhibition of protein phosphatases, its effects on the repair kinetic of 2AAF-induced DNA damage were also investigated with the UDS assay. The results showed that OA did not interact with the DNA-repair process involved in in vitro UDS in rat hepatocytes. We concluded that OA failed to induce direct DNA damage but acted principally by altering the chromosome number, which could contribute to its carcinogenic effect.

  19. 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.

  20. Bioluminescent assay for human lymphocyte blast transformation.

    PubMed

    Bulanova, E G; Budagyan, V M; Romanova, N A; Brovko LYu; Ugarova, N N

    1995-05-01

    One of the basic tests of in vitro evaluation of immune cell functional activity is a proliferative response of lymphocytes on the action of external stimuli such as mitogenic lectines, antigens, etc. We compared two methods used to assess the lymphocyte functional status. (1) [3H]thymidine incorporation and (2) bioluminescence for determination of intracellular ATP in blast cells. Comparison has been done for healthy donors and patients with proven low immunological status. The proposed bioluminescent method for evaluation of the proliferative response was shown to be sensitive enough for diagnostic purposes. This method allows one to process a large number of samples at the same time and correlates highly with the radionuclide test use hazardous radioactive materials.

  1. Performance of a MALDI-TOF MS-based imipenem hydrolysis assay incorporating zinc sulfate.

    PubMed

    Knox, James; Palombo, Enzo

    2017-03-01

    A MALDI-TOF MS(1)-based imipenem hydrolysis assay was modified by adding ZnSO4. This improved detection of metallo-β-lactamase producing strains without compromising detection of other carbapenemase types. Using 129 genetically characterized Gram-negative bacilli, the sensitivity and specificity were 98.5% (95% confidence interval [CI]: 91.9-99.7%) and 100% (95% CI: 94.3-100%), respectively.

  2. Diagnostic and genetic studies on fibrin-stabilizing factor with a new assay based on amine incorporation.

    PubMed

    Lorand, L; Urayama, T; De Kiewiet, J W; Nossel, H L

    1969-06-01

    Fibrinoligase, the fibrin cross-linking enzyme, transiently appearing during the course of coagulation in normal blood, was shown to catalyze the incorporation of a fluorescent amine, monodansylcadaverine [or N-(5-aminopentyl)-5-dimethylamino-1-naphthalene-sulfonamide] into casein. The reaction provided the basis of a sensitive fluorimetric method for measuring the activity of the enzyme (and also of similar other transpeptidases, such as transglutaminase). In tests involving plasma, certain difficulties had to be overcome which were mainly due to the fact that the enzyme itself does not occur in citrated plasma. Only its precursor (fibrin-stabilizing factor or factor XIII) is present, still requiring limited proteolytic activation by thrombin. Thus, in order to measure amine incorporation with plasma as a source of the factor, thrombin must be added. This necessitated a differential desensitization of the intrinsic fibrinogen so that the latter could not clot and could not thereby interfere with amine incorporation. Also, the thrombin-inactivating capacity of plasma had to be saturated to enable full conversion of the factor to the transpeptidase. Concentrations of casein, monodansylcadaverine, calcium, and hydrogen ions were chosen to permit almost maximal velocity of amine incorporation. A linear relationship with regard to plasma concentration could be obtained only under such conditions. No similar assay is presently available for quantitatively evaluating fibrin-stabilizing factor levels in plasma.The amine incorporation test was applied to a clinical case of hereditary total fibrin-stabilizing factor deficiency. The effect of transfusion therapy was studied, and some of the patient's relatives were examined. Whereas a paternal aunt and uncle gave values well within the normal range, a brother and the mother proved to be partially deficient and could be considered as heterozygous carriers. The father appeared to have a reduced level of fibrin

  3. 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

  4. Measurement of suppressor activity of T CD4⁺CD25⁺ T reg cells using bromodeoxyuridine incorporation assay.

    PubMed

    Avalos-Martínez, Claudia E; Rodríguez-Alba, Juan Carlos; Berrón-Ruiz, Laura; Romero-Ramírez, Hector; Santos-Argumedo, Leopoldo; Jiménez-Zamudio, Luis Antonio; Domínguez-López, Maria Lilia; Vega-López, Armando; García-Latorre, Ethel

    2013-01-01

    The suppressor effect of T regulatory lymphocytes in co-cultures with T effector cells obtained by magnetic columns from healthy donors and activated by CD3/CD28 was measured by a proliferation assay using BrdU incorporation and an ELISA test. Tritiated thymidine incorporation was used as a reference since it is the gold standard for proliferation assays. Both methods were used simultaneously in the same samples in order to compare them. Correlation between them was statistically significant (p < 0.001). The purification using magnetic columns was very efficient since CD4⁺CD25⁺ cells were also FOXP3⁺ therefore; they were identified as suppressor T cells. The use of BrdU incorporation in suppression assays is an excellent method that avoids the use of radioactive contaminating materials.

  5. A versatile quantitation platform based on platinum nanoparticles incorporated volumetric bar-chart chip for highly sensitive assays.

    PubMed

    Wang, Yuzhen; Zhu, Guixian; Qi, Wenjin; Li, Ying; Song, Yujun

    2016-11-15

    Platinum nanoparticles incorporated volumetric bar-chart chip (PtNPs-V-Chip) is able to be used for point-of-care tests by providing quantitative and visualized readout without any assistance from instruments, data processing, or graphic plotting. To improve the sensitivity of PtNPs-V-Chip, hybridization chain reaction was employed in this quantitation platform for highly sensitive assays that can detect as low as 16 pM Ebola Virus DNA, 0.01ng/mL carcinoembryonic antigen (CEA), and the 10 HER2-expressing cancer cells. Based on this amplified strategy, a 100-fold decrease of detection limit was achieved for DNA by improving the number of platinum nanoparticle catalyst for the captured analyte. This quantitation platform can also distinguish single base mismatch of DNA hybridization and observe the concentration threshold of CEA. The new strategy lays the foundation for this quantitation platform to be applied in forensic analysis, biothreat detection, clinical diagnostics and drug screening.

  6. Incorporating expert judgments in utility evaluation of bacteroidales qPCR assays for microbial source tracking in a drinking water source.

    PubMed

    Åström, Johan; Pettersson, Thomas J R; Reischer, Georg H; Norberg, Tommy; Hermansson, Malte

    2015-02-03

    Several assays for the detection of host-specific genetic markers of the order Bacteroidales have been developed and used for microbial source tracking (MST) in environmental waters. It is recognized that the source-sensitivity and source-specificity are unknown and variable when introducing these assays in new geographic regions, which reduces their reliability and use. A Bayesian approach was developed to incorporate expert judgments with regional assay sensitivity and specificity assessments in a utility evaluation of a human and a ruminant-specific qPCR assay for MST in a drinking water source. Water samples from Lake Rådasjön were analyzed for E. coli, intestinal enterococci and somatic coliphages through cultivation and for human (BacH) and ruminant-specific (BacR) markers through qPCR assays. Expert judgments were collected regarding the probability of human and ruminant fecal contamination based on fecal indicator organism data and subjective information. Using Bayes formula, the conditional probability of a true human or ruminant fecal contamination given the presence of BacH or BacR was determined stochastically from expert judgments and regional qPCR assay performance, using Beta distributions to represent uncertainties. A web-based computational tool was developed for the procedure, which provides a measure of confidence to findings of host-specific markers and demonstrates the information value from these assays.

  7. Use of an aqueous soluble tetrazolium/formazan assay to measure viability and proliferation of lymphokine-dependent cell lines.

    PubMed

    Buttke, T M; McCubrey, J A; Owen, T C

    1993-01-04

    A new tetrazolium compound, MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt), has recently been described which in the presence of phenazine methosulfate (PMS) is reduced by living cells to yield a formazan product that can be assayed colorimetrically. An important advantage of MTS/PMS over other tetrazolium dyes (e.g., MTT) is the aqueous solubility of the reduced formazan product which eliminates the need for detergent solubilization or organic solvent extraction steps. Its advantages over XTT/PMS, another tetrazolium which yields a water-soluble formazan product, include the absorbance range of color produced (515-580 nm as opposed to 450 nm), the rapidity of color development, and the storage stability of the MTS/PMS reagent solution. In the present study, MTS/PMS was used to assay viability and proliferation of the IL-2-dependent HT-2 and CTLL-2 cell lines and the IL-3-dependent FDC-P1 and FL5.12 cell lines. With each cell line, the amount of formazan product was time-dependent and proportional to the number of viable cells. Furthermore, with both HT-2 and CTLL-2 cells it was found that cultures could be simultaneously labeled with MTS/PMS and [3H]thymidine, with relatively little effect of the dye on uptake of the latter. This feature was further capitalized upon in studies with FDC-P1 cells, in which the co-addition of MTS/PMS and [3H]thymidine was used to distinguish between cell viability and proliferation.

  8. Incorporation of a fluorescent guanosine analog into oligonucleotides and its application to a real time assay for the HIV-1 integrase 3'-processing reaction.

    PubMed Central

    Hawkins, M E; Pfleiderer, W; Mazumder, A; Pommier, Y G; Balis, F M

    1995-01-01

    We have synthesized a highly fluorescent (quantum yield 0.88) guanosine analog, (3-methyl-8-(2-deoxy-beta-D-ribofuranosyl) isoxanthopterin (3-Mi) in a dimethoxytrityl, phosphoramidite protected form, which can be site-specifically inserted into oligonucleotides through a 3',5'-phosphodiester linkage using an automated DNA synthesizer. Fluorescence is partially quenched within an oligonucleotide and the degree of quench is a function of the fluorophore's proximity to purines and its position in the oligonucleotide. As an example of the potential utility of this class of fluorophores, we developed a continuous assay for HIV-1 integrase 3'-processing reaction by incorporating 3-MI at the cleavage site in a double-stranded oligonucleotide identical to the U5 terminal sequence of the HIV genome. Integrase cleaves the 3'-terminal dinucleotide containing the fluorophore, resulting in an increase in fluorescence which can be monitored on a spectrofluorometer. Substitution of the fluorophore for guanosine at the cleavage site does not inhibit integrase activity. This assay is specific for the 3'-processing reaction. The change in fluorescence intensity is linear over time and proportional to the rate of the reaction. This assay demonstrates the potential utility of this new class of fluorophore for continuous monitoring of protein/DNA interactions. PMID:7659509

  9. Assay to detect chemically induced DNA repair in rat spermatocytes

    SciTech Connect

    Working, P.K.; Butterworth, B.E.

    1984-01-01

    An in vivo/in vitro DNA repair assay has been developed to quantitate chemically induced unscheduled DNA synthesis (UDS) in rat spermatocytes utilizing autoradiography. Male Fischer-344 rats were treated by i.p. injection or gavage with a variety of genotoxic agents dissolved in dimethyl sulfoxide, corn oil, or water. At selected times after treatment, spermatocytes were isolated by trypsin digestion of testes and cultured for 24 hr in the presence of /sup 3/H-thymidine. The direct-acting genotoxicants methyl methanesulfonate (MMS) and ethyl methanesulfonate and the chemotherapeutic agent cyclophosphamide (CPA) produced positive UDS responses in spermatocyes isolated l hr after i.p. injection. Other known genotoxicants--including dimethylnitrosamine, aflatoxin B/sub 1/, 2-acetylaminofluorene, 2, 6-dinitrotoluene, and l,6-dinitropyrene--failed to induce UDS, even with routes of administration and at times of exposure known to produce a positive response in hepatocytes. These results demonstrate that the in vivo/in vitro spermatocyte DNA repair assay may be useful as a predictive screen for germ cell mutagens.

  10. Anti-proliferative activity of oral anti-hyperglycemic agents on human vascular smooth muscle cells: thiazolidinediones (glitazones) have enhanced activity under high glucose conditions

    PubMed Central

    Little, Peter J; Osman, Narin; de Dios, Stephanie T; Cemerlang, Nelly; Ballinger, Mandy; Nigro, Julie

    2007-01-01

    Background Inhibition of vascular smooth muscle cell (vSMC) proliferation by oral anti-hyperglycemic agents may have a role to play in the amelioration of vascular disease in diabetes. Thiazolidinediones (TZDs) inhibit vSMC proliferation but it has been reported that they anomalously stimulate [3H]-thymidine incorporation. We investigated three TZDs, two biguanides and two sulfonylureas for their ability of inhibit vSMC proliferation. People with diabetes obviously have fluctuating blood glucose levels thus we determined the effect of media glucose concentration on the inhibitory activity of TZDs in a vSMC preparation that grew considerably more rapidly under high glucose conditions. We further explored the mechanisms by which TZDs increase [3H]-thymidine incorporation. Methods VSMC proliferation was investigated by [3H]-thymidine incorporation into DNA and cell counting. Activation and inhibition of thymidine kinase utilized short term [3H]-thymidine uptake. Cell cycle events were analyzed by FACS. Results VSMC cells grown for 3 days in DMEM with 5% fetal calf serum under low (5 mM glucose) and high (25 mM glucose) increased in number by 2.5 and 4.7 fold, respectively. Rosiglitazone and pioglitazone showed modest but statistically significantly greater inhibitory activity under high versus low glucose conditions (P < 0.05 and P < 0.001, respectively). We confirmed an earlier report that troglitazone (at low concentrations) causes enhanced incorporation of [3H]-thymidine into DNA but did not increase cell numbers. Troglitazone inhibited serum mediated thymidine kinase induction in a concentration dependent manner. FACS analysis showed that troglitazone and rosiglitazone but not pioglitazone placed a slightly higher percentage of cells in the S phase of a growing culture. Of the biguanides, metformin had no effect on proliferation assessed as [3H]-thymidine incorporation or cell numbers whereas phenformin was inhibitory in both assays albeit at high concentrations

  11. Aluminum ions stimulate mitosis in murine cells in tissue culture.

    PubMed

    Jones, T R; Antonetti, D L; Reid, T W

    1986-01-01

    Addition of aluminum to the culture medium of Nakano mouse lens epithelial (NMLE) cells and Swiss 3T3K cells induced both 3H-thymidine incorporation and mitosis. This is in contrast to other metal ions such as vanadium, which, at concentrations high enough to increase 3H-thymidine incorporation, actually inhibits mitosis (Jones and Reid, J Cell Physiol 121:199, 1984). Aluminum concentrations between 20 microM and 50 microM were most effective. The 3T3 cells respond to aluminum with a 7.6-fold increase, and NMLE cells respond with a 21-fold increase in 3H-thymidine incorporation. DNA synthesis in NMLE cells was also found to be synergistically stimulated by aluminum and low concentrations of insulin (4.5 X 10(-8) M). A 3.25-hr incubation with 50 microM aluminum was sufficient to induce 50% of maximum 3H-thymidine incorporation during the 40-hr assay. Aluminum-stimulated 3H-thymidine incorporation is inhibited by hydroxyurea, and aluminum causes an increase in cell number. Also, by sedimentation equilibrium analysis of the product of aluminum-stimulated DNA synthesis it was found that a single copy of DNA was synthesized following addition of aluminum to quiescent cells. These facts indicate that aluminum induces both S-phase DNA synthesis and mitosis. However, only 48% of the NMLE cells found to be labeled with DNA went on to divide. In contrast, although only a small percentage of 3T3 cells were found to be labeled after aluminum treatment, all of these cells appeared to go through mitosis.

  12. 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

  13. Purification of human platelet-derived growth factor

    SciTech Connect

    Raines, E.W.; Ross, R.

    1985-01-01

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

  14. 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

  15. 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.

  16. Relative Impact of Incorporating Pharmacokinetics on Predicting In Vivo Hazard and Mode of Action from High-Throughput In Vitro Toxicity Assays

    EPA Science Inventory

    The use of high-throughput in vitro assays has been proposed to play a significant role in the future of toxicity testing. In this study, rat hepatic metabolic clearance and plasma protein binding were measured for 59 ToxCast phase I chemicals. Computational in vitro-to-in vivo e...

  17. Refinement and optimisation of the rat CFU-GM assay to incorporate the use of cryopreserved bone-marrow cells for in vitro toxicology applications.

    PubMed

    Pessina, Augusto; Bonomi, Arianna; Baglio, Carolina; Cavicchini, Loredana; Gribaldo, Laura

    2009-09-01

    The colony-forming unit-granulocyte-macrophage (CFU-GM) assay has been validated for testing drug haematotoxicity (with both mouse bone-marrow and human cord blood cells) and for predicting in vivo human Maximal Tolerated Dose (MTD) values by extrapolating in vivo data on mouse toxicity. The rat CFU-GM assay is widely used for its capability to evaluate in vitro haematotoxicity, but no standardised procedure suitable for data comparison has been developed. A validated rat CFU-GM assay is needed for many reasons - not least because the rat is the most commonly-used species for the in vivo testing of toxicants. This report describes the refinement and optimisation of a standardised protocol for entering into the prevalidation phase of test development. The sensitivity of rat progenitors to granulocyte-macrophage-colony stimulating factor (GM-CSF), the correlation between the number of cells seeded and the number of colonies obtained, the role of mesenchymal cells on CFU-GM proliferation and the performance of the assay, and the effects of using different types of plastic dishes and sources of cytokines, are described. A standard operating procedure (SOP) based on the use of cryopreserved progenitors has been generated, to be applied to the in vitro toxicity testing of compounds. This SOP dramatically reduces the number of rats used and increases the homogeneity of the data obtained.

  18. Hereditary orotic aciduria, Lesch-Nyhan syndrome, and xeroderma pigmentosum probed by herpes simplex virus: /sup 125/I-iododeoxycytidine incorporation as an assay for viral growth. [Human fibroblasts

    SciTech Connect

    Campisi, J.; Hafner, J.; Boorstein, R.; Pardee, A.B.

    1983-01-01

    /sup 125/I-Iododeoxycytidine (/sup 125/IdC) incorporation into acid-insoluble material was a sensitive, rapid, and quantitative assay for the growth of herpes simplex virus type 1 (HSV-1) in human fibroblasts. Cellular utilization of the isotope was 10 to 25% of the incorporation by infected cells and could be 80% inhibited by tetrahydrouridine (THU). Viral utilization was inhibited by acycloguanosine, thioguanine (TG), and cytosine arabinoside. Isotope was incorporated equally well by growing or quiescent infected cells. HSV-1 was used to probe the metabolic capabilities of three mutant human fibroblast strains. /sup 125/IdC incorporation quantitatively measured the ability of the virus to grow in these cells. Viral /sup 125/IdC incorporation was sensitive to TG in normal fibroblasts but showed a 8- to 10-fold greater resistance to TG in fibroblasts derived from patients with Lesch-Nyhan syndrome (LN). Similarly, the growth of ultraviolet irradiated HSV-1 in normal fibroblasts was 5-fold greater than in fibroblasts derived from patients with xeroderma pigmentosum. In fibroblasts derived from patients with hereditary orotic aciduria, viral /sup 125/IdC incorporation was sensitive to adenosine (AD) at concentrations which were slightly stimulatory in normal fibroblasts. This was a 2-fold difference in AD sensitivity, which the radioassay reliably and quantitatively documented. HSV-1 infected cells could be individually identified by their incorporated /sup 125/IdC; such cells had blackened nuclei in autoradiograms prepared 12 hr after infection. Normal cells infected in the presence of TG had many fewer labeled nuclei than LN cells similarly infected in the presence of the drug. (JMT)

  19. 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.

  20. 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.

  1. Characterization of the action of epidermal growth factor in three different biological systems

    SciTech Connect

    Woost, P.G.

    1986-01-01

    In general, these studies characterized in each system one or more of the following aspects of EGF's biological action: EGF binding, EGF-induced phosphorylation, or EGF-stimulated DNA synthesis. In human corneal fibroblasts (HCF), EGF binds to specific, saturable, high affinity receptors and stimulated (/sup 3/H)-thymidine incorporation, and in vitro measurement of DNA synthesis, approximately 2.5 fold above control cultures, with maximal stimulation occurring at approximately 1 nM EGF. In addition, fibroblasts growth factor (FGF) and insulin stimulated maximal (/sup 3/H)-thymidine incorporation in HCF at approximately 100 ng ml/sup -1/ and 1 nM, respectively. In combination with dexamethasone (5 nM), the stimulation of (/sup 3/H)-thymidine incorporation by EGF was maintained. However, dexamethasone abolished the stimulatory action of FGF. Highly purified preparations of human placental outer cell membranes and intracellular organelles were assayed for functional EGF binding proteins (EGF-receptors). Functionality of a protein was determined by covalent labeling with (/sup 125/I)-EGF and EGF-stimulated autophosphorylation.

  2. TumorNext: A comprehensive tumor profiling assay that incorporates high resolution copy number analysis and germline status to improve testing accuracy

    PubMed Central

    Gray, Phillip N.; Vuong, Huy; Tsai, Pei; Lu, Hsaio-Mei; Mu, Wenbo; Hsuan, Vickie; Hoo, Jayne; Shah, Swati; Uyeda, Lisa; Fox, Susanne; Patel, Harshil; Janicek, Mike; Brown, Sandra; Dobrea, Lavinia; Wagman, Lawrence; Plimack, Elizabeth; Mehra, Ranee; Golemis, Erica A.; Bilusic, Marijo; Zibelman, Matthew; Elliott, Aaron

    2016-01-01

    The development of targeted therapies for both germline and somatic DNA mutations has increased the need for molecular profiling assays to determine the mutational status of specific genes. Moreover, the potential of off-label prescription of targeted therapies favors classifying tumors based on DNA alterations rather than traditional tissue pathology. Here we describe the analytical validation of a custom probe-based NGS tumor panel, TumorNext, which can detect single nucleotide variants, small insertions and deletions in 142 genes that are frequently mutated in somatic and/or germline cancers. TumorNext also detects gene fusions and structural variants, such as tandem duplications and inversions, in 15 frequently disrupted oncogenes and tumor suppressors. The assay uses a matched control and custom bioinformatics pipeline to differentiate between somatic and germline mutations, allowing precise variant classification. We tested 170 previously characterized samples, of which > 95% were formalin-fixed paraffin embedded tissue from 8 different cancer types, and highlight examples where lack of germline status may have led to the inappropriate prescription of therapy. We also describe the validation of the Affymetrix OncoScan platform, an array technology for high resolution copy number variant detection for use in parallel with the NGS panel that can detect single copy amplifications and hemizygous deletions. We analyzed 80 previously characterized formalin-fixed paraffin-embedded specimens and provide examples of hemizygous deletion detection in samples with known pathogenic germline mutations. Thus, the TumorNext combined approach of NGS and OncoScan potentially allows for the identification of the “second hit” in hereditary cancer patients. PMID:27626691

  3. Electric fields and proliferation in a chronic wound model

    SciTech Connect

    Goldman, R.; Pollack, S.

    1996-12-31

    A wound model for decubitus and leg ulcers consisting of human dermal fibroblasts in type 1 collagen dermal equivalent matrix (DEM) was exposed in vitro to electric fields similar to postulated endogenous fields in wounds. After an 8--10 day maturation period, conductivity of DEM samples was determined. Then, DEM samples were mounted in overall windows equidistant between Ag/AgCl agar electrodes in exposure chambers containing serum-free medium. A known low-frequency sinusoidal current was then applied for 12 h, and the average electric field amplitude was calculated in the region of the cells. After a 6 h hiatus, {sup 3}H-thymidine was introduced for 6 h. This was followed by assay. Over a series of trials, field amplitude ranged from 18 to 1,000 mV/m at frequencies of 10 and 100 Hz. Proliferation was measured by total DNA and {sup 3}H-thymidine incorporation. Results indicated that a narrow amplitude window between 37 and 50 mV/meter at 10 Hz yielded increases in proliferation: at maximum (41 mV/m), there was a 70% increase in total DNA (P < .01). Increases occurred in {sup 3}H-thymidine incorporation at 41--50 mV/m but not at other amplitudes (P < .05). Increases in total DNA at 41 mV/m occurred at 10 Hz but not 100 Hz (P < .01). {sup 3}H-thymidine incorporation was in agreement (P < .05). Response was also a function of cell density within matrix. Proliferation occurred in the same amplitude and frequency ranges in which endogenous fields are expected to occur.

  4. Antimicrobial assays of natural extracts and their inhibitory effect against Listeria innocua and fish spoilage bacteria, after incorporation into biopolymer edible films.

    PubMed

    Iturriaga, L; Olabarrieta, I; de Marañón, I Martínez

    2012-08-01

    The antimicrobial activity of twelve natural extracts was tested against two fish spoilage bacteria (Pseudomonas fluorescens and Aeromonas hydrophila/caviae) and Listeria innocua, in order to assess their potential utilization in the preservation and safety of minimally processed fish products. After a screening of the active extracts by agar diffusion and vapour diffusion methods, oregano and thyme essential oils and citrus extract were selected. The minimum inhibitory concentration (MIC) of the selected extracts was determined by disc diffusion method against target bacteria and at two temperatures: bacteria's optimal growth temperature (30 °C or 37 °C) and refrigeration temperature (4 °C). Due to its better solubility, lack of odour and greater inhibitory effect obtained against L. innocua at refrigerated temperature, citrus extract was selected and incorporated at 1% (v/v) into different biopolymer film forming solutions (gelatin, methyl cellulose and their blend 50:50 w/w). The antimicrobial activity of the developed films was then evaluated, just after preparation of the films and after one month of storage at 43±3% relative humidity and 24±3 °C. Regardless of the biopolymer matrix, all the developed films showed antimicrobial activity against the target bacteria. The most sensitive bacterium towards active films was L. innocua while P. fluorescens appeared as the most resistant one, in accordance with the previously performed antimicrobial tests for pure extracts. The differences in activity of the films between the tested two temperatures were not significant except for L. innocua, for which three times higher inhibition diameters were observed at refrigerated temperature. The inhibitory effectiveness of the films against the tested strains was maintained regardless of the biopolymer matrix for at least one month. Therefore, these edible films show potential for their future use in fresh fish fillets preservation.

  5. [Inhibition of human breast cancer cell line BCap-37 by flavonoid extract of wheat germ in vitro].

    PubMed

    Xu, G; Zhao, X; Zhao, L; Xu, H

    1999-05-30

    Cell growth and proliferation were measured by microculture tetrazolium(MTT) assay, cell colony-forming assay and the synthesis of DNA by 3H-thymidine incorporation. Flavonoid extract of wheat germ resulted to a dose-dependent, time-dependent growth inhibition, reduction of colony and 3H-thymiding incorporation in DNA of human breast cancer cell BCap-37. These findings indicated that the flavonoid extract of wheat germ can inhibit tumor cell growth and proliferation by blocking DNA synthesis in vitro.

  6. 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

  7. Human B cell function in normal individuals of various ages. 1. In vitro enumeration of pokeweed-induced peripheral blood lymphocyte immunoglobulin-synthesizing cells and the comparison of the results with numbers of peripheral B and T cells, mitogen responses, and levels of serum immunoglobulins.

    PubMed Central

    Nagel, J E; Crest, F J; Adler, W H

    1981-01-01

    The effect of age on the in vitro generation of immunoglobulin-secreting cells in pokeweed mitogen-stimulated cultures was examined using a staphylococcal protein A plaque assay. Although there was no statistically significant decrease with age in the numbers of plaque-forming cells, subjects whose cells failed to produce immunoglobulin were four times more common amongst individuals over 55 years of age. Simultaneously-measured T and B lymphocyte numbers. 3H-thymidine incorporation by mitogen-stimulated cultures, and serum immunoglobulins were comparable in both the young and the aged. PMID:7035035

  8. Effects of cathepsin D inhibitor from Vicia sativa L. seed hulls on human skin fibroblasts and breast cancer cells (in vitro studies).

    PubMed

    Roszkowska-Jakimiec, W; Wołczyński, S; Chlabicz, M

    2004-01-01

    Cathepsin D is a lysosomal protease which plays an important role in cancer invasion and metastasis. There are known inhibitors of that enzyme, such as pepstatin and potato inhibitor. In this study, we examined effects of the cathepsin D inhibitor from Vicia sativa L. seed hulls on cell cultures of human skin fibroblasts and breast cancer cells. There is no effect of the D-cathepsin inhibitor from Vicia sativa L. seed hulls on the proliferative activity of either human skin fibroblasts or breast cancer cells, measured by the [3H] thymidine incorporation assay.

  9. 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.

  10. Evaluation of a Test Article in the Salmonella typhimurium/Escherichia coli Plate Incorporation Mutation Assay in the Presence and Absence of Induced Rat Liver S-9. Test Article: N,N,N’,N’-tetramethyl ethanediamine (TMEDA)

    DTIC Science & Technology

    2008-06-12

    Chemical Company St. Louis, MO 63178 Storage Conditions: Room Temperature Lot No.: 46081638 Expiration Dates: February 9,2012 November 20, 2012 Lot...1983 STORAGE: At or below -70°C INDUCING AGENT(s): Aroclor 1254 ( Monsanto KL615), 500 mglkg i.p. BIOCHEMISTRY: -PROTEIN 38.9 mglm! Assayed

  11. Decrease in toxic potential of mixed tensides maintained below the critical micelle concentration: an in vitro study.

    PubMed

    Benassi, L; Bertazzoni, G; Magnoni, C; Rinaldi, M; Fontanesi, C; Seidenari, S

    2003-01-01

    Sodium lauryl sulphate (SLS) is an anionic tenside widely utilized in commercial topical preparations that may cause skin irritation. It has been shown that the barrier damage caused by SLS in vivo is lower when SLS is used in combination with other tensides which are able to reduce the critical micelle concentration (CMC). The aim of our study was to evaluate if the cytotoxic effect of SLS is reduced by the association with different tensides also at concentrations well below the CMC. Normal human keratinocytes from plastic surgery were grown in serum-free medium. At subconfluency, the cells were treated with SLS at a dose of 0.0025% in combination with cocamidopropyl betaine, Tween 20 and Tween 80 at the minimum toxic dose. Following tenside treatment, the culture medium was changed, and after 24 h the cells were collected for (3)H-thymidine incorporation, the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay and neutral red (NR) uptake. The cytotoxic effect on normal human keratinocytes, as evaluated by (3)H-thymidine incorporation, MTT assay and NR uptake, was significantly decreased by the combination with all the tested tensides. The correlation between cytotoxicity and physical properties was also studied by a conductimetric assay to investigate the mechanism involved in this toxicity reduction.

  12. Topoisomerase Assays

    PubMed Central

    Nitiss, John L.; Soans, Eroica; Rogojina, Anna; Seth, Aman; Mishina, Margarita

    2012-01-01

    Topoisomerases are nuclear enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of topoisomerases: type I, which makes single-stranded cuts in DNA, and type II enzymes, which cut and pass double-stranded DNA. DNA topoisomerases are important targets of approved and experimental anti-cancer agents. The protocols described in this unit are of assays used to assess new chemical entities for their ability to inhibit both forms of DNA topoisomerase. Included are an in vitro assay for topoisomerase I activity based on relaxation of supercoiled DNA and an assay for topoisomerase II based on the decatenation of double-stranded DNA. The preparation of mammalian cell extracts for assaying topoisomerase activity is described, along with a protocol for an ICE assay for examining topoisomerase covalent complexes in vivo and an assay for measuring DNA cleavage in vitro. PMID:22684721

  13. Differences in the Detection of BrdU/EdU Incorporation Assays Alter the Calculation for G1, S, and G2 Phases of the Cell Cycle in Trypanosomatids.

    PubMed

    da Silva, Marcelo Santos; Muñoz, Paula Andrea Marin; Armelin, Hugo Aguirre; Elias, Maria Carolina

    2017-03-04

    Trypanosomatids are the etiologic agents of various infectious diseases in humans. They diverged early during eukaryotic evolution and have attracted attention as peculiar models for evolutionary and comparative studies. Here, we show a meticulous study comparing the incorporation and detection of the thymidine analogs BrdU and EdU in Leishmania amazonensis, Trypanosoma brucei, and Trypanosoma cruzi to monitor their DNA replication. We used BrdU- and EdU-incorporated parasites with the respective standard detection approaches: Indirect Immunofluorescence to detect BrdU after standard denaturation (2 M HCl) and "click" chemistry to detect EdU. We found a discrepancy between these two thymidine analogs due to the poor detection of BrdU, which is reflected on the estimative of the duration of the cell cycle phases G1, S, and G2. To solve this discrepancy, we increase the exposure of incorporated BrdU using different concentrations of HCl. Using a new value for HCl concentration, we re-estimated the phases G1, S, G2+M, and cytokinesis durations, confirming the values found by this approach using EdU. In conclusion, we suggest that the studies using BrdU with standard detection approach, not only in trypanosomatids but also in others cell types, should be reviewed to ensure an accurate estimation of DNA replication monitoring. This article is protected by copyright. All rights reserved.

  14. Lectin binding as a probe of proliferative and differentiative phases in primary monolayer cultures of cutaneous keratinocytes

    SciTech Connect

    Ku, W.W.; Bernstein, I.A. )

    1988-04-01

    The surface of cells in the cutaneous epidermis of the newborn rat exhibits a discrete change in lectin-binding specificity from Griffonia simplicifolia I-B4 (GS I-B4), specific for {alpha}-D-galactosyl residues, to Ulex europeus agglutinin I (UEA), specific for {alpha}-L-fucose, as the cell leaves the basal layer and differentiates. Primary monolayer cultures of rat keratinocytes maintained in low Ca{sup 2+} medium exhibited a characteristic unimodal pattern in the ratio of bound UEA to bound GS I-B4 (UEA/B4 ratio) over a 7-day culture period as determined by a quantitative fluorometric assay. Estimation of DNA synthesis showed (a) a higher ({sup 3}H)thymidine incorporation when the UEA/B4 ratio was low and (b) a steady but lower incorporation between Days 3 and 4, coincident with the higher UEA/B4 ratio. Autoradiographic results further showed that cells stained intensely with UEA failed to incorporate ({sup 3}H)thymidine into their nuclei. Overall, the results suggest that (a) the increase in the UEA/B4 ratio between Days 2 and 4 reflects the progression of a proportion of the cells in the monolayer to an early spinous cell stage, the ultimate fate of which is desquamation into the medium and (b) the decrease in the UEA/B4 ratio between Days 5 and 7 reflects a consequent proliferative response to this loss of cells.

  15. Enzyme assays.

    PubMed

    Reymond, Jean-Louis; Fluxà, Viviana S; Maillard, Noélie

    2009-01-07

    Enzyme assays are analytical tools to visualize enzyme activities. In recent years a large variety of enzyme assays have been developed to assist the discovery and optimization of industrial enzymes, in particular for "white biotechnology" where selective enzymes are used with great success for economically viable, mild and environmentally benign production processes. The present article highlights the aspects of fluorogenic and chromogenic substrates, sensors, and enzyme fingerprinting, which are our particular areas of interest.

  16. Metronidazole decreases viability of DLD-1 colorectal cancer cell line.

    PubMed

    Sadowska, Anna; Krętowski, Rafał; Szynaka, Beata; Cechowska-Pasko, Marzanna; Car, Halina

    2013-10-01

    The aim of our study was to evaluate the impact of metronidazole (MTZ) on DLD-1 colorectal cancer cell (CRC) line. Toxicity of MTZ was determined by MTT test. Cells were incubated with MTZ used in different concentrations for 24, 48, and 72 hours. The effect of MTZ on DNA synthesis was measured as [3H]-thymidine incorporation. The morphological changes in human DLD-1 cell line were defined by transmission electron microscope OPTON 900. The influence of MTZ on the apoptosis of DLD-1 cell lines was detected by flow cytometry and fluorescence microscopy, while cell concentration, volume, and diameter were displayed by Scepter Cell Counter from Millipore. Our results show that cell viability was diminished in all experimental groups in comparison with the control, and the differences were statistically significant. We did not find any significant differences in [3H]-thymidine incorporation in all experimental groups and times of observation. Cytofluorimetric assays demonstrated a statistically significant increase of apoptotic rate in MTZ concentrations 10 and 50 μg/mL after 24 hours; 0.1, 10, 50, and 250 μg/mL after 48 hours; and in all concentrations after 72 hours compared with control groups. In the ultrastructural studies, necrotic or apoptotic cells were occasionally seen. In conclusion, MTZ affects human CRC cell line viability. The reduction of cell viability was consistent with the apoptotic test.

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

    SciTech Connect

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

    1985-07-01

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

  18. VUV modification promotes endothelial cell proliferation on PTFE vascular grafts

    NASA Astrophysics Data System (ADS)

    Cezeaux, J. L.; Romoser, C. E.; Benson, R. S.; Buck, C. K.; Sackman, J. E.

    1998-05-01

    Small diameter (⩽6 mm ID ) synthetic vascular grafts, used as lower-limb vessel replacements in patients without suitable autologous saphenous veins, have a failure rate of 53% after 4 yr. Graft failure is due to thrombosis and intimal hyperplasia, an increase in smooth muscle cells in the lumen of the vessel which leads to progressive closing and ultimate occlusion of the vessel. In an effort to increase patency rates of synthetic grafts, investigators have seeded vascular grafts with endothelial cells prior to implantation in an attempt to control both thrombosis and smooth muscle proliferation. This technique has been successful for the development of an endothelial monolayer in animal trials, but has met with limited success in humans. The hydrophobicity, low surface energy, and weak electrical charge of expanded polytetrafluoroethylene (ePTFE) provides conditions which are not optimal for endothelial cell attachment. The purpose of this study is to evaluate the effect of vacuum ultraviolet (VUV) modification of ePTFE on endothelial cell adhesion and proliferation. Pieces of ePTFE graft material were exposed to 10, 20 or 40 W VUV radiation for 10, 20 or 40 min using a UV excimer lamp. Prior to cell adhesion and proliferation experiments, the grafts pieces were autoclaved and cut into pledgets. Half of the pledgets were precoated with fibronectin ( 20 μg/ml). Cell adhesion was measured by seeding 3H-thymidine labeled human umbilical vein endothelial cells (HUVEC) onto the pledgets for 60 min. The pledgets were then washed and the remaining radioactivity assayed using scintillation counting. For the cell proliferation experiments, pledgets were seeded with unlabeled HUVEC which were allowed to adhere to the graft material for 18 h. The cells were then exposed to 3H-thymidine ( 1 μCi/ml) for approximately 48 h and then washed to remove any unincorporated 3H-thymidine. Incorporation of 3H-thymidine was measured using scintillation counting. Four replicate

  19. Measuring ribonucleotide incorporation into DNA in vitro and in vivo.

    PubMed

    Clausen, Anders R; Williams, Jessica S; Kunkel, Thomas A

    2015-01-01

    Ribonucleotides are incorporated into genomes by DNA polymerases, they can be removed, and if not removed, they can have deleterious and beneficial consequences. Here, we describe an assay to quantify stable ribonucleotide incorporation by DNA polymerases in vitro, and an assay to probe for ribonucleotides in each of the two DNA strands of the yeast nuclear genome.

  20. Measuring Ribonucleotide Incorporation into DNA In Vitro and In Vivo

    PubMed Central

    Clausen, Anders R.; Williams, Jessica S.; Kunkel, Thomas A.

    2017-01-01

    Ribonucleotides are incorporated into genomes by DNA polymerases, they can be removed, and if not removed, they can have deleterious and beneficial consequences. Here, we describe an assay to quantify stable ribonucleotide incorporation by DNA polymerases in vitro, and an assay to probe for ribonucleotides in each of the two DNA strands of the yeast nuclear genome. PMID:25916710

  1. Diverse effects of three furocoumarins on human lymphocyte proliferation

    SciTech Connect

    Arslan, P.; Cantini, M.; Cossarizza, A.; Franceschi, C.; Dall'acqua, F.

    1989-01-01

    The biological effects of three furocoumarins on the proliferation of human normal peripheral blood lymphocytes have been investigated. Mitogen-stimulated human lymphocytes were assayed in vitro by measuring /sup 3/H-thymidine (/sup 3/H-TdR) incorporation in the presence and in the absence of 15-30 /mu/M 3-carbethoxypsoralen (3-CPs), trimethylangelicin (TMA) and psoralen (PSR) with and without UV-A irradiation. The three furocoumarins differ in their ability to form mono- and bi-functional adducts with DNA pyrimidine furocoumarin doses and short times of UV-A irradiation used in the present study, 3-CPs did not affect /sup 3/H-TdR incorporation in PHA-stimulated human lymphocytes. TMA strongly inhibited /sup 3/H-TdR incorporation, while, unexpectedly, PSR increased /sup 3/H-TdR incorporation in the absence of irradiation, likely acting, under these experimental conditions, as a co-mitogen.

  2. Growth cone collapse assay.

    PubMed

    Cook, Geoffrey M W; Jareonsettasin, Prem; Keynes, Roger J

    2014-01-01

    The growth cone collapse assay has proved invaluable in detecting and purifying axonal repellents. Glycoproteins/proteins present in detergent extracts of biological tissues are incorporated into liposomes, added to growth cones in culture and changes in morphology are then assessed. Alternatively purified or recombinant molecules in aqueous solution may be added directly to the cultures. In both cases after a defined period of time (up to 1 h), the cultures are fixed and then assessed by inverted phase contrast microscopy for the percentage of growth cones showing a collapsed profile with loss of flattened morphology, filopodia, and lamellipodia.

  3. Hexosaminidase assays.

    PubMed

    Wendeler, Michaela; Sandhoff, Konrad

    2009-11-01

    beta-Hexosaminidases (EC 3.2.1.52) are lysosomal enzymes that remove terminal beta-glycosidically bound N-acetylglucosamine and N-acetylgalactosamine residues from a number of glycoconjugates. Reliable assay systems are particularly important for the diagnosis of a family of lysosomal storage disorders, the GM2 gangliosidoses that result from inherited beta-hexosaminidase deficiency. More recently, aberrant hexosaminidase levels have also been found to be associated with a variety of inflammatory diseases. Apart from patient testing and carrier screening, practical in vitro assays are indispensable for the characterization of knock-out mice with potentially altered hexosaminidase activities, for detailed structure-function studies aimed at elucidating the enzymatic mechanism, and to characterize newly described enzyme variants from other organisms. The purpose of this article is to discuss convenient hexosaminidase assay procedures for these and other applications, using fluorogenic or chromogenic artificial substrates as well as the physiological glycolipid substrate GM2. Attempts are also made to provide an overview of less commonly used alternative techniques and to introduce recent developments enabling high-throughput screening for enzyme inhibitors.

  4. Control of cell proliferation in human glioma by glucocorticoids.

    PubMed

    Freshney, R I; Sherry, A; Hassanzadah, M; Freshney, M; Crilly, P; Morgan, D

    1980-06-01

    Survival and proliferation of cell cultures from human anaplastic astrocytomas were shown to be enhanced by glucocorticoids with an optimal concentration of approximately 2.5 x 10(-5)M (10 micrograms/ml). The stimulation of proliferation was only observed in a clonal growth assay and was reversed as the size of individual colonies reached approximately 50 cells. Above this size, and in regular monolayer cultures, glucocorticoids were found to inhibit cell proliferation as measured by direct cell counting and incorporation of [3H] thymidine. Cultures grown to maximum cell densities in non-limiting medium conditions reached a lower terminal cell density, and had a reduced labelling index with [3H] thymidine in the presence of glucocorticoids. Although there was little difference between the actions of beta-methasone, dexamethasone and ethyl prednisolone, methyl prednisolone was found to be more effective, both in terms of stimulation of clonal growth and inhibition of growth at high cell densities. There was no evidence of cytotoxicity with glucocorticoids up to 5 x 10(-5)M (20 micrograms/ml) and it is suggested that glucocorticoids act via a normal regulatory process, perhaps enhancing cell-cell recognition.

  5. Control of cell proliferation in human glioma by glucocorticoids.

    PubMed Central

    Freshney, R. I.; Sherry, A.; Hassanzadah, M.; Freshney, M.; Crilly, P.; Morgan, D.

    1980-01-01

    Survival and proliferation of cell cultures from human anaplastic astrocytomas were shown to be enhanced by glucocorticoids with an optimal concentration of approximately 2.5 x 10(-5)M (10 micrograms/ml). The stimulation of proliferation was only observed in a clonal growth assay and was reversed as the size of individual colonies reached approximately 50 cells. Above this size, and in regular monolayer cultures, glucocorticoids were found to inhibit cell proliferation as measured by direct cell counting and incorporation of [3H] thymidine. Cultures grown to maximum cell densities in non-limiting medium conditions reached a lower terminal cell density, and had a reduced labelling index with [3H] thymidine in the presence of glucocorticoids. Although there was little difference between the actions of beta-methasone, dexamethasone and ethyl prednisolone, methyl prednisolone was found to be more effective, both in terms of stimulation of clonal growth and inhibition of growth at high cell densities. There was no evidence of cytotoxicity with glucocorticoids up to 5 x 10(-5)M (20 micrograms/ml) and it is suggested that glucocorticoids act via a normal regulatory process, perhaps enhancing cell-cell recognition. Images Fig. 2 Fig. 3 PMID:7426310

  6. Cytotoxic and antiproliferative effect of tepary bean lectins on C33-A, MCF-7, SKNSH, and SW480 cell lines.

    PubMed

    Valadez-Vega, Carmen; Morales-González, José A; Sumaya-Martínez, María Teresa; Delgado-Olivares, Luis; Cruz-Castañeda, Areli; Bautista, Mirandeli; Sánchez-Gutiérrez, Manuel; Zuñiga-Pérez, Clara

    2014-07-07

    For many years, several studies have been employing lectin from vegetables in order to prove its toxic effect on various cell lines. In this work, we analyzed the cytotoxic, antiproliferative, and post-incubatory effect of pure tepary bean lectins on four lines of malignant cells: C33-A; MCF-7; SKNSH, and SW480. The tests were carried out employing MTT and 3[H]-thymidine assays. The results showed that after 24 h of lectin exposure, the cells lines showed a dose-dependent cytotoxic effect, the effect being higher on MCF-7, while C33-A showed the highest resistance. Cell proliferation studies showed that the toxic effect induced by lectins is higher even when lectins are removed, and in fact, the inhibition of proliferation continues after 48 h. Due to the use of two techniques to analyze the cytotoxic and antiproliferative effect, differences were observed in the results, which can be explained by the fact that one technique is based on metabolic reactions, while the other is based on the 3[H]-thymidine incorporated in DNA by cells under division. These results allow concluding that lectins exert a cytotoxic effect after 24 h of exposure, exhibiting a dose-dependent effect. In some cases, the cytotoxic effect is higher even when the lectins are eliminated, however, in other cases, the cells showed a proliferative effect.

  7. Immunological aspects of the common food colorants, amaranth and tartrazine.

    PubMed

    Koutsogeorgopoulou, L; Maravelias, C; Methenitou, G; Koutselinis, A

    1998-02-01

    We describe a sensitive and reproducible microassay model using human peripheral blood lymphocytes (PBL) for discrimination between the cytotoxic and immunosuppressive effects of food colorants such as amaranth and tartrazine. The cytotoxic effects of a wide range of concentrations of these substances were studied on human PBL by the colorimetric in vitro cytotoxicity assays, neutral red uptake (NR) and thiazolyl blue tetrazolium bromide (MTT). The immunotoxic properties of these 2 substances were determined by a [3H]-thymidine DNA incorporation assay on phytohemagglutinin stimulated or non-stimulated lymphocytes, as well as by a Cr51 release Natural Killer assays. The results showed clear immunosuppressive effects from the 2 substances tested, although the concentrations chosen for this study proved to be non-cytotoxic by NR and MTT cytotoxic endpoints.

  8. 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.

  9. Activity and mode of action of acridine compounds against Leishmania donovani.

    PubMed Central

    Mesa-Valle, C M; Castilla-Calvente, J; Sanchez-Moreno, M; Moraleda-Lindez, V; Barbe, J; Osuna, A

    1996-01-01

    In the present work, we have assayed both the in vitro and in vivo action of two acridine compounds against Leishmania donovani. As part of this effort, we have studied the possible action mechanism of these compounds at the ultrastructural and biochemical levels and in relation to the synthesis of macromolecules. The two acridinones inhibit the in vitro growth of the promastigote forms of L. donovani at the highest concentration assayed (100 micrograms/ml). The in vivo results indicate that both compounds reduce the number of amastigotes per gram of spleen, and decrease parasitism, by more than 40%. With respect to the action mechanism, both compounds inhibit the incorporation of [3H]thymidine, inducing alterations at the ultrastructural level in the DNA and mitochondria. Alterations are also caused in the enzymes of the Krebs cycle. PMID:8851593

  10. DNA synthesis in mouse brown adipose tissue is under. beta. -adrenergic control

    SciTech Connect

    Rehnmark, S.; Nedergaard, J. )

    1989-02-01

    The rate of DNA synthesis in mouse brown adipose tissue was followed with injections of ({sup 3}H)thymidine. Cold exposure led to a large increase in the rate of ({sup 3}H)thymidine incorporation, reaching a maximum after 8 days, after which the activity abruptly ceased. A series of norepinephrine injections was in itself able to increase ({sup 3}H)thymidine incorporation. When norepinephrine was injected in combination with the {alpha}-adrenergic antagonist phentolamine or with the {beta}-adrenergic antagonist propranolol, the stimulation was fully blocked by propranolol. It is suggested that stimulation of DNA synthesis in brown adipose tissue is a {beta}-adrenergically mediated process and that the tissue is an interesting model for studies of physiological control of DNA synthesis.

  11. Permanent hair dye-incorporated hyaluronic acid nanoparticles.

    PubMed

    Lee, Hye-Young; Jeong, Young-Il; Kim, Da-Hye; Choi, Ki-Choon

    2013-01-01

    We prepared p-phenylenediamine (PDA)-incorporated nanoparticles using hyaluronic acid (HA). PDA-incorporated HA nanoparticles have spherical shapes and sizes were less than 300 nm. The results of FT-IR spectra indicated that PDA-incorporated HA nanoparticles were formed by ion-complex formation between amine group of PDA and carboxyl group of HA. Furthermore, powder-X-ray diffractogram (XRD) measurement showed that intrinsic crystalline peak of PDA disappeared by formation of nanoparticle with HA at XRD measurement. These results indicated that PDA-incorporated HA nanoparticles were formed by ion-complex formation. At drug release study, the higher PDA contents induced faster release rate from nanoparticles. PDA-incorporated nanoparticles showed reduced intrinsic toxicity against HaCaT human keratinocyte cells at MTT assay and apoptosis assay. We suggest that PDA-incorporated HA nanoparticles are promising candidates for novel permanent hair dye.

  12. High-frequency low-level diode laser irradiation promotes proliferation and migration of primary cultured human gingival epithelial cells.

    PubMed

    Ejiri, Kenichiro; Aoki, Akira; Yamaguchi, Yoko; Ohshima, Mitsuhiro; Izumi, Yuichi

    2014-07-01

    In periodontal therapy, the use of low-level diode lasers has recently been considered to improve wound healing of the gingival tissue. However, its effects on human gingival epithelial cells (HGECs) remain unknown. The aim of the present study was to examine whether high-frequency low-level diode laser irradiation stimulates key cell responses in wound healing, proliferation and migration, in primary cultured HGECs in vitro. HGECs were derived from seven independent gingival tissue specimens. Cultured HGECs were exposed to a single session of high-frequency (30 kHz) low-level diode laser irradiation with various irradiation time periods (fluence 5.7-56.7 J/cm(2)). After 20-24 h, cell proliferation was evaluated by WST-8 assay and [(3)H]thymidine incorporation assay, and cell migration was monitored by in vitro wound healing assay. Further, phosphorylation of the mitogen-activated protein kinase (MAPK) pathways after irradiation was investigated by Western blotting. The high-frequency low-level irradiation significantly increased cell proliferation and [(3)H]thymidine incorporation at various irradiation time periods. Migration of the irradiated cells was significantly accelerated compared with the nonirradiated control. Further, the low-level diode laser irradiation induced phosphorylation of MAPK/extracellular signal-regulated protein kinase (ERK) at 5, 15, 60, and 120 min after irradiation. Stress-activated protein kinases/c-Jun N-terminal kinase and p38 MAPK remained un-phosphorylated. The results show that high-frequency low-level diode laser irradiation promotes HGEC proliferation and migration in association with the activation of MAPK/ERK, suggesting that laser irradiation may accelerate gingival wound healing.

  13. Dominance of the Senescent Phenotype in Heterokaryons Between Replicative and Post-Replicative Human Fibroblast-Like Cells

    PubMed Central

    Norwood, Thomas H.; Pendergrass, William R.; Sprague, Curtis A.; Martin, George M.

    1974-01-01

    In heterokaryons between senescent and young diploid fibroblast-like cells, dominance of the former with respect to nuclear DNA synthesis (incorporation of [3H]thymidine) was demonstrated. For identification of the respective partners, double-layer autoradiography was used after the old cells were labeled with [3H]methionine and the young cells were labeled with [14C]thymidine. Synchrony of nuclear labeling (i.e., all nuclei in a cell labeled with [3H]thymidine) was observed in the majority of di- and polykaryons during the second and third of three 24-hr periods of labeling with [3H]thymidine. The results are compatible with either terminal differentiation or error theories of clonal senescence. Images PMID:4366757

  14. Effect of an antimitotic agent colchicine on thioacetamide hepatotoxicity.

    PubMed Central

    Mangipudy, R S; Rao, P S; Mehendale, H M

    1996-01-01

    In an earlier study we established that timely and adequate tissue repair response following the administration of a six-fold dose-range of thioacetamide (TA; 50, 150, and 300 mg/kg) prevented progression of injury and led to recovery and animal survival. Delayed and attenuated repair response after the 600 mg/kg TA dose resulted in a marked progression of injury and 100% lethality. The objective of the present study was to further scrutinize this concept in an experimental protocol in which we hypothesized that a selective ablation of the tissue repair response should lead to lethality from the nonlethal, moderately toxic doses of 150 and 300 mg/kg TA. In this study we investigated the effect of the antimitotic agent colchicine (CLC, 1 mg/kg) on the outcome of TA hepatotoxicity. Male Sprague-Dawley rats (175-225 g) were injected intraperitoneally (ip) with 150 and 300 mg/kg TA. We assessed liver injury by serum enzyme elevations and histopathology. Tissue regeneration response was measured by 3H-thymidine incorporation into hepatonuclear DNA and by proliferating cell nuclear antigen (PCNA) assay. S-Phase stimulation, as indicated by 3H-thymidine incorporation, was noted at 36 and 48 hr following the administration of 150 mg/kg TA, whereas with the 300 mg/kg TA S-phase stimulation was elicited at 48 hr following treatment. Therefore, two doses of CLC (30 hr and 42 hr, 1 mg/kg, ip) were administered to the 150 mg/kg treated group while a single dose of CLC (42 hr, 1 mg/kg, ip) was administered to the 300 mg/kg group. CLC treatment resulted in 100% lethality in both groups. Thus, CLC administration converted nonlethal doses into lethal doses. The 150 mg/kg TA dose was then chosen to further investigate the underlying mechanism. Rats treated with TA alone recovered from injury by 36-48 hr while CLC treatment resulted in a progression of injury as indicated by serum enzyme elevation and histopathology. Tissue repair, as evidenced by 3H-thymidine incorporation and PCNA

  15. Human cell mediated immunity to porins from Salmonella typhi.

    PubMed

    Blanco, F; Isibasi, A; Raúl González, C; Ortiz, V; Paniagua, J; Arreguín, C; Kumate, J

    1993-01-01

    The current studies were undertaken to assess the role of the porins and outer membrane proteins (OMP) in the human immune response to Salmonella typhi 9, 12 Vi:d. Experiments were performed to determinate the lymphocyte activation response to porins in individuals who had been vaccinated against typhoid fever. 10 healthy volunteers were studied before and 10 days after oral or subcutaneous immunisation. Five patients with typhoid fever were also studied. Lymphocyte activation was measured by the 3H thymidine incorporation assay. Individuals with typhoid fever as well as those immunised with oral vaccine responded well to porins and outer membrane proteins, as opposed to those immunised with the subcutaneous vaccine. These results suggest that the porins and OMP play a role in the cellular immune response against Salmonella typhi.

  16. Induction of unscheduled DNA synthesis in suspensions of rat hepatocytes by an environmental toxicant, 3,3'4,4'-tetrachloroazobenzene.

    PubMed

    Hsia, M T; Kreamer, B L

    1979-04-01

    Unscheduled DNA synthesis was induced by 3,3'4,4'-tetrachloroazobenzene (TCAB)) in freshly isolated suspensions of rat hepatocytes. A dose-dependent response was demonstrated. Hepatocellular DNA was obtained after the chloroform-isoamyl alchohol-phenol extraction of the isolated nuclei. The induction of unscheduled DNA synthesis was measured by the incorporation of [3H]-thymidine in the presence of hydroxyurea as determined by the scintillation counting assay. DNA repair data obtained in this study on benzo[a]pyrene and methyl methanesulfonate are comparable to a previous report using primary cultures of hepatocytes and cesium chloride gradients. Hence, the present method offers promise as a rapid and sensitive screen for chemical carcinogens.

  17. Effects of hormones and drugs on cartilage repair.

    PubMed

    Franchimont, P; Bassleer, C; Henrotin, Y

    1989-08-01

    When cartilage is attacked, either by an excess of charges or by biochemical agents, its morphological and functional impairment is associated with a local homeostatic reaction; this includes a proliferative response (assessed by 3H-thymidine incorporation) and a stimulation of proteoglycan and type II collagen (II coll) synthesis. This homeostatic reaction may be studied in vitro using tridimensional culture of human chondrocytes. Cartilage clusters are formed after 4 days' culture and chondrocytes multiply for the first 15 days of culture. Furthermore, human cartilage proteoglycans and II coll, assayed by specific radioimmunoassays, are released into the culture medium and constitute the new matrix of the clusters. Moreover, it appears that these human chondrocytes are targets for several hormones capable of stimulating a proliferative response without affecting proteoglycan and II coll synthesis.

  18. Effects of hydroxyapatite and Biostite on osteogenic induction of hMSC.

    PubMed

    Marinucci, Lorella; Balloni, Stefania; Becchetti, Ennio; Bistoni, Giovanni; Calvi, Edoardo Maria; Lumare, Eleonora; Ederli, Filippo; Locci, Paola

    2010-03-01

    When isolated from the iliac crest human mesenchymal stem cells (hMSC) differentiate into osteoblast-like cells with appropriate stimulation in culture. This in vitro study tested the hypothesis that Biostite and hydroxyapatite (HA) affect proliferation and differentiation of hMSC into osteoblastic cells. Cell proliferation was determined by measuring 3H-thymidine incorporation into DNA and typical markers of osteoblastic phenotype were determined by RT-PCR assay. No differences emerged in cell proliferation cultures with Biostite or hydroxyapatite (HA), but gene expression analysis revealed higher expression of collagen,alkaline phosphatase (ALP), osteopontin and bone sialoprotein (BSP) in the presence of Biostite. TGFb2 production, as assessed by an Elisa kit, and Runx2 expression by RT-PCR, were greater in Biostite cultures, suggesting Biostite provides a better environment for hMSC differentiation into osteoblasts and is, potentially, a more promising bone-filling material than HA.

  19. Growth factors with heparin binding affinity in human synovial fluid

    SciTech Connect

    Hamerman, D.; Taylor, S.; Kirschenbaum, I.; Klagsbrun, M.; Raines, E.W.; Ross, R.; Thomas, K.A.

    1987-12-01

    Synovial effusions were obtained from the knees of 15 subjects with joint trauma, menisceal or ligamentous injury, or osteoarthritis. Heparin-Sepharose affinity chromatography of these synovial fluids revealed, in general, three major peaks of mitogenic activity as measured by incorporation of /sup 3/H-thymidine into 3T3 cells. Gradient elution patterns showed activities at 0.5M NaCl, which is characteristic of platelet derived growth factor, and at 1.1 M NaCl and 1.6M NaCl, indicative of acidic and basic fibroblast growth factors, respectively. The identities of these mitogenic fractions were confirmed by specific immunologic and receptor-binding assays. The presence of platelet derived, acidic and basic fibroblast growth factors in the synovial fluid may contribute to wound healing in the arthritic joint.

  20. 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.

  1. 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.

  2. INFLUENCE OF LIGHT ON BACTERIOPLANKTON PRODUCTION AND RESPIRATION IN A SUBTROPICAL CORAL REEF

    EPA Science Inventory

    The influence of sunlight on bacterioplankton production (14C-leucine (Leu) and 3H-thymidine (TdR) incorporation; changes in cell abundances) and O2 consumption was investigated in a shallow subtropical coral reef located near Key Largo, Florida. Quartz (light) and opaque (dark) ...

  3. Neurotensin enhances estradiol induced DNA synthesis in immature rat uterus

    SciTech Connect

    Mistry, A.; Vijayan, E.

    1985-05-27

    Systemic administration of Neurotensin, a tridecapeptide, in immature rats treated with estradiol benzoate significantly enhances uterine DNA synthesis as reflected by the incorporation of /sup 3/H-thymidine. The peptide may have a direct action on the uterus. Substance P, a related peptide, had no effect on uterine DNA synthesis. 18 references, 4 tables.

  4. RAS - Screens & Assays

    Cancer.gov

    A primary goal of the RAS Initiative is to develop assays for RAS activity, localization, and signaling and adapt those assays so they can be used for finding new drug candidates. Explore the work leading to highly validated screening protocols.

  5. Assays of Serum Testosterone.

    PubMed

    Herati, Amin S; Cengiz, Cenk; Lamb, Dolores J

    2016-05-01

    The diagnosis of male hypogonadism depends on an assessment of the clinical signs and symptoms of hypogonadism and serum testosterone level. Current clinical laboratory testosterone assay platforms include immunoassays and mass spectrometry. Despite significant advances to improve the accuracy and precision of the currently available assays, limited comparability exists between assays at the lower and upper extremes of the testosterone range. Because of this lack of comparability, there is no current gold standard assay for the assessment of total testosterone levels.

  6. A lateral electrophoretic flow diagnostic assay.

    PubMed

    Lin, Robert; Skandarajah, Arunan; Gerver, Rachel E; Neira, Hector D; Fletcher, Daniel A; Herr, Amy E

    2015-03-21

    Immunochromatographic assays are a cornerstone tool in disease screening. To complement existing lateral flow assays (based on wicking flow) we introduce a lateral flow format that employs directed electrophoretic transport. The format is termed a "lateral e-flow assay" and is designed to support multiplexed detection using immobilized reaction volumes of capture antigen. To fabricate the lateral e-flow device, we employ mask-based UV photopatterning to selectively immobilize unmodified capture antigen along the microchannel in a barcode-like pattern. The channel-filling polyacrylamide hydrogel incorporates a photoactive moiety (benzophenone) to immobilize capture antigen to the hydrogel without a priori antigen modification. We report a heterogeneous sandwich assay using low-power electrophoresis to drive biospecimen through the capture antigen barcode. Fluorescence barcode readout is collected via a low-resource appropriate imaging system (CellScope). We characterize lateral e-flow assay performance and demonstrate a serum assay for antibodies to the hepatitis C virus (HCV). In a pilot study, the lateral e-flow assay positively identifies HCV+ human sera in 60 min. The lateral e-flow assay provides a flexible format for conducting multiplexed immunoassays relevant to confirmatory diagnosis in near-patient settings.

  7. Laminar shear stress stimulates vascular smooth muscle cell apoptosis via the Akt pathway.

    PubMed

    Fitzgerald, Tamara N; Shepherd, Benjamin R; Asada, Hidenori; Teso, Desarom; Muto, Akihito; Fancher, Tiffany; Pimiento, Jose M; Maloney, Stephen P; Dardik, Alan

    2008-08-01

    Vascular smooth muscle cells (SMC) may be directly exposed to blood flow after an endothelial-denuding injury. It is not known whether direct exposure of SMC to shear stress reduces SMC turnover and contributes to the low rate of restenosis after most vascular interventions. This study examines if laminar shear stress inhibits SMC proliferation or stimulates apoptosis. Bovine aortic SMC were exposed to arterial magnitudes of laminar shear stress (11 dynes/cm(2)) for up to 24 h and compared to control SMC (0 dynes/cm(2)). SMC density was assessed by cell counting, DNA synthesis by (3)[H]-thymidine incorporation, and apoptosis by TUNEL staining. Akt, caspase, bax, and bcl-2 phosphorylation were assessed by Western blotting; caspase activity was also measured with an in vitro assay. Analysis of variance was used to compare groups. SMC exposed to laminar shear stress had a 38% decrease in cell number (n = 4, P = 0.03), 54% reduction in (3)[H]-thymidine incorporation (n = 3, P = 0.003), and 15-fold increase in TUNEL staining (n = 4, P < 0.0001). Akt phosphorylation was reduced by 67% (n = 3, P < 0.0001), whereas bax/bcl-2 phosphorylation was increased by 1.8-fold (n = 3, P = 0.01). Caspase-3 activity was increased threefold (n = 5, P = 0.03). Pretreatment of cells with ZVAD-fmk or wortmannin resulted in 42% increased cell retention (n = 3, P < 0.01) and a fourfold increase in apoptosis (n = 3, P < 0.04), respectively. Cells transduced with constitutively-active Akt had twofold decreased apoptosis (n = 3, P < 0.002). SMC exposed to laminar shear stress have decreased proliferation and increased apoptosis, mediated by the Akt pathway. These results suggest that augmentation of SMC apoptosis may be an alternative strategy to inhibit restenosis after vascular injury.

  8. Effect of irradiation on DNA synthesis, NBN gene expression and chromosomal stability in cells with NBN mutations

    PubMed Central

    Nowak, Jerzy; Świątek-Kościelna, Bogna; Kałużna, Ewelina M.; Rembowska, Jolanta; Dzikiewicz-Krawczyk, Agnieszka; Zawada, Mariola

    2017-01-01

    Introduction The NBN gene product is part of the MRE11/RAD50/NBN complex, which plays an essential role in genomic stability. In the study we try to answer the question what is the effect of irradiation on DNA synthesis, NBN gene expression and chromosomal stability in cells with homozygous c.657-661del, and heterozygous c.657-661del, p.I171V and p.R215W NBN gene mutations. Material and methods Immortalized B-lymphocytes with NBN gene mutations were X-ray irradiated at doses of 1, 2, 5 and 8 Gy/min. Radioresistant DNA synthesis rate and the percentage of cells in phase S was analyzed by 3H thymidine and BrdU incorporation assays. NBN gene expression was quantified by real-time PCR with TaqMan fluorescent probe. Results Increasing the irradiation dose resulted in gradual decrease of 3H thymidine incorporation in all cells, but significantly only in homo- and heterozygous c.657-661del cells (p-values < 0.0001). After irradiation the relative expression of NBN was significantly higher in homozygous c.657-661del and heterozygous p.R215W cells as compared to heterozygous c.657-661del, p.I171V and control cells (p < 0.01). All cells with NBN gene mutations showed significantly higher total number of chromosomal aberrations per metaphase as compared to control cells, with the highest number of aberrations in homozygous c.657-661del cells (p < 0.001). Conclusions The results obtained indicate that homozygous c.657-661del mutation affects cell sensitivity to irradiation. Moreover, homozygous variant is associated with disturbance in the activation of cell cycle checkpoints and with defects in DNA repair. In turn, heterozygous c.657-661del, p.R215W and p.I171V mutations do not substantially alter the radiosensitivity. PMID:28261280

  9. Colorimetric protein assay techniques.

    PubMed

    Sapan, C V; Lundblad, R L; Price, N C

    1999-04-01

    There has been an increase in the number of colorimetric assay techniques for the determination of protein concentration over the past 20 years. This has resulted in a perceived increase in sensitivity and accuracy with the advent of new techniques. The present review considers these advances with emphasis on the potential use of such technologies in the assay of biopharmaceuticals. The techniques reviewed include Coomassie Blue G-250 dye binding (the Bradford assay), the Lowry assay, the bicinchoninic acid assay and the biuret assay. It is shown that each assay has advantages and disadvantages relative to sensitivity, ease of performance, acceptance in the literature, accuracy and reproducibility/coefficient of variation/laboratory-to-laboratory variation. A comparison of the use of several assays with the same sample population is presented. It is suggested that the most critical issue in the use of a chromogenic protein assay for the characterization of a biopharmaceutical is the selection of a standard for the calibration of the assay; it is crucial that the standard be representative of the sample. If it is not possible to match the standard with the sample from the perspective of protein composition, then it is preferable to use an assay that is not sensitive to the composition of the protein such as a micro-Kjeldahl technique, quantitative amino acid analysis or the biuret assay. In a complex mixture it might be inappropriate to focus on a general method of protein determination and much more informative to use specific methods relating to the protein(s) of particular interest, using either specific assays or antibody-based methods. The key point is that whatever method is adopted as the 'gold standard' for a given protein, this method needs to be used routinely for calibration.

  10. DISTRIBUTED AMPLIFIER INCORPORATING FEEDBACK

    DOEpatents

    Bell, P.R. Jr.

    1958-10-21

    An improved distributed amplifier system employing feedback for stabilization is presented. In accordance with the disclosed invention, a signal to be amplified is applled to one end of a suitable terminated grid transmission line. At intervals along the transmission line, the signal is fed to stable, resistance-capacitance coupled amplifiers incorporating feedback loops therein. The output current from each amplifier is passed through an additional tube to minimize the electrostatic capacitance between the tube elements of the last stage of the amplifier, and fed to appropriate points on an output transmission line, similar to the grid line, but terminated at the opposite (input) end. The output taken from the unterminated end of the plate transmission line is proportional to the input voltage impressed upon the grid line.

  11. Absolute nuclear material assay

    DOEpatents

    Prasad, Manoj K [Pleasanton, CA; Snyderman, Neal J [Berkeley, CA; Rowland, Mark S [Alamo, CA

    2012-05-15

    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

  12. Absolute nuclear material assay

    DOEpatents

    Prasad, Manoj K.; Snyderman, Neal J.; Rowland, Mark S.

    2010-07-13

    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

  13. Nepal CRS project incorporates.

    PubMed

    1983-01-01

    The Nepal Contraceptive Retail Sales (CRS) Project, 5 years after lauching product sales in June 1978, incorporated as a private, nonprofit company under Nepalese management. The transition was finalized in August 1983. The Company will work through a cooperative agreement with USAID/Kathmandu to complement the national family planning goals as the program continues to provide comtraceptives through retail channels at subsidized prices. Company objectives include: increase contraceptive sales by at least 15% per year; make CRS cost effective and move towards self sufficiency; and explore the possibility of marketing noncontraceptive health products to improve primary health care. After only5 years the program can point to some impressive successes. The number of retial shops selling family planning products increased from 100 in 1978 to over 8000, extending CRS product availability to 66 of the country's 75 districts. Retail sales have climbed dramatically in the 5-year period, from Rs 46,817 in 1978 to Rs 271,039 in 1982. Sales in terms of couple year protection CYP) have grown to 24,451 CYP(1982), a 36% increase over 1980 CYP. Since the beginning of the CRS marketing program, total distribution of contraceptives--through both CRS and the Family Planning Maternal and Child Haelth (FP/MCH) Project--has been increasing. While the FP/MCH program remains the largest distributor,contribution of CRS Products is increasing, indicating that CRS is creating new product acceptors. CRS market share in 1982 was 43% for condoms and 16% for oral contraceptives (OCs). CRS markets 5 products which are subsidized in order to be affordable to consumers as well as attractive to sellers. The initial products launched in June 1978 were Gulaf standard dose OCs and Dhaal lubricated colored condoms. A less expensive lubricates, plain Suki-Dhaal condom was introduced in June 1980 in an attempt to reach poorer rural populations, but rural distribution costs are excessive and Suki

  14. CPTAC Assay Portal: a repository of targeted proteomic assays

    SciTech Connect

    Whiteaker, Jeffrey R.; Halusa, Goran; Hoofnagle, Andrew N.; Sharma, Vagisha; MacLean, Brendan; Yan, Ping; Wrobel, John; Kennedy, Jacob; Mani, DR; Zimmerman, Lisa J.; Meyer, Matthew R.; Mesri, Mehdi; Rodriguez, Henry; Abbateillo, Susan E.; Boja, Emily; Carr, Steven A.; Chan, Daniel W.; Chen, Xian; Chen, Jing; Davies, Sherri; Ellis, Matthew; Fenyo, David; Hiltket, Tara; Ketchum, Karen; Kinsinger, Christopher; Kuhn, Eric; Liebler, Daniel; Lin, De; Liu, Tao; Loss, Michael; MacCoss, Michael; Qian, Weijun; Rivers, Robert; Rodland, Karin D.; Ruggles, Kelly; Scott, Mitchell; Smith, Richard D.; Thomas, Stefani N.; Townsend, Reid; Whiteley, Gordon; Wu, Chaochao; Zhang, Hui; Zhang, Zhen; Paulovich, Amanda G.

    2014-06-27

    To address these issues, the Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as a public repository of well-characterized quantitative, MS-based, targeted proteomic assays. The purpose of the CPTAC Assay Portal is to facilitate widespread adoption of targeted MS assays by disseminating SOPs, reagents, and assay characterization data for highly characterized assays. A primary aim of the NCI-supported portal is to bring together clinicians or biologists and analytical chemists to answer hypothesis-driven questions using targeted, MS-based assays. Assay content is easily accessed through queries and filters, enabling investigators to find assays to proteins relevant to their areas of interest. Detailed characterization data are available for each assay, enabling researchers to evaluate assay performance prior to launching the assay in their own laboratory.

  15. Lateral flow assays

    PubMed Central

    Koczula, Katarzyna M.

    2016-01-01

    Lateral flow assays (LFAs) are the technology behind low-cost, simple, rapid and portable detection devices popular in biomedicine, agriculture, food and environmental sciences. This review presents an overview of the principle of the method and the critical components of the assay, focusing on lateral flow immunoassays. This type of assay has recently attracted considerable interest because of its potential to provide instantaneous diagnosis directly to patients. The range and interpretation of results and parameters used for evaluation of the assay will also be discussed. The main advantages and disadvantages of LFAs will be summarized and relevant future improvements to testing devices and strategies will be proposed. Finally, the major recent advances and future diagnostic applications in the LFA field will be explored. PMID:27365041

  16. Cell viability assays: introduction.

    PubMed

    Stoddart, Martin J

    2011-01-01

    The measurement of cell viability plays a fundamental role in all forms of cell culture. Sometimes it is the main purpose of the experiment, such as in toxicity assays. Alternatively, cell viability can be used to -correlate cell behaviour to cell number, providing a more accurate picture of, for example, anabolic -activity. There are wide arrays of cell viability methods which range from the most routine trypan blue dye exclusion assay to highly complex analysis of individual cells, such as using RAMAN microscopy. The cost, speed, and complexity of equipment required will all play a role in determining the assay used. This chapter aims to provide an overview of many of the assays available today.

  17. Tube-Forming Assays.

    PubMed

    Brown, Ryan M; Meah, Christopher J; Heath, Victoria L; Styles, Iain B; Bicknell, Roy

    2016-01-01

    Angiogenesis involves the generation of new blood vessels from the existing vasculature and is dependent on many growth factors and signaling events. In vivo angiogenesis is dynamic and complex, meaning assays are commonly utilized to explore specific targets for research into this area. Tube-forming assays offer an excellent overview of the molecular processes in angiogenesis. The Matrigel tube forming assay is a simple-to-implement but powerful tool for identifying biomolecules involved in angiogenesis. A detailed experimental protocol on the implementation of the assay is described in conjunction with an in-depth review of methods that can be applied to the analysis of the tube formation. In addition, an ImageJ plug-in is presented which allows automatic quantification of tube images reducing analysis times while removing user bias and subjectivity.

  18. Doped colorimetric assay liposomes

    DOEpatents

    Charych, Deborah; Stevens, Raymond C.

    2001-01-01

    The present invention provides compositions comprising colorimetric assay liposomes. The present invention also provides methods for producing colorimetric liposomes and calorimetric liposome assay systems. In preferred embodiments, these calorimetric liposome systems provide high levels of sensitivity through the use of dopant molecules. As these dopants allow the controlled destabilization of the liposome structure, upon exposure of the doped liposomes to analyte(s) of interest, the indicator color change is facilitated and more easily recognized.

  19. Multiple log potash assay

    NASA Astrophysics Data System (ADS)

    Hill, D. G.

    1993-10-01

    A five-mineral multiple-log potash assay technique has been successfully applied to evaluate potash-rich intervals in evaporite sequences. The technique is able to distinguish economic potash minerals from non-economic potash minerals and from other non-potash radioactive minerals. It can be applied on location, using a programmable calculator or microcomputer, providing near real-time logs of potash mineral concentrations. Log assay values show good agreement with core wet chemistry analyses.

  20. SNAP Assay Technology.

    PubMed

    O'Connor, Thomas P

    2015-12-01

    The most widely used immunoassay configuration is the enzyme-linked immunosorbent assay (ELISA) because the procedure produces highly sensitive and specific results and generally is easy to use. By definition, ELISAs are immunoassays used to detect a substance (typically an antigen or antibody) in which an enzyme is attached (conjugated) to one of the reactants and an enzymatic reaction is used to amplify the signal if the substance is present. Optimized ELISAs include several steps that are performed in sequence using a defined protocol that typically includes application of sample and an enzyme-conjugated antibody or antigen to an immobilized reagent, followed by wash and enzyme reaction steps. The SNAP assay is an in-clinic device that performs each of the ELISA steps in a timed sequential fashion with little consumer interface. The components and mechanical mechanism of the assay device are described. Detailed descriptions of features of the assay, which minimize nonspecific binding and enhance the ability to read results from weak-positive samples, are given. Basic principles used in assays with fundamentally different reaction mechanisms, namely, antigen-detection, antibody-detection, and competitive assays are given. Applications of ELISA technology, which led to the development of several multianalyte SNAP tests capable of testing for up to 6 analytes using a single-sample and a single-SNAP device are described.

  1. Serum selenium assay following serum ferritin assay

    SciTech Connect

    Stevens, R.G.; Morris, J.S.; Hann, H.L.; Pulsipher, B.; Stahlhut, M.W.

    1986-08-01

    Stored serum samples can be an important research resource into the etiology of cancer. These sera cannot be replaced and should therefore be used to best advantage. In previous epidemiologic studies, only single serum constituents have been assayed in individual serum samples. For example, serum ferritin has been examined in samples stored for as long as 10 years at -20C for a possible relation with general mortality (1) and cancer death (2). Ferritin is the tissue iron-storage protein and is therefore subject to denaturation. Serum selenium has also been examined in relation to cancer risk in a prospective manner by using stored frozen serum samples (3, 4). The interactions of a variety of serum factors in relation to cancer risk would be a desirable research goal, except that the amounts of serum typically available in frozen serum banks are less than 1 ml. It was the purpose of this investigation to determine if a radioimmunoassay for ferritin affected a subsequent neutron activation assay for selenium on the same 0.1 ml serum sample.

  2. Homogeneous, bioluminescent proteasome assays.

    PubMed

    O'Brien, Martha A; Moravec, Richard A; Riss, Terry L; Bulleit, Robert F

    2015-01-01

    Protein degradation is mediated predominantly through the ubiquitin-proteasome pathway. The importance of the proteasome in regulating degradation of proteins involved in cell-cycle control, apoptosis, and angiogenesis led to the recognition of the proteasome as a therapeutic target for cancer. The proteasome is also essential for degrading misfolded and aberrant proteins, and impaired proteasome function has been implicated in neurodegerative and cardiovascular diseases. Robust, sensitive assays are essential for monitoring proteasome activity and for developing inhibitors of the proteasome. Peptide-conjugated fluorophores are widely used as substrates for monitoring proteasome activity, but fluorogenic substrates can exhibit significant background and can be problematic for screening because of cellular autofluorescence or interference from fluorescent library compounds. Furthermore, fluorescent proteasome assays require column-purified 20S or 26S proteasome (typically obtained from erythrocytes), or proteasome extracts from whole cells, as their samples. To provide assays more amenable to high-throughput screening, we developed a homogeneous, bioluminescent method that combines peptide-conjugated aminoluciferin substrates and a stabilized luciferase. Using substrates for the chymotrypsin-like, trypsin-like, and caspase-like proteasome activities in combination with a selective membrane permeabilization step, we developed single-step, cell-based assays to measure each of the proteasome catalytic activities. The homogeneous method eliminates the need to prepare individual cell extracts as samples and has adequate sensitivity for 96- and 384-well plates. The simple "add and read" format enables sensitive and rapid proteasome assays ideal for inhibitor screening.

  3. SIGMA RECEPTOR BINDING ASSAYS

    PubMed Central

    CHU, UYEN B.; RUOHO, ARNOLD E.

    2016-01-01

    Sigma receptors belong to a class of small molecule-regulated, primarily endoplasmic reticulum (ER) membrane-associated receptors, of which there are two subtypes: the Sigma-1 receptor (S1R) and the Sigma-2 receptor (S2R). Both S1R and S2R bind to a number of drugs including antipsychotic, haloperidol, and the opioid analgesic, (+)-pentazocine. Sigma receptors are implicated in multiple disease pathologies associated with the nervous system including diseases affecting motor control such as Amyotrophic Lateral Sclerosis (ALS) and Alzeimher's disease. This unit describes methods for the pharmacological characterization of S1R and S2R using radioligand-binding assays. In the first section, radioligand saturation binding assay to determine receptor densities and competitive inhibition assays to characterize affinities of novel compounds are presented for S1R using the selective S1R ligand, [3H]-(+)-pentazocine. The second section describes radioligand saturation binding assay and competitive inhibition assays for the S2R using a non-selective S1R and S2R ligand, [3H]-1,3-di(2-tolyl)guanidine ([3H]-DTG). PMID:26646191

  4. Rover waste assay system

    SciTech Connect

    Akers, D.W.; Stoots, C.M.; Kraft, N.C.; Marts, D.J.

    1997-11-01

    The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched {sup 235}U contaminated piping, tank sections, and debris from the Rover nuclear rocket fuel processing facility at the Idaho Chemical Processing Plant. A scanning system translates a NaI(Tl) detector/collimator system over the structural components where both relative and calibrated measurements for {sup 137}Cs are made. Uranium-235 concentrations are in operation and is sufficiently automated that most functions are performed by the computer system. These functions include system calibration, problem identification, collimator control, data analysis, and reporting. Calibration of the system was done through a combination of measurements on calibration standards and benchmarked modeling. A description of the system is presented along with the methods and uncertainties associated with the calibration and analysis of the system for components from the Rover facility. 4 refs., 2 figs., 4 tabs.

  5. Clonogenic Assay: Adherent Cells

    PubMed Central

    Rafehi, Haloom; Orlowski, Christian; Georgiadis, George T.; Ververis, Katherine; El-Osta, Assam; Karagiannis, Tom C.

    2011-01-01

    The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant

  6. Multiplex Flow Assays

    PubMed Central

    2016-01-01

    Lateral flow or dipstick assays (e.g., home pregnancy tests), where an analyte solution is drawn through a porous membrane and is detected by localization onto a capture probe residing at a specific site on the flow strip, are the most commonly and extensively used type of diagnostic assay. However, after over 30 years of use, these assays are constrained to measuring one or a few analytes at a time. Here, we describe a completely general method, in which any single-plex lateral flow assay is transformed into a multiplex assay capable of measuring an arbitrarily large number of analytes simultaneously. Instead of identifying the analyte by its localization onto a specific geometric location in the flow medium, the analyte-specific capture probe is identified by its association with a specific optically encoded region within the flow medium. The capture probes for nucleic acids, antigens, or antibodies are attached to highly porous agarose beads, which have been encoded using multiple lanthanide emitters to create a unique optical signature for each capture probe. The optically encoded capture probe-derivatized beads are placed in contact with the analyte-containing porous flow medium and the analytes are captured onto the encoded regions as the solution flows through the porous medium. To perform a multiplex diagnostic assay, a solution comprising multiple analytes is passed through the flow medium containing the capture probe-derivatized beads, and the captured analyte is treated with a suitable fluorescent reporter. We demonstrate this multiplex analysis technique by simultaneously measuring DNA samples, antigen–antibody pairs, and mixtures of multiple nucleic acids and antibodies. PMID:27819063

  7. Fluorometric assay for aflatoxins

    SciTech Connect

    Chakrabarti, A.G.

    1984-11-01

    The method that is now widely adopted by the government laboratories for the assay of individual aflatoxin components (B/sub 1/, B/sub 2/, G/sub 1/, and G/sub 2/) utilizes a TLC technique. The extraction and clean-up steps of this technique were further researched but the method is still time consuming. It is, therefore, very important to develop a rapid and accurate assay technique for aflatoxins. The current research proposes a technique which utilizes a Turner Fluorometer.

  8. CTL ELISPOT assay.

    PubMed

    Ranieri, Elena; Popescu, Iulia; Gigante, Margherita

    2014-01-01

    Enzyme-linked immune absorbent spot (Elispot) is a quantitative method for measuring relevant parameters of T cell activation. The sensitivity of Elispot allows the detection of low-frequency antigen-specific T cells that secrete cytokines and effector molecules, such as granzyme B and perforin. Cytotoxic T cell (CTL) studies have taken advantage with this high-throughput technology by providing insights into quantity and immune kinetics. Accuracy, sensitivity, reproducibility, and robustness of Elispot resulted in a wide range of applications in research as well as in diagnostic field. Actually, CTL monitoring by Elispot is a gold standard for the evaluation of antigen-specific T cell immunity in clinical trials and vaccine candidates where the ability to detect rare antigen-specific T cells is of relevance for immune diagnostic. The most utilized Elispot assay is the interferon-gamma (IFN-γ) test, a marker for CD8(+) CTL activation, but Elispot can also be used to distinguish different subsets of activated T cells by using other cytokines such as T-helper (Th) 1-type cells (characterized by the production of IFN-γ, IL-2, IL-6, IL-12, IL-21, and TNF-α), Th2 (producing cytokines like IL-4, IL-5, IL-10, and IL-13), and Th17 (IL-17) cells. The reliability of Elispot-generated data, by the evaluation of T cell frequency recognizing individual antigen/peptide, is the core of this method currently applied widely to investigate specific immune responses in cancer, infections, allergies, and autoimmune diseases. The Elispot assay is competing with other methods measuring single-cell cytokine production, e.g., intracellular cytokine by FACS or Miltenyi cytokine secretion assay. Other types of lymphocyte frequency and function assays include limiting dilution assay (LDA), cytotoxic T cell assay (CTL), and tetramer staining. Compared with respect to sensitivity the Elispot assay is outranking other methods to define frequency of antigen-specific lymphocytes. The method

  9. Lateral flow strip assay

    SciTech Connect

    Miles, Robin R; Benett, William J; Coleman, Matthew A; Pearson, Francesca S; Nasarabadi, Shanavaz L

    2011-03-08

    A lateral flow strip assay apparatus comprising a housing; a lateral flow strip in the housing, the lateral flow strip having a receiving portion; a sample collection unit; and a reagent reservoir. Saliva and/or buccal cells are collected from an individual using the sample collection unit. The sample collection unit is immersed in the reagent reservoir. The tip of the lateral flow strip is immersed in the reservoir and the reagent/sample mixture wicks up into the lateral flow strip to perform the assay.

  10. Assays for calcitonin receptors

    SciTech Connect

    Teitelbaum, A.P.; Nissenson, R.A.; Arnaud, C.D.

    1985-01-01

    The assays for calcitonin receptors described focus on their use in the study of the well-established target organs for calcitonin, bone and kidney. The radioligand used in virtually all calcitonin binding studies is /sup 125/I-labelled salmon calcitonin. The lack of methionine residues in this peptide permits the use of chloramine-T for the iodination reaction. Binding assays are described for intact bone, skeletal plasma membranes, renal plasma membranes, and primary kidney cell cultures of rats. Studies on calcitonin metabolism in laboratory animals and regulation of calcitonin receptors are reviewed.

  11. Cascade enzyme-linked immunosorbent assay (CELISA).

    PubMed

    Lee, Young-mi; Jeong, Yujin; Kang, Hyo Jin; Chung, Sang J; Chung, Bong Hyun

    2009-10-15

    Immunoassays are representative biochemical detection methods. Among them, sandwich-type immunoassays, typified by sandwich ELISA, have used in disease diagnosis or biochemical detection with high target selectivity. Horseradish peroxidase and alkaline phosphatase have been typically used for signal amplification in ELISA. Recently developed sandwich-type immunoassays such as biobarcode immunoassays, immuno-PCR, and immuno-RCA have improved sensitivity by changing mainly the signal amplification method. To develop a novel amplification method in ELISA, an enzyme-cascading system was incorporated into an ELISA, and the new assay is termed a cascading enzyme-linked immunosorbent assay (CELISA). This CELISA includes a trypsinogen-enterokinase combination as the cascading enzyme system, and was used to detect alpha-fetoprotein (AFP), which is a liver cancer marker, and prostate-specific antigen (PSA). Using a colorimetric reagent for signal generation, CELISA had 0.1-10pM limits-of-detection for AFP and PSA in whole human serum and assay buffers, depending on the platform, well plate, or microbead type used. This study represents the first example that incorporated an enzyme cascading step in an ELISA system, resulting in successful signal amplification with sensitive detection of pathogenic antigens in serum.

  12. Sigma Receptor Binding Assays.

    PubMed

    Chu, Uyen B; Ruoho, Arnold E

    2015-12-08

    Sigma receptors, both Sigma-1(S1R) and Sigma-2 (S2R), are small molecule-regulated, primarily endoplasmic reticulum (ER) membrane-associated sites. A number of drugs bind to sigma receptors, including the antipsychotic haloperidol and (+)-pentazocine, an opioid analgesic. Sigma receptors are implicated in many central nervous system disorders, in particular Alzheimer's disease and conditions associated with motor control, such as Amyotrophic Lateral Sclerosis (ALS). Described in this unit are radioligand binding assays used for the pharmacological characterization of S1R and S2R. Methods detailed include a radioligand saturation binding assay for defining receptor densities and a competitive inhibition binding assay employing [³H]-(+)-pentazocine for identifying and characterizing novel ligands that interact with S1R. Procedures using [³H]-1,3-di(2-tolyl)guanidine ([³H]-DTG), a nonselective sigma receptor ligand, are described for conducting a saturation binding and competitive inhibition assays for the S2R site. These protocols are of value in drug discovery in identifying new sigma ligands and in the characterization of these receptors.

  13. New oligosaccharyltransferase assay method.

    PubMed

    Kohda, Daisuke; Yamada, Masaki; Igura, Mayumi; Kamishikiryo, Jun; Maenaka, Katsumi

    2007-11-01

    We developed a new in vitro assay for oligosaccharyltransferase (OST), which catalyzes the transfer of preassembled oligosaccharides on lipid carriers onto asparagine residues in polypeptide chains. The asparagine residues reside in the sequon, Asn-X-Thr/Ser, where X can be any amino acid residue except Pro. We demonstrate the potency of our assay using the OST from yeast. In our method, polyacrylamide gel electrophoresis is used to separate the glycopeptide products from the peptide substrates. The substrate peptide is fluorescently labeled and the formation of glycopeptides is analyzed by fluorescence gel imaging. Two in vitro OST assay methods are now widely used, but both the methods depend on previous knowledge of the oligosaccharide moiety: One method uses lectin binding as the separation mechanism and the other method uses biosynthetically or chemoenzymatically synthesized lipid-linked oligosaccharides as donors. N-linked protein glycosylation is found in all three domains of life, but little is known about the N-glycosylation in Archaea. Thus, our new assay, which does not require a priori knowledge of the oligosaccharides, will be useful in such cases. Indeed, we have detected the OST activity in the membrane fraction from a hyperthermophilic archaeon, Pyrococcus furiosus.

  14. Kinetic tetrazolium microtiter assay

    NASA Technical Reports Server (NTRS)

    Pierson, Duane L. (Inventor); Stowe, Raymond P. (Inventor); Koeing, David W. (Inventor)

    1992-01-01

    A method for conducting an in vitro cell assay using a tetrazolium indicator is disclosed. The indicator includes a nonionic detergent which solubilizes a tetrazolium reduction product in vitro and has low toxicity for the cells. The incubation of test cells in the presence of zolium bromide and octoxynol (TRITON X-100) permits kinetics of the cell metabolism to be determined.

  15. Instrument for assaying radiation

    DOEpatents

    Coleman, Jody Rustyn; Farfan, Eduardo B.

    2016-03-22

    An instrument for assaying radiation includes a flat panel detector having a first side opposed to a second side. A collimated aperture covers at least a portion of the first side of the flat panel detector. At least one of a display screen or a radiation shield may cover at least a portion of the second side of the flat panel detector.

  16. Overview of Plant Incorporated Protectants

    EPA Pesticide Factsheets

    When assessing the potential risks of genetically engineered plant-incorporated protectants, EPA requires extensive studies examining numerous factors. Learn more about the history and process for regulating PIPs.

  17. Comparison of luminescence ADP production assay and radiometric scintillation proximity assay for Cdc7 kinase.

    PubMed

    Takagi, Toshimitsu; Shum, David; Parisi, Monika; Santos, Ruth E; Radu, Constantin; Calder, Paul; Rizvi, Zahra; Frattini, Mark G; Djaballah, Hakim

    2011-09-01

    Several assay technologies have been successfully adapted and used in HTS to screen for protein kinase inhibitors; however, emerging comparative analysis studies report very low hit overlap between the different technologies, which challenges the working assumption that hit identification is not dependent on the assay method of choice. To help address this issue, we performed two screens on the cancer target, Cdc7-Dbf4 heterodimeric protein kinase, using a direct assay detection method measuring [(33)P]-phosphate incorporation into the substrate and an indirect method measuring residual ADP production using luminescence. We conducted the two screens under similar conditions, where in one, we measured [(33)P]-phosphate incorporation using scintillation proximity assay (SPA), and in the other, we detected luminescence signal of the ATP-dependent luciferase after regenerating ATP from residual ADP (LUM). Surprisingly, little or no correlation were observed between the positives identified by the two methods; at a threshold of 30% inhibition, 25 positives were identified in the LUM screen whereas the SPA screen only identified two positives, Tannic acid and Gentian violet, with Tannic acid being common to both. We tested 20 out of the 25 positive compounds in secondary confirmatory study and confirmed 12 compounds including Tannic acid as Cdc7-Dbf4 kinase inhibitors. Gentian violet, which was only positive in the SPA screen, inhibited luminescence detection and categorized as a false positive. This report demonstrates the strong impact in detection format on the success of a screening campaign and the importance of carefully designed confirmatory assays to eliminate those compounds that target the detection part of the assay.

  18. The corneal pocket assay.

    PubMed

    Ziche, Marina; Morbidelli, Lucia

    2015-01-01

    The cornea in most species is physiologically avascular, and thus this assay allows the measurement of newly formed vessels. The continuous monitoring of neovascular growth in the same animal allows the evaluation of drugs acting as suppressors or stimulators of angiogenesis. Under anesthesia a micropocket is produced in the cornea thickness and the angiogenesis stimulus (tumor tissue, cell suspension, growth factor) is placed into the pocket in order to induce vascular outgrowth from the limbal capillaries. Neovascular development and progression can be modified by the presence of locally released or applied inhibitory factors or by systemic treatments. In this chapter the experimental details of the avascular cornea assay, the technical challenges, and advantages and disadvantages in different species are discussed. Protocols for local drug treatment and tissue sampling for histology and pharmacokinetic profile are reported.

  19. Kinetic Tetrazolium Microtiter Assay

    NASA Technical Reports Server (NTRS)

    Pierson, Duane L.; Stowe, Raymond; Koenig, David

    1993-01-01

    Kinetic tetrazolium microtiter assay (KTMA) involves use of tetrazolium salts and Triton X-100 (or equivalent), nontoxic, in vitro color developer solubilizing colored metabolite formazan without injuring or killing metabolizing cells. Provides for continuous measurement of metabolism and makes possible to determine rate of action of antimicrobial agent in real time as well as determines effective inhibitory concentrations. Used to monitor growth after addition of stimulatory compounds. Provides for kinetic determination of efficacy of biocide, greatly increasing reliability and precision of results. Also used to determine relative effectiveness of antimicrobial agent as function of time. Capability of generating results on day of test extremely important in treatment of water and waste, disinfection of hospital rooms, and in pharmaceutical, agricultural, and food-processing industries. Assay also used in many aspects of cell biology.

  20. Automated filtration capture immunoelectrochemical assay of bacteria

    NASA Astrophysics Data System (ADS)

    Brewster, Jeffrey D.

    1999-01-01

    An automated system for filtration capture and immunoelectrochemical detection of bactria in liquid samples is described. The detector incorporates a porous electrode in contact with the filter, rather than the solid electrode used previously, to allow sample and reagent solutions to be delivered in a flowing stream. This eliminated the need for manual assembly of the electrode and filter for each assay and allowed repetitive assays on a single filter/electrode. The electrochemical response of the novel gold grid electrode under static and flow conditions was found to be consistent with theory for a planar electrode operating in laminar flow conditions. A computer-controlled fluid handling system was coupled to the detector for delivery of samples and reagents at controlled flow rates and times. The combination of flow detector and fluid handling system allows for automation of the previous assay protocol as well as providing new operating modes with enhanced background rejection and improved sensitivity. The use of these operating modes is demonstrated by a simple assay for Escherichia coli O157:H7 with virtually no background current.

  1. B cell helper assays.

    PubMed

    Abrignani, Sergio; Tonti, Elena; Casorati, Giulia; Dellabona, Paolo

    2009-01-01

    Activation, proliferation and differentiation of naïve B lymphocytes into memory B cells and plasma cells requires engagement of the B cell receptor (BCR) coupled to T-cell help (1, 2). T cells deliver help in cognate fashion when they are activated upon recognition of specific MHC-peptide complexes presented by B cells. T cells can also deliver help in a non-cognate or bystander fashion, when they do not find specific MHC-peptide complexes on B cells and are activated by alternative mechanisms. T-cell dependent activation of B cells can be studied in vitro by experimental models called "B cell helper assays" that are based on the co-culture of B cells with activated T cells. These assays allow to decipher the molecular bases for productive T-dependent B cell responses. We show here examples of B cell helper assays in vitro, which can be reproduced with any subset of T lymphocytes that displays the appropriate helper signals.

  2. Periodontal tissue regeneration with PRP incorporated gelatin hydrogel sponges.

    PubMed

    Nakajima, Dai; Tabata, Yasuhiko; Sato, Soh

    2015-10-20

    Gelatin hydrogels have been designed and prepared for the controlled release of the transforming growth factor (TGF-b1) and the platelet-derived growth factor (PDGF-BB). PRP (Platelet rich plasma) contains many growth factors including the PDGF and TGF-b1. The objective of this study was to evaluate the regeneration of periodontal tissue following the controlled release of growth factors in PRP. For the periodontal ligament cells and osteoblast, PRP of different concentrations was added. The assessment of DNA, mitochondrial activity and ALP activity were measured. To evaluate the TGF-β1 release from PRP incorporated gelatin sponge, amounts of TGF-β1 in each supernatant sample were determined by the ELISA. Transplantation experiments to prepare a bone defect in a rat alveolar bone were an implanted gelatin sponge incorporated with different concentration PRP. In DNA assay and MTT assay, after the addition of PRP to the periodontal ligament cells and osteoblast, the cell count and mitochondrial activity had increased the most in the group with the addition of 5  ×  PRP. In the ALP assay, after the addition of PRP to the periodontal ligament cells, the cell activity had increased the most in the group with the addition of 3  ×  PRP. In the transplantation, the size of the bone regenerated in the defect with 3  ×  PRP incorporated gelatin sponge was larger than that of the other group.

  3. ABCs of DNA aptamer and related assay development.

    PubMed

    Sharma, Tarun Kumar; Bruno, John G; Dhiman, Abhijeet

    This review is intended to guide the novice in aptamer research and development to understand virtually all of the aptamer development options and currently available assay modalities. Aptamer development topics range from discussions of basic and advanced versions of Systematic Evolution of Ligands by EXponential Enrichment (SELEX) and SELEX variations involving incorporation of exotic unnatural nucleotides to expand library diversity for even greater aptamer affinity and specificity to improved next generation methods of DNA sequencing, screening and tracking aptamer development throughout the SELEX process and characterization of lead aptamer candidates. Aptamer assay development topics include descriptions of various colorimetric and fluorescent assays in microplates or on membranes including homogeneous beacon and multiplexed Fluorescence Resonance Energy Transfer (FRET) assays. Finally, a discussion of the potential for marketing successful aptamer-based assays or test kits is included.

  4. Estimates of bacterial growth from changes in uptake rates and biomass.

    PubMed Central

    Kirchman, D; Ducklow, H; Mitchell, R

    1982-01-01

    Rates of nucleic acid synthesis have been used to examine microbiol growth in natural waters. These rates are calculated from the incorporation of [3H]adenine and [3H]thymidine for RNA and DNA syntheses, respectively. Several additional biochemical parameters must be measured or taken from the literature to estimate growth rates from the incorporation of the tritiated compounds. We propose a simple method of estimating a conversion factor which obviates measuring these biochemical parameters. The change in bacterial abundance and incorporation rates of [3H]thymidine was measured in samples from three environments. The incorporation of exogenous [3H]thymidine was closely coupled with growth and cell division as estimated from the increase in bacterial biomass. Analysis of the changes in incorporation rates and initial bacterial abundance yielded a conversion factor for calculating bacterial production rates from incorporation rates. Furthermore, the growth rate of only those bacteria incorporating the compound can be estimated. The data analysis and experimental design can be used to estimate the proportion of nondividing cells and to examine changes in cell volumes. PMID:6760812

  5. Host thin films incorporating nanoparticles

    NASA Astrophysics Data System (ADS)

    Qureshi, Uzma

    The focus of this research project was the investigation of the functional properties of thin films that incorporate a secondary nanoparticulate phase. In particular to assess if the secondary nanoparticulate material enhanced a functional property of the coating on glass. In order to achieve this, new thin film deposition methods were developed, namely use of nanopowder precursors, an aerosol assisted transport technique and an aerosol into atmospheric pressure chemical vapour deposition system. Aerosol assisted chemical vapour deposition (AACVD) was used to deposit 8 series of thin films on glass. Five different nanoparticles silver, gold, ceria, tungsten oxide and zinc oxide were tested and shown to successfully deposit thin films incorporating nanoparticles within a host matrix. Silver nanoparticles were synthesised and doped within a titania film by AACVD. This improved solar control properties. A unique aerosol assisted chemical vapour deposition (AACVD) into atmospheric pressure chemical vapour deposition (APCVD) system was used to deposit films of Au nanoparticles and thin films of gold nanoparticles incorporated within a host titania matrix. Incorporation of high refractive index contrast metal oxide particles within a host film altered the film colour. The key goal was to test the potential of nanopowder forms and transfer the suspended nanopowder via an aerosol to a substrate in order to deposit a thin film. Discrete tungsten oxide nanoparticles or ceria nanoparticles within a titanium dioxide thin film enhanced the self-cleaning and photo-induced super-hydrophilicity. The nanopowder precursor study was extended by deposition of zinc oxide thin films incorporating Au nanoparticles and also ZnO films deposited from a ZnO nanopowder precursor. Incorporation of Au nanoparticles within a VO: host matrix improved the thermochromic response, optical and colour properties. Composite VC/TiC and Au nanoparticle/V02/Ti02 thin films displayed three useful

  6. Selenium incorporation using recombinant techniques

    SciTech Connect

    Walden, Helen

    2010-04-01

    An overview of techniques for recombinant incorporation of selenium and subsequent purification and crystallization of the resulting labelled protein. Using selenomethionine to phase macromolecular structures is common practice in structure determination, along with the use of selenocysteine. Selenium is consequently the most commonly used heavy atom for MAD. In addition to the well established recombinant techniques for the incorporation of selenium in prokaryal expression systems, there have been recent advances in selenium labelling in eukaryal expression, which will be discussed. Tips and things to consider for the purification and crystallization of seleno-labelled proteins are also included.

  7. 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.

  8. 17 CFR 260.7a-30 - Identification of material incorporated; form of incorporation.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... incorporated; form of incorporation. 260.7a-30 Section 260.7a-30 Commodity and Securities Exchanges SECURITIES... Incorporation by Reference § 260.7a-30 Identification of material incorporated; form of incorporation. In each case of incorporation by reference, the matter incorporated shall be clearly identified in...

  9. Influence of grape pomace extract incorporation on chitosan films properties.

    PubMed

    Ferreira, Andreia S; Nunes, Cláudia; Castro, Alichandra; Ferreira, Paula; Coimbra, Manuel A

    2014-11-26

    Chitosan has been studied as a renewable polymer to form edible films allowing the incorporation of functional compounds. The aim of this work was to evaluate the effects in the chitosan films properties of the incorporation of grape pomace extracts: 0.15% of hot water extract (mainly polysaccharides), 0.15 and 0.3% of chloroform extract (wax), and 0.3 and 0.75% of n-hexane extract (oil). The evaluation of the surface morphology revealed that the films with the aqueous extract had the most homogeneous and smoother topography. The incorporation of higher proportion of wax and oil led to changes in mechanical properties of the films, namely lower resistance and stiffness. The chitosan-based films with 0.75% oil demonstrated a 75% decrease of solubility in water, due to their hydrophobicity, as confirmed by the contact angle and surface free energy measurements. The hydrophobic films showed higher antioxidant capacity in organic medium (ABTS and DPPH assays) whereas the most hydrophilic films showed an improvement in FRAP and reducing power assays. Therefore, all the chitosan-based films prepared by incorporation of these grape pomace extracts are promising for food shelf life extension.

  10. Radon assay for SNO+

    SciTech Connect

    Rumleskie, Janet

    2015-12-31

    The SNO+ experiment will study neutrinos while located 6,800 feet below the surface of the earth at SNOLAB. Though shielded from surface backgrounds, emanation of radon radioisotopes from the surrounding rock leads to back-grounds. The characteristic decay of radon and its daughters allows for an alpha detection technique to count the amount of Rn-222 atoms collected. Traps can collect Rn-222 from various positions and materials, including an assay skid that will collect Rn-222 from the organic liquid scintillator used to detect interactions within SNO+.

  11. Radon assay for SNO+

    NASA Astrophysics Data System (ADS)

    Rumleskie, Janet

    2015-12-01

    The SNO+ experiment will study neutrinos while located 6,800 feet below the surface of the earth at SNOLAB. Though shielded from surface backgrounds, emanation of radon radioisotopes from the surrounding rock leads to back-grounds. The characteristic decay of radon and its daughters allows for an alpha detection technique to count the amount of Rn-222 atoms collected. Traps can collect Rn-222 from various positions and materials, including an assay skid that will collect Rn-222 from the organic liquid scintillator used to detect interactions within SNO+.

  12. Incorporating Argumentation through Forensic Science

    ERIC Educational Resources Information Center

    Wheeler, Lindsay B.; Maeng, Jennifer L.; Smetana, Lara K.

    2014-01-01

    This article outlines how to incorporate argumentation into a forensic science unit using a mock trial. Practical details of the mock trial include: (1) a method of scaffolding students' development of their argument for the trial, (2) a clearly outlined set of expectations for students during the planning and implementation of the mock…

  13. Zebrafish Assays of Ciliopathies

    PubMed Central

    Zaghloul, Norann A.; Katsanis, Nicholas

    2013-01-01

    In light of the growing list of human disorders associated with their dysfunction, primary cilia have recently come to attention as being important regulators of developmental signaling pathways and downstream processes. These organelles, present on nearly every vertebrate cell type, are highly conserved structures allowing for study across a range of species. Zebrafish, in particular, have emerged as useful organisms in which to explore the consequences of ciliary dysfunction and to model human ciliopathies. Here, we present a range of useful techniques that allow for investigation of various aspects of ciliary function. The described assays capitalize on the hallmark gastrulation defects associated with ciliary defects as well as relative ease of visualization of cilia in whole-mount embryos. Further, we describe our recently developed assay for querying functionality of human gene variants in live developing embryos. Finally, a current catalog of known zebrafish ciliary mutant lines is included. The techniques presented here provide a basic toolkit for in vivo investigation of both the biological and genetic mechanisms underlying a growing class of human diseases. PMID:21951534

  14. RAS - Screens & Assays - Drug Discovery

    Cancer.gov

    The RAS Drug Discovery group aims to develop assays that will reveal aspects of RAS biology upon which cancer cells depend. Successful assay formats are made available for high-throughput screening programs to yield potentially effective drug compounds.

  15. Biosensors: Viruses for ultrasensitive assays

    NASA Astrophysics Data System (ADS)

    Donath, Edwin

    2009-04-01

    A three-dimensional assay based on genetically engineered viral nanoparticles and nickel nanohairs can detect much lower levels of protein markers associated with heart attacks than conventional assays.

  16. Chemotaxis: Under Agarose Assay.

    PubMed

    Brazill, Derrick

    2016-01-01

    The unicellular eukaryote Dictyostelium discoideum represents a superb model for examining chemotaxis. Under vegetative conditions, the amoebae are chemotactically responsive to pterins, such as folate. Under starved conditions, they lose their sensitivity to pterins, and become chemotactically responsive to cAMP. As an NIH model system, Dictyostelium offers a variety of advantages in studying chemotaxis, including its conservation of mammalian signaling pathways, its ease of growth, and its genetic tractability. In this chapter, we describe the use of the under agarose chemotaxis assay to identify proteins involved in controlling motility and directional sensing in Dictyostelium discoideum. Given the similarities between Dictyostelium and mammalian cells, this allows us to dissect the conserved pathways involved in eukaryotic chemotaxis.

  17. Selenium incorporation using recombinant techniques.

    PubMed

    Walden, Helen

    2010-04-01

    Using selenomethionine to phase macromolecular structures is common practice in structure determination, along with the use of selenocysteine. Selenium is consequently the most commonly used heavy atom for MAD. In addition to the well established recombinant techniques for the incorporation of selenium in prokaryal expression systems, there have been recent advances in selenium labelling in eukaryal expression, which will be discussed. Tips and things to consider for the purification and crystallization of seleno-labelled proteins are also included.

  18. Survival assays using Caenorhabditis elegans

    PubMed Central

    Park, Hae-Eun H.; Jung, Yoonji; Lee, Seung-Jae V.

    2017-01-01

    Caenorhabditis elegans is an important model organism with many useful features, including rapid development and aging, easy cultivation, and genetic tractability. Survival assays using C. elegans are powerful methods for studying physiological processes. In this review, we describe diverse types of C. elegans survival assays and discuss the aims, uses, and advantages of specific assays. C. elegans survival assays have played key roles in identifying novel genetic factors that regulate many aspects of animal physiology, such as aging and lifespan, stress response, and immunity against pathogens. Because many genetic factors discovered using C. elegans are evolutionarily conserved, survival assays can provide insights into mechanisms underlying physiological processes in mammals, including humans. PMID:28241407

  19. A fluorescent microsphere-based method for assay of multiple analytes in plasma.

    PubMed

    Bernhard, Oliver K; Mathias, Rommel A; Barnes, Thomas W; Simpson, Richard J

    2011-01-01

    Measurement of multiple analytes can provide increased sensitivity and specificity for the detection and management of disease. The enzyme-linked immunosorbent assay (ELISA) is currently the "gold standard" for protein quantification; however, individual assays for each analyte must be performed, placing demand on sample volume. On the contrary, multiplex assays using microsphere-based technologies allow for multiple analytes to be simultaneously assayed within a single sample. Here, we present a protocol for the preparation and development of a multiple-analyte assay in human plasma using the BioPlex 200 platform (Bio-Rad), which incorporates xMAP technology (Luminex).

  20. Cell Proliferation and Cytotoxicity Assays.

    PubMed

    Adan, Aysun; Kiraz, Yağmur; Baran, Yusuf

    Cell viability is defined as the number of healthy cells in a sample and proliferation of cells is a vital indicator for understanding the mechanisms in action of certain genes, proteins and pathways involved cell survival or death after exposing to toxic agents. Generally, methods used to determine viability are also common for the detection of cell proliferation. Cell cytotoxicity and proliferation assays are generally used for drug screening to detect whether the test molecules have effects on cell proliferation or display direct cytotoxic effects. Regardless of the type of cell-based assay being used, it is important to know how many viable cells are remaining at the end of the experiment. There are a variety of assay methods based on various cell functions such as enzyme activity, cell membrane permeability, cell adherence, ATP production, co-enzyme production, and nucleotide uptake activity. These methods could be basically classified into different categories: (I) dye exclusion methods such as trypan blue dye exclusion assay, (II) methods based on metabolic activity, (III) ATP assay, (IV) sulforhodamine B assay, (V) protease viability marker assay, (VI) clonogenic cell survival assay, (VII) DNA synthesis cell proliferation assays and (V) raman micro-spectroscopy. In order to choose the optimal viability assay, the cell type, applied culture conditions, and the specific questions being asked should be considered in detail. This particular review aims to provide an overview of common cell proliferation and cytotoxicity assays together with their own advantages and disadvantages, their methodologies, comparisons and intended purposes.

  1. Coagulation assays and anticoagulant monitoring.

    PubMed

    Funk, Dorothy M Adcock

    2012-01-01

    Anticoagulant therapy, including conventional agents and a variety of new oral, fast-acting drugs, is prescribed for millions of patients annually. Each anticoagulant varies in its effect on routine and specialty coagulation assays and each drug may require distinct laboratory assay(s) to measure drug concentration or activity. This review provides an overview of the assorted assays that can measure anticoagulant drug concentration or activity and includes key assay interferences. The effect of these conventional and new anticoagulant agents on specialty coagulation assays used to evaluate for bleeding or clotting disorders, and whether this impact is physiological or factitious, is included. Also provided is a short review of superwarfarin poisoning and features distinguishing this from warfarin overdose. Knowledge of clinically significant pearls and pitfalls pertinent to coagulation assays in relation to anticoagulant therapy are important to optimize patient care.

  2. Quantification of histoautoradiographic evidence of DNA repair synthesis in the liver.

    PubMed

    Hochmann, J; Stambergová, H

    1988-01-01

    Histoautoradiography was used to detect dimethylnitrosamine-induced 3H-thymidine incorporation in vivo into G phase hepatocytes. A description of the standard procedure for counting the grains as well as the mode of mathematical evaluation is presented. The results exhibited higher sensitivity than those in the investigation of the DNA repair synthesis by means of a scintillation counter using the method of detection of hydroxyurea-resistant incorporation of 3H-thymidine. Thus it was possible to simplify the investigation by lowering the number of evaluated cells. A suitable compromise between precision and laboriousness will probably be achieved by counting 20 hepatocytes per animal. In case that there are striking differences between the experimental and the control group, a qualitative conclusion may be drawn even without counting the grains.

  3. Rapid anterograde spread of premitotic activity along degenerating cat sciatic nerve.

    PubMed

    Oaklander, A L; Miller, M S; Spencer, P S

    1987-01-01

    Peripheral nerve transection triggers a series of phenotypic alterations in Schwann cells distal to the site of injury. Mitosis is one of the earliest and best characterized of these responses, although the mechanism by which axonal damage triggers this critical event is unknown. This study examines the appearance and spatio-temporal spread of premitotic activity in distal stumps of transected cat tibial nerves. Premitotic activity was determined by measuring incorporation of [3H]thymidine (a marker of DNA synthesis during the S-phase of the cell cycle) into consecutive segments of desheathed tibial nerve. Incorporation of [3H]thymidine spread proximo-distally within distal nerve stumps between 3 and 4 days posttransection with an apparent velocity of at least 199 +/- 67 mm/day. This suggests that anterograde fast axonal transport may directly or indirectly be associated with the Schwann cell mitotic response to axon transection.

  4. Limenin, a defensin-like peptide with multiple exploitable activities from shelf beans.

    PubMed

    Wong, Jack H; Ng, T B

    2006-05-01

    From the seeds of the shelf bean, an antifungal peptide with a molecular mass of 6.5 kDa was isolated. The isolation procedure comprised affinity chromatography on Affi-gel blue gel, ion exchange chromatography on Mono S, and gel filtration on Superdex 75. The peptide was adsorbed on Affi-gel blue gel and Mono S. It potently suppressed mycelial growth in Botrytis cinerea, Fusarium oxysporum, and Mycosphaerella arachidicola with an IC(50) of 2.9, 2.1, and 0.34 microM, respectively. It exerted antibacterial activity toward several bacterial species with an IC(50) approximating 100 microM. [Methyl-(3)H]-thymidine incorporation into isolated mouse splenocytes was stimulated. [Methyl-(3)H]-thymidine incorporation into M1 (myeloma) and L1210 (leukemia) cells was inhibited. The peptide reduced the activity of HIV-1 reverse transcriptase and also inhibited translation in a cell-free rabbit reticulocyte lysate system.

  5. Effect of cholinomimetics and adrenomimetics on proliferation of mouse B lymphocytes during primary immune response to protein antigen

    SciTech Connect

    Ado, A.D.; Dontsov, V.I.; Gol'dshtein, M.M.

    1985-12-01

    The aim of this investigation was to study the effect of neurotransmitters on proliferation of B lymphocytes induced by specific antigen. Experiments were carried out on female mice. To estimate proliferative activity, lymphocytes enriched with B cells were incubated in medium 199 for 2 h at 37 degrees C in a dose of 2.10/sup 6/-5.10/sup 6/ cells with 2 microCi of /sup 3/H-(methyl)-thymidine. The effect of acetylcholine on incorporation of /sup 3/H-thymidine into B lymphocytes of mice immunized with different doses of antigen during culture is shown. Discordance of effects of adrenalin and acetylcholine on incorporation of /sup 3/H-thymidine into B lymphocytes of mice immunized with different doses of ovalbumin is also shown.

  6. Effects of proposed adipogenic factors in fetal swine sera upon preadipocyte development

    SciTech Connect

    Ramsay, T.G.; Hausman, G.J.; Martin, R.J.

    1986-03-01

    Genetic obesity has been detected in fetal pigs which suggests primary factors that cause the obesity develop prenatally. Growth hormone and thyroid hormones have been implicated as regulatory factors in fetal serum for preadipocyte differentiation. This experiment examined effects of growth hormone (GH) and thyroxine (T4) addition upon preadipocyte proliferation and differentiation when supplemented to deficient fetal pig sea. Hormones were added to decapitated fetal pig (Decap) sera to concentrations present in intact littermate (Reference) sera. Primary stromal-vascular cell cultures were prepared from rat inguinal adipose tissue. Cells were incubated with 5% decap or reference sera and hormones in media 199 during: days 1 to 5 for a /sup 3/H-thymidine incorporation assay; days 1 to 15 for assay of ..cap alpha..-glycerol phosphate dehydrogenase; days 5 to 14 for a complete differentiation assay. Decap sera promoted less proliferation and enzyme differentiation than reference sera with no effect of GH addition. GH reduced detection of lipid accumulating cells on percol density gradients by 81%. T4 addition stimulated preadipocyte multiplication and produced a 30% increase in completely differentiated preadipocytes. These results indicate thyroid hormones are important components of fetal sera for regulation of preadipocyte development, whereas GH may only affect cellular metabolism.

  7. Heparin fragments inhibit human vascular smooth muscle cell proliferation in vitro

    SciTech Connect

    Selden, S.C.; Johnson, W.V.; Maciag, T.

    1986-03-01

    The authors have examined the effect of heparin on human abdominal aortic smooth muscle cell growth. Cell proliferation was inhibited by more than 90% at a concentration of 20 ..mu..g/ml in a 12 day growth assay using heparin from Sigma, Upjohn or Calbiochem. Additionally, 200 ..mu..g/ml Upjohn heparin inhibits /sup 3/H-thymidine incorporation by 50% in short term assays using serum or purified platelet-derived growth factor (25-100ng/ml) to initiate the cell cycle. Homogeneous size classes of heparin fragments were prepared by nitrous acid cleavage and BioGel P-10 filtration chromatography. Deca-, octa-, hexa-, tetra-, and di-saccharides inhibited proliferation by 90% at concentrations of 280, 320, 260, 180 and 100 ..mu..g/ml, respectively, in a 12 day growth assay. These data confirm the work of Castellot et.al. and extend the range of inhibitory fragments down to the tetra- and di-saccharide size. These data suggest, therefore, that di-saccharide subunit of heparin is sufficient to inhibit vascular smooth muscle cell proliferation. The authors are now examining the role of the anhydromannose moiety on the reducing end of the nitrous acid generated fragments as a possible mediator of heparin-induced inhibition of vascular smooth muscle cell proliferation.

  8. Incorporating Spirituality in Primary Care.

    PubMed

    Isaac, Kathleen S; Hay, Jennifer L; Lubetkin, Erica I

    2016-06-01

    Addressing cultural competency in health care involves recognizing the diverse characteristics of the patient population and understanding how they impact patient care. Spirituality is an aspect of cultural identity that has become increasingly recognized for its potential to impact health behaviors and healthcare decision-making. We consider the complex relationship between spirituality and health, exploring the role of spirituality in primary care, and consider the inclusion of spirituality in existing models of health promotion. We discuss the feasibility of incorporating spirituality into clinical practice, offering suggestions for physicians.

  9. Incorporation of additives into polymers

    DOEpatents

    McCleskey, T. Mark; Yates, Matthew Z.

    2003-07-29

    There has been invented a method for incorporating additives into polymers comprising: (a) forming an aqueous or alcohol-based colloidal system of the polymer; (b) emulsifying the colloidal system with a compressed fluid; and (c) contacting the colloidal polymer with the additive in the presence of the compressed fluid. The colloidal polymer can be contacted with the additive by having the additive in the compressed fluid used for emulsification or by adding the additive to the colloidal system before or after emulsification with the compressed fluid. The invention process can be carried out either as a batch process or as a continuous on-line process.

  10. Biomolecular Interaction Assay

    SciTech Connect

    Bruckner-Lea, Cindy J.; Brown, L; Holman, David A.; Olson, Lydia; Grate, Jay W.

    2000-12-29

    Understanding the binding interactions of complexes of multiple proteins is an important area of medical research since many biological signaling pathways involve multiple protein complexes. A number of sensor technologies have been adapted to monitoring biomolecular interactions. Acoustic wave devices such as flexural plate wave devices, surface transverse waves, and quartz crystal microbalances detect the mass increase observed upon binding of a solution biomolecule to a surface bound biomolecule. However, these devices will also respond to changes in viscosity, temperature, liquid density, and viscoelastic effects, which may confound the interpretation of observed signals. Nonspecific binding is indistinguishable from specific binding. Several techniques for refractive index sensing, such as planar wave guides and surface plasmon resonance (SPR), can also be used to observe biomolecular interactions localized at a surface. Again, nonspecific binding is indistinguishable from specific binding. In addition, the derivatized surface must be very thin and uniform to obtain adequate sensitivity and reproducibility, and the technique is not suited for monitoring large multiple protein complexes since the measurement sensitivity decreases rapidly with distance from the sensor surface. All of these techniques use planar surfaces that are difficult to prepare and characterize, and must be prepared fresh for each assay.

  11. Assay for calcium channels

    SciTech Connect

    Glossmann, H.; Ferry, D.R.

    1985-01-01

    This chapter focuses on biochemical assays for Ca/sup 2 +/-selective channels in electrically excitable membranes which are blocked in electrophysiological and pharmacological experiments by verapamil, 1,4-dihydropyridines, diltiazen (and various other drugs), as well as inorganic di- or trivalent cations. The strategy employed is to use radiolabeled 1,4-dihydropyridine derivatives which block calcium channels with ED/sub 50/ values in the nanomolar range. Although tritiated d-cis-diltiazem and verapamil can be used to label calcium channels, the 1,4-dihydropyridines offer numerous advantages. The various sections cover tissue specificity of channel labeling, the complex interactions of divalent cations with the (/sup 3/H)nimodipine-labeled calcium channels, and the allosteric regulation of (/sup 3/H)nimodipine binding by the optically pure enantiomers of phenylalkylamine and benzothiazepine calcium channel blockers. A comparison of the properties of different tritiated 1,4-dihydropyridine radioligands and the iodinated channel probe (/sup 125/I)iodipine is given.

  12. The interaction of signal transduction pathways in FRTL5 thyroid follicular cells: Studies with stable expression of beta 2-adrenergic receptors

    SciTech Connect

    Tsuzaki, S.; Cone, R.D.; Frazier, A.L.; Moses, A.C. )

    1991-03-01

    Multiple signal transduction pathways interact in FRTL5 cells to promote thyroid follicular cell differentiated function and cell proliferation. In these cells, TSH is a tissue-specific mitogen that promotes DNA synthesis primarily through activation of adenylate cyclase. To further test the role of adenylate cyclase in regulating cell growth and differentiated function we have introduced into FRTL5 the human beta 2-adrenergic receptor (BAR) complementary DNA and have studied the ability of isoproterenol, alone and in combination with insulin-like growth factor I (IGF-I), to stimulate cAMP accumulation, iodide transport, (3H)thymidine incorporation into DNA, and cell growth. Wild-type FRTL5 were infected with a PLJ retroviral construct containing the BAR in either a sense (FRTL BAR) or antisense (FRTL RBAR) orientation, and cell populations were selected on the basis of resistance to the antibiotic geneticin. FRTL BAR expressed approximately 1.3 x 10(5) high affinity binding sites per cell for the beta 2-specific ligand, CGP-12177, while neither FRTL5 wild-type nor RBAR cells demonstrated any specific binding. FRTL BAR had significantly higher levels of intracellular cAMP, (3H)thymidine incorporation, and iodide uptake in the absence of added isoproterenol than FRTL RBAR or wild-type cells. In FRTL BAR, but not RBAR cells, isoproterenol stimulated a dose-dependent accumulation of cAMP, iodide uptake, (3H)thymidine incorporation, and cell growth. FRTL BAR and RBAR cells were equally responsive to TSH and to IGF-I. Isoproterenol enhanced the ability of IGF-I to stimulate (3H)thymidine incorporation in BAR but not RBAR cells. Isoproterenol partially inhibited the ability of TSH to stimulate cAMP generation and DNA synthesis.

  13. Biomedical applications of digital autoradiography with a MWPC

    SciTech Connect

    Bellazzini, R.; Betti, G.; Del Guerra, A.; Massai, M.M.; Ragadini, M.; Spandre, G.; Tonelli, G.; Venturi, R.; Zito, F.

    1982-05-01

    A Multiwire Proportional Chamber (MWPC) was used as a ..beta../sup -/ radioactivity detector in biological and medical applications. Two different kinds of experiments were performed: the study of variations in the ability of cell clones to incorporate a radioactive precursor of DNA biosynthesis (/sup 3/H-thymidine) and the regional carbohydrate consumption in myocardial tissue by means of a deposit tracer of glucose metabolism (/sup 3/H-deoxyglucose).

  14. A preparative suspension culture system permitting quantitation of anchorage-independent growth by direct radiolabeling of cellular DNA.

    PubMed

    Assoian, R K; Boardman, L A; Drosinos, S

    1989-02-15

    We have developed a hybrid methylcellulose/agar suspension culture system which permits long-term colony formation of transformed mesenchymal cells. In contrast to traditional agar suspensions, our system allows for recovery of cells and direct biochemical analysis of anchorage-independent growth. The ability to readily radiolabel cellular macromolecules in these preparative cultures permits a quantitative and objective analysis of colony formation by incorporation of [3H]thymidine into newly synthesized DNA.

  15. Stimulation of DNA and Collagen Synthesis by Autologous Growth Factor in Cultured Fetal Rat Calvaria

    NASA Astrophysics Data System (ADS)

    Canalis, Ernesto; Peck, William A.; Raisz, Lawrence G.

    1980-11-01

    Conditioned medium derived from organ or cell cultures prepared from 19- to 21-day fetal rat calvaria stimulated the incorporation of [3H]proline into collagen and of [3H]thymidine into DNA in organ cultures of the same tissue. Addition of cortisol enhanced the effect on collagen but not on DNA synthesis. These effects appeared to be due to a nondialyzable and heat-stable growth factor.

  16. DNA repair synthesis in the rat retina following in vivo exposure to 300-nm radiation

    SciTech Connect

    Rapp, L.M.; Jose, J.G.; Pitts, D.G.

    1985-03-01

    Quantitative autoradiography was used to study the incorporation of /sup 3/H-thymidine into the retina of albino rats following in vivo exposure to 300-nm radiation. Relative to background labeling in unexposed eyes, there was 8-20 times as much label per unit area in the outer nuclear layer, inner nuclear layer, and ganglion cells of 300-nm exposed retinas. The photoreceptor inner segments also showed thymidine labeling in both control and exposed retinas.

  17. Cell migration and invasion assays.

    PubMed

    Moutasim, Karwan A; Nystrom, Maria L; Thomas, Gareth J

    2011-01-01

    A number of in vitro assays have been developed to study tumor cell motility. Historically, assays have been mainly monocellular, where carcinoma cells are studied in isolation. Scratch assays can be used to study the collective and directional movement of populations of cells, whereas two chamber assays lend themselves to the analysis of chemotactic/haptotactic migration and cell invasion. However, an inherent disadvantage of these assays is that they grossly oversimplify the complex process of invasion, lacking the tumor structural architecture and stromal components. Organotypic assays, where tumor cells are grown at an air/liquid interface on gels populated with stromal cells, are a more physiologically relevant method for studying 3-dimensional tumor invasion.

  18. Evaluation of Five Jet Fuels in the Salmonella-Escherichia coli / Microsome Plate Incorporation Assay

    DTIC Science & Technology

    2010-09-01

    provide data relating to the test substance’s health effects, environmental effects, or environmental fate testing regulated by the U.S. Environmental...procedures will be consistent with the Office of Prevention, Pesticides and Toxic Substances (OPPTS), Health Effects Test Guidelines, 870.5100 (U.S...Mutat. Res. 455: 29-60, 2000. U.S. Environmental Protection Agency (EPA). Office of Prevention, Pesticides and Toxic Substances (OPPTS), Health

  19. Enzymatic incorporation and utilization of an emissive 6-azauridine.

    PubMed

    Hopkins, Patrycja A; McCoy, Lisa S; Tor, Yitzhak

    2017-01-18

    To display favorable fluorescent properties, the non-emissive native nucleosides need to be modified. Here we present a motif that relies on conjugating 5-membered aromatic heterocycles (e.g., thiophene) to a 6-azapyrimidine (1,2,4-triazine) core. Synthetic accessibility and desirable photophysical properties make these nucleosides attractive candidates for enzymatic incorporation and biochemical assays. While 6-azauridine triphosphate is known to be poorly tolerated by polymerases in RNA synthesis, we illustrate that conjugating a thiophene ring at position 5 overcomes such limitations, facilitating its T7 RNA polymerase-mediated in vitro transcription incorporation into RNA constructs. We further show that the modified transcripts can be ligated to longer oligonucleotides to form singly modified RNAs, as illustrated for an A-site hairpin model RNA construct, which was employed to visualize aminoglycoside antibiotics binding.

  20. Neurotensin induces hyperplasia of the pancreas and growth of the gastric antrum in rats.

    PubMed

    Feurle, G E; Müller, B; Rix, E

    1987-01-01

    We investigated whether chronic subcutaneous infusion of neurotensin during 14 days would affect pancreatic and gastric growth of rats. In another experiment, neurotensin (836 pmol/kg) was injected intraperitoneally three times a day for three days in 12 rats. Thereafter, pancreatic DNA and in vitro incorporation of 3H-thymidine into pancreatic DNA was determined. Long term infusion of 282 pmol/kg neurotensin induced an increase of pancreatic weight, DNA, and pancreatic polypeptide, whereas pancreatic protein, RNA, amylase and lipase contents were not increased. In relation to DNA, even these parameters were significantly depressed. Insulin remained unchanged. Neurotensin, therefore, caused hyperplasia of the pancreas. Intraperitoneal injection of neurotensin induced an increase of pancreatic DNA content and stimulated 3H-thymidine incorporation into DNA, whereas caerulein only augmented 3H-thymidine incorporation. Moreover, long term neurotensin infusion led to a rise in protein concentration and an increase in the thickness of the gastric antrum; antral DNA concentration was insignificantly stimulated. Neurotensin, therefore, can act as a trophic factor on pancreas and gastric antrum of the rat.

  1. Chlorinated dibenzo-P-dioxins and dibenzofurans and the human immune system (2) In vitro proliferation of lymphocytes from workers with quantified moderately-increased body burdens

    SciTech Connect

    Neubert, R.; Maskow, L.; Delgado, I.

    1995-11-01

    Lymphocyte proliferation responses were studied in workers with moderately increased body burdens of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin and other polychlorinated dibenzo-p-dioxin and other polyclorinated dibenzo-p-dioxins and dibenzofurans (PCDDs/PCDFs), calculated as International Toxicity Equivalencies [I-TE]. Mitogens (pokeweed mitogen [PWM], phytohemagglutinine [PHA], concanavalin A [Con A], as well as anti-human monoclonal antibody against CD3 were used as proliferation stimulators in vitro. Additionally, the feasibility of using the lymphocyte response to tetanus toxoid was assessed, and the response to this recall-antigen was included in this trial. No decrease in the capacity of {sup 3}H-thymidine incorporation was observed with any of the proliferation stimulators in the group of volunteers with the increased TCDD-body burden when compared with volunteers exhibiting TCDD-concentrations in blood flat within the reference range. Regression analysis revealed a slight trend towards an increase for {sup 3}H-thymidine incorporation during the stimulation with PHA only. It can be concluded from our data that moderates increases in the TCDD- or I-TE-body burdens do not induce any medically significant changes in the capacity for proliferation of lymphocytes, measured as {sub 3}H=thymidine incorporation. 25 refs., 5 figs., 3 tabs.

  2. Incorporation of heparin into biomaterials.

    PubMed

    Sakiyama-Elbert, Shelly E

    2014-04-01

    This review provides an overview of the incorporation of heparin into biomaterials with a focus on drug delivery and the use of heparin-based biomaterials for self-assembly of polymer networks. Heparin conjugation to biomaterials was originally explored to reduce the thrombogenicity of materials in contact with blood. Many of the conjugation strategies that were developed for these applications are still popular today for other applications. More recently heparin has been conjugated to biomaterials for drug delivery applications. Many of the delivery approaches have taken advantage of the ability of heparin to bind to a wide variety of growth factors, protecting them from degradation and potentiating interactions with cell surface receptors. More recently, the use of heparin as a base polymer for scaffold fabrication has also been explored, often utilizing non-covalent binding of heparin with peptides or proteins to promote self-assembly of hydrogel networks. This review will highlight recent advances in each of these areas.

  3. Click-iT assay with improved DNA distribution histograms.

    PubMed

    Hamelik, Ronald M; Krishan, Awtar

    2009-10-01

    The Click-iT Assay developed and commercialized by Invitrogen is based on incorporation of a new 5-bromo-2'-deoxyuridine analog, 5-ethynyl-2'-deoxyuridine (EdU) into newly synthesized DNA and its recognition by azide dyes via a copper mediated "click" reaction. This relatively convenient and useful procedure depends on fixation of cells with paraformaldehyde and staining of the DNA with 7-aminoactinomycin-D (7-AAD). Both of these procedures result in DNA histograms with broad coefficients of variation (CV's). In this report, we have shown that after EdU incorporation, nuclei isolated by lysis can be incubated with the Click-iT Assay and stained with propidium iodide for generation of DNA histograms with low CV's. This modified procedure results in better DNA histograms by replacing 7-AAD with propidium iodide and also saves processing time by eliminating the fixation and permeabilization steps.

  4. Click-iT proliferation assay with improved DNA histograms.

    PubMed

    Krishan, Awtar; Hamelik, Ronald M

    2010-04-01

    The Click-iT EdU cell proliferation assay (Invitrogen) for detection of replicating cells is based on incorporation of EdU into newly synthesized DNA and its recognition by azide dyes via a copper mediated "click" reaction. In the protocol provided by Invitrogen, cells are fixed with paraformaldehyde and stained with 7-aminoactinomycin D (7-AAD) for DNA content analysis. Both of these procedures result in DNA histograms with a broad coefficient of variation. We have modified this protocol and show that after EdU incorporation, nuclei isolated by hypotonic lysis of cells can be directly labeled using the Click-iT Alexa Fluor 488 Assay kit and stained with propidium iodide. This modified procedure using isolated nuclei and propidium iodide staining results in DNA histograms with better resolution (lower coefficient of variation of the G(1) peak) and shorter processing time by eliminating the fixation and permeabilization steps.

  5. Comparison of the Third Wave Invader Human Papillomavirus (HPV) Assay and the Digene HPV Hybrid Capture 2 Assay for Detection of High-Risk HPV DNA▿

    PubMed Central

    Ginocchio, C. C.; Barth, D.; Zhang, F.

    2008-01-01

    This study compared the clinical performance of the Digene Hybrid Capture 2 (HC2) assay to that of a prototype Third Wave Invader human papillomavirus (HPV) (IHPV) analyte-specific reagent-based assay for the detection of oncogenic or “high-risk” (HR) HPV DNA using liquid-based cytology specimens. In total, 821 ThinPrep vials were tested using both assays. In accordance with the type-specific probes contained within each test, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for the IHPV assay were 95.9%, 97.6%, 97.5%, and 96.1%, respectively, and those for the HC2 assay were 98.1%, 86.2%, 87.1%, and 97.9%. Overall, the sensitivity and NPV were comparable between the assays, but the IHPV assay demonstrated a better specificity and PPV, since the IHPV assay had fewer false-positive HR HPV results. The incorporation of an internal control to evaluate the cellularity of the test material is an important feature of the IHPV assay and should reduce the risk of false-negative results due to insufficient sample collection rather than the lack of HR HPV DNA. An additional benefit of the IHPV assay was the smaller sample volume required (1 ml versus 4 ml for the HC2 assay). PMID:18367578

  6. Nuclear ploidy of neonatal rat livers: effects of two hepatic carcinogens (mirex and dimethylnitrosamine)

    SciTech Connect

    Carlson, J.; Abraham, R.

    1985-01-01

    The effect of two hepatic carcinogens, dimethylnitrosamine (DMN) (genotoxic) and mirex (epigenetic), on polyploidization in 12-d-old neonatal rats was investigated by Coulter counteranalysis and (/sup 3/H) thymidine uptake in isolated hepatic nuclear classes. DMN disturbed the normal ploidy development in the neonatal liver and the proportion of nuclei in the ploidy classes by inducing the premature formation of a significant population of tetraploids with a concommitant reduction in diploids. A great proportion of the replicative activity was present in tetraploid nuclei as measured by the incorporation of (/sup 3/H) thymidine. The labeling index and number of mitoses were also increased. In contrast to DMN, mirex had no influence on polyploidization. The neonatal rats used in these studies thus offer an opportunity to investigate in vivo the mode of action of genotoxic versus epigenetic compounds with reference to their effect on DNA.

  7. Opioid-dependent growth of glial cultures: Suppression of astrocyte DNA synthesis by met-enkephalin

    SciTech Connect

    Stiene-Martin, A.; Hauser, K.F. )

    1990-01-01

    The action of met-enkephalin on the growth of astrocytes in mixed-glial cultures was examined. Primary, mixed-glial cultures were isolated from 1 day-old mouse cerebral hemispheres and continuously treated with either basal growth media, 1 {mu}M met-enkephalin, 1 {mu}M met-enkephalin plus the opioid antagonist naloxone, or naloxone alone. Absolute numbers of neural cells were counted in unstained preparations, while combined ({sup 3}H)-thymidine autoradiography and glial fibrillary acid protein (GFAP) immunocytochemistry was performed to identify specific changes in astrocytes. When compared to control and naloxone treated cultures, met-enkephalin caused a significant decrease in both total cell numbers, and in ({sup 3}H)-thymidine incorporation by GFAP-positive cells with flat morphology. These results indicate that met-enkephalin suppresses astrocyte growth in culture.

  8. Investigation of the pharmacokinetics and mechanism of the action of Eleutherococcus glycosides. VIII. Investigation of the activation of natural killers by Eleutherococcus extract

    SciTech Connect

    Barenboim, G.M.; Sterlina, A.G.; Bebyakova, N.V.; Ribokas, A.A.; Fuks, B.B.

    1987-06-01

    The authors studied whether there is activation of the cells of natural resistance in intact mice that have received Eleutherococcus extract (EE) as well as how EE influences the activity of the normal killers, proliferation of splenocytes of mice, and the synthesis of lymphokines (interferon, interleukin-2). The proliferation of splenocytes with concanavalin A was estimated according to the incorporation of (/sup 3/H)thymidine in a 96-well plate. Titration of interleukin-2 was performed on three-day concanavalin A partially in the presence of (/sup 3/H)thymidine. Splenocytes of intact mice were cultured for five days in the presence of various doses of Eleutherococcus preparation in RPMI-1640 medium with 10% fetal serum then used as effectors in the membranotoxic text and the level of proliferation was determined.

  9. From Antenna to Assay

    PubMed Central

    Moore, Evan G.; Samuel, Amanda P. S.; Raymond, Kenneth N.

    2009-01-01

    Conspectus Ligand-sensitized, luminescent lanthanide(III) complexes are of considerable importance because their unique photophysical properties (microsecond to millisecond lifetimes, characteristic and narrow emission bands, and large Stokes shifts) make them well suited as labels in fluorescence-based bioassays. The long-lived emission of lanthanide(III) cations can be temporally resolved from scattered light and background fluorescence to vastly enhance measurement sensitivity. One challenge in this field is the design of sensitizing ligands that provide highly emissive complexes with sufficient stability and aqueous solubility for practical applications. In this Account, we give an overview of some of the general properties of the trivalent lanthanides and follow with a summary of advances made in our laboratory in the development of highly luminescent Tb(III) and Eu(III) complexes for applications in biotechnology. A focus of our research has been the optimization of these compounds as potential commercial agents for use in Homogeneous Time-Resolved Fluorescence (HTRF) technology. Our approach involves developing high-stability octadentate Tb(III) and Eu(III) complexes that rely on all-oxygen donor atoms and using multi-chromophore chelates to increase molar absorptivity; earlier examples utilized a single pendant chromophore (that is, a single “antenna”). Ligands based on 2-hydroxyisophthalamide (IAM) provide exceptionally emissive Tb(III) complexes with quantum yield values up to ∼60% that are stable at the nanomolar concentrations required for commercial assays. Through synthetic modification of the IAM chromophore and time-dependent density functional theory (TD-DFT) calculations, we have developed a method to predict absorption and emission properties of these chromophores as a tool to guide ligand design. Additionally, we have investigated chiral IAM ligands that yield Tb(III) complexes possessing both high quantum yield values and strong

  10. 49 CFR 572.180 - Incorporated materials.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 7 2014-10-01 2014-10-01 false Incorporated materials. 572.180 Section 572.180... Test Dummy, 50th Percentile Adult Male § 572.180 Incorporated materials. (a) The following materials... approved the materials incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part...

  11. 49 CFR 572.80 - Incorporated materials.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 7 2014-10-01 2014-10-01 false Incorporated materials. 572.80 Section 572.80... Incorporated materials. The drawings and specifications referred to in § 572.81(a) that are not set forth in full are hereby incorporated in this part by reference. These materials are thereby made part of...

  12. 49 CFR 572.30 - Incorporated materials.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 7 2011-10-01 2011-10-01 false Incorporated materials. 572.30 Section 572.30....30 Incorporated materials. (a) The drawings and specifications referred to in this regulation that... Federal Register has approved the materials incorporated by reference. For materials subject to...

  13. 49 CFR 572.30 - Incorporated materials.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 7 2012-10-01 2012-10-01 false Incorporated materials. 572.30 Section 572.30....30 Incorporated materials. (a) The drawings and specifications referred to in this regulation that... Federal Register has approved the materials incorporated by reference. For materials subject to...

  14. 49 CFR 572.30 - Incorporated materials.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 7 2010-10-01 2010-10-01 false Incorporated materials. 572.30 Section 572.30....30 Incorporated materials. (a) The drawings and specifications referred to in this regulation that... Federal Register has approved the materials incorporated by reference. For materials subject to...

  15. 49 CFR 572.30 - Incorporated materials.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 7 2013-10-01 2013-10-01 false Incorporated materials. 572.30 Section 572.30....30 Incorporated materials. (a) The drawings and specifications referred to in this regulation that... Federal Register has approved the materials incorporated by reference. For materials subject to...

  16. 49 CFR 572.190 - Incorporated materials.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 7 2013-10-01 2013-10-01 false Incorporated materials. 572.190 Section 572.190... Test Dummy, Small Adult Female § 572.190 Incorporated materials. (a) The following materials are hereby... Register approved the materials incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR...

  17. 49 CFR 572.80 - Incorporated materials.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 7 2010-10-01 2010-10-01 false Incorporated materials. 572.80 Section 572.80... Incorporated materials. The drawings and specifications referred to in § 572.81(a) that are not set forth in full are hereby incorporated in this part by reference. These materials are thereby made part of...

  18. 49 CFR 587.5 - Incorporated materials.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 7 2010-10-01 2010-10-01 false Incorporated materials. 587.5 Section 587.5... Barrier § 587.5 Incorporated materials. (a) The drawings and specifications referred to in this regulation that are not set forth in full are hereby incorporated in this part by reference. These materials...

  19. 49 CFR 572.80 - Incorporated materials.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 7 2011-10-01 2011-10-01 false Incorporated materials. 572.80 Section 572.80... Incorporated materials. The drawings and specifications referred to in § 572.81(a) that are not set forth in full are hereby incorporated in this part by reference. These materials are thereby made part of...

  20. 49 CFR 572.80 - Incorporated materials.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 7 2013-10-01 2013-10-01 false Incorporated materials. 572.80 Section 572.80... Incorporated materials. The drawings and specifications referred to in § 572.81(a) that are not set forth in full are hereby incorporated in this part by reference. These materials are thereby made part of...

  1. 49 CFR 587.5 - Incorporated materials.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 7 2011-10-01 2011-10-01 false Incorporated materials. 587.5 Section 587.5... Barrier § 587.5 Incorporated materials. (a) The drawings and specifications referred to in this regulation that are not set forth in full are hereby incorporated in this part by reference. These materials...

  2. 49 CFR 587.5 - Incorporated materials.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 7 2013-10-01 2013-10-01 false Incorporated materials. 587.5 Section 587.5... Barrier § 587.5 Incorporated materials. (a) The drawings and specifications referred to in this regulation that are not set forth in full are hereby incorporated in this part by reference. These materials...

  3. 49 CFR 572.180 - Incorporated materials.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 7 2013-10-01 2013-10-01 false Incorporated materials. 572.180 Section 572.180... Test Dummy, 50th Percentile Adult Male § 572.180 Incorporated materials. (a) The following materials... approved the materials incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part...

  4. 49 CFR 572.80 - Incorporated materials.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 7 2012-10-01 2012-10-01 false Incorporated materials. 572.80 Section 572.80... Incorporated materials. The drawings and specifications referred to in § 572.81(a) that are not set forth in full are hereby incorporated in this part by reference. These materials are thereby made part of...

  5. Numeral Incorporation in Japanese Sign Language

    ERIC Educational Resources Information Center

    Ktejik, Mish

    2013-01-01

    This article explores the morphological process of numeral incorporation in Japanese Sign Language. Numeral incorporation is defined and the available research on numeral incorporation in signed language is discussed. The numeral signs in Japanese Sign Language are then introduced and followed by an explanation of the numeral morphemes which are…

  6. Nondestructive boxed transuranic (TRU) waste assay systems

    NASA Astrophysics Data System (ADS)

    Caldwell, John T.; Jones, Stephanie A.; Lucero, Randy F.

    1999-01-01

    A brief history of boxed waste assay systems (primarily those developed at Los Alamos National Laboratory) is presented. The characteristics and design process involved with current generation systems--as practiced by BII--are also discussed in some detail. Finally, a specific boxed waste assay system and acceptance test results are presented. This system was developed by BII and installed at the Waste Receiving and Packaging (WRAP) facility in Hanford, Washington in early 1997. The WRAP system combines imaging passive/active neutron (IPAN) techniques with gamma- ray energy analysis (GEA) to assay crates up to 2.5 m X 2.5 m X 6.5 m in size. (Systems that incorporate both these methodologies are usually denoted IPAN/GEA types.) Two separate gamma-ray measurements are accomplished utilizing 16 arrayed NaI detectors and a moveable HPGe detector, while 3He detectors acquire both active and passive neutron data. These neutron measurements use BII's proprietary imaging methodology. Acceptance testing of the system was conducted at Hanford in January 1998. The system's operating performance was evaluated based on accuracy and sensitivity requirements for three different matrix types. Test results indicate an average 13% active mode accuracy for 10 nCi/g loadings of Pu waste and 5% passive mode accuracy for 10 g loadings of Pu waste. Sensitivity testing demonstrated an active mode lower limit of detection of less than 5 nCi/g of 239Pu for the medium matrix and less than 20 pCi/g of fission and activation products at 3(sigma) above background.

  7. Transporter assays and assay ontologies: useful tools for drug discovery.

    PubMed

    Zdrazil, Barbara; Chichester, Christine; Zander Balderud, Linda; Engkvist, Ola; Gaulton, Anna; Overington, John P

    2014-06-01

    Transport proteins represent an eminent class of drug targets and ADMET (absorption, distribution, metabolism, excretion, toxicity) associated genes. There exists a large number of distinct activity assays for transport proteins, depending on not only the measurement needed (e.g. transport activity, strength of ligand–protein interaction), but also due to heterogeneous assay setups used by different research groups. Efforts to systematically organize this (divergent) bioassay data have large potential impact in Public-Private partnership and conventional commercial drug discovery. In this short review, we highlight some of the frequently used high-throughput assays for transport proteins, and we discuss emerging assay ontologies and their application to this field. Focusing on human P-glycoprotein (Multidrug resistance protein 1; gene name: ABCB1, MDR1), we exemplify how annotation of bioassay data per target class could improve and add to existing ontologies, and we propose to include an additional layer of metadata supporting data fusion across different bioassays.

  8. Effects of Cissus quadrangularis on the proliferation, differentiation and matrix mineralization of human osteoblast like SaOS-2 cells.

    PubMed

    Muthusami, Sridhar; Senthilkumar, Krishnamoorthy; Vignesh, Chandragandan; Ilangovan, Ramachandran; Stanley, Jone; Selvamurugan, Nagarajan; Srinivasan, Narasimhan

    2011-04-01

    Osteoporosis is a public health problem which is associated with significant morbidity and mortality. The repair of bone defect is still a big challenge for orthopedic surgeons. Traditional use of Cissus quadrangularis (C. quadrangularis) in the treatment of bone disorders has been documented. The present study was employed to delineate the effects of ethanolic extract of C. quadrangularis on the proliferation, differentiation and matrix mineralization of human osteoblast like SaOS-2 cells. Lactate dehydrogenase assayed in the conditioned medium of control and C. quadrangularis treated cells did not differ significantly indicating that ethanolic extract of C. quadrangularis is nontoxic to osteoblastic cells. [(3)H] Thymidine incorporation assay revealed that C. quadrangularis treatment has increased the DNA synthesis of human osteoblastic SaOS-2 cells indicating increased proliferation of these cells. The data on alizarin red and ALP staining revealed increased matrix mineralization of human osteoblast like SaOS-2 cells. The study also revealed that the anabolic actions of ethanolic extract of C. quadrangularis in human osteoblast like cells are mediated through increased mRNA and protein expression of Runx2, a key transcription factor involved in the regulation of bone matrix proteins. Chromatin immunoprecipitation analysis revealed increased transcriptional activity of Runx2 on the promoter of osteocalcin after C. quadrangularis treatment. These results indicate positive regulation of C. quadrangularis on the proliferation, differentiation, and matrix mineralization of human osteoblast like SaOS-2 cells.

  9. Role of polypeptide growth factors in phenotypic transformation of normal rat kidney cells

    SciTech Connect

    van Zoelen, E.J.J.; van Oostwaard, T.M.J.; de Laat, S.W.

    1988-01-05

    A serum-free assay has been established for studying the role of polypeptide growth factors in inducing loss of density-dependent inhibition of growth of normal rat kidney (NRK) cells. The process has been characterized by measuring the time course of (/sup 3/H)thymidine incorporation into confluent, quiescent NRK cultures stimulated by defined polypeptide growth factors, in combination with cell counting studies, increases in DNA content, and cell cycle analysis by means of a fluorescence-activated cell sorter. It is shown that none of the growth factors tested is able to induce loss of density-dependent inhibition of growth by itself, but strong synergism was observed when combinations of growth factors were tested. None of the above factors was found to be essential, however, since any combination of three of the above four growth factors strongly induced the process. Strong parallels were observed between the growth factor requirements for inducing loss of density-dependent inhibition of growth under serum-free conditions and the requirements for induction of anchorage-independent proliferation under growth factor-defined assay conditions. This indicates that most likely the same cellular processes underlie these two aspects of phenotypic transformation, although data indicate that anchorage-independent proliferation may be a more restricted property of phenotypic transformation than loss of density dependence of proliferation. It is concluded that phenotypic transformation of NRK cells does not require specific polypeptide growth factors, but reflects the ability of these cells to respond to multiple growth factors.

  10. Metal-based paullones as putative CDK inhibitors for antitumor chemotherapy.

    PubMed

    Schmid, Wolfgang F; John, Roland O; Mühlgassner, Gerhard; Heffeter, Petra; Jakupec, Michael A; Galanski, Markus; Berger, Walter; Arion, Vladimir B; Keppler, Bernhard K

    2007-12-13

    Paullones constitute a class of potent cyclin-dependent kinase inhibitors. To overcome the insufficient solubility and bioavailability, which hamper their potential medical application, we aim at the development of metal-based derivatives. Two types of paullone ligands, L (1) - L (3) and L (4) , with different locations of metal-binding sites, were prepared. They were found to form organometallic complexes of the general formula [M (II)Cl(eta (6)- p-cymene)L]Cl ( 1- 4, L = L (1) - L (4) ; a, M = Ru; b, M = Os). The complexes were characterized by X-ray crystallography, one- and two-dimensional NMR spectroscopy and other physical methods. Complexes 1- 3, with a coordinated amidine unit, were found to undergo E/ Z isomerization in solution. The reaction was studied by NMR spectroscopy, and activation parameters Delta H (double dagger) and Delta S (double dagger) were determined. Antiproliferative activity in the low micromolar range was observed in vitro in three human cancer cell lines by means of MTT assays. (3)H-Thymidine incorporation assays revealed the compounds to lower the rate of DNA synthesis, and flow cytometric analyses showed cell cycle arrest mainly in G 0/ G 1 phase.

  11. PKC-dependent extracellular signal-regulated kinase 1/2 pathway is involved in the inhibition of Ib on AngiotensinII-induced proliferation of vascular smooth muscle cells

    SciTech Connect

    Wang Yu; Yan Tianhua; Wang Qiujuan Wang Wei; Xu Jinyi; Wu Xiaoming; Ji Hui

    2008-10-10

    AngiotensinII (AngII) induces vascular smooth muscle cell (VSMC) proliferation, which plays an important role in the development and progression of hypertension. AngII-induced cellular events have been implicated, in part, in the activation of protein kinase C (PKC) and extracellular signal-regulated kinases 1/2 (ERK1/2). In the present study, we investigated the effect of Ib, a novel nonpeptide AngII receptor type 1 (AT{sub 1}) antagonist, on the activation of PKC and ERK1/2 in VSMC proliferation induced by AngII. MTT, and [{sup 3}H]thymidine incorporation assay showed that AngII-induced VSMC proliferation was inhibited significantly by Ib. The specific binding of [{sup 125}I]AngII to AT{sub 1} receptors was blocked by Ib in a concentration-dependent manner with IC{sub 50} value of 0.96 nM. PKC activity assay and Western blot analysis demonstrated that Ib significantly inhibited the activation of PKC and phosphorylation of ERK1/2 induced by AngII, respectively. Furthermore, AngII-induced ERK1/2 activation was obviously blocked by GF109203X, a PKC inhibitor. These findings suggest that the suppression of Ib on AngII-induced VSMC proliferation may be attributed to its inhibitory effect on PKC-dependent ERK1/2 pathway.

  12. Carfilzomib Inhibits Constitutive NF-κB Activation in Mantle Cell Lymphoma B Cells and Leads to the Induction of Apoptosis.

    PubMed

    Zhang, Yong-Li; Guang, Matthew Ho Zhi; Zhuo, Hui-Qin; Min, Xiang-Hui; Yao, Qin; Gu, Ai-Qi; Wu, Sheng-Hua; Zhang, Dai-Bo; Lu, Jing-Yuan; Chen, Yan; Chen, Yu-Han; Zhang, Ke-Jie

    2017-01-01

    Mantle cell lymphoma (MCL) remains incurable and new treatments are needed, especially in the relapsed/refractory setting. We therefore investigated the effects of carfilzomib, a novel, long-acting, second-generation proteasome inhibitor, in MCL cells. Eight established MCL cell lines and freshly isolated primary MCL cells were treated with carfilzomib. Cell proliferation was assessed by a 3H-thymidine incorporation assay. Cell apoptosis was evaluated by flow cytometry with annexin V and propidium iodide. Electrophoresis mobility shift (EMSA), Western blot, and luciferase assays were used to analyze NF-κB activation and related signaling proteins. Carfilzomib inhibited growth and induced apoptosis in both established MCL cell lines and freshly isolated primary MCL cells in a dose-dependent manner. In contrast, carfilzomib was less toxic to normal peripheral blood mononuclear cells from healthy individuals. The carfilzomib-induced apoptosis of MCL cells occurred in a caspase-dependent manner through both intrinsic and extrinsic caspase pathways. In addition, carfilzomib inhibited constitutive activation of the NF-κB signaling cascade, both in MCL cell lines and primary MCL cells, by completely blocking the phosphorylation of IκBα. Our results demonstrate that carfilzomib can induce growth arrest and apoptosis in MCL cells and that the mechanism may involve the NF-κB signaling pathway.

  13. Upregulation of B-cell translocation gene 2 by epigallocatechin-3-gallate via p38 and ERK signaling blocks cell proliferation in human oral squamous cell carcinoma cells.

    PubMed

    Lee, Jehn-Chuan; Chung, Li-Chuan; Chen, Yu-Jen; Feng, Tsui-Hsia; Chen, Wen-Tsung; Juang, Horng-Heng

    2015-05-01

    Oral squamous cell carcinoma (OSCC) is a well-known malignancy that accounts for the majority of oral cancers. B-cell translocation gene 2 (BTG2) is an important regulator of cell cycle dynamics in cancer cells. However, the role of BTG2 in OSCC cells and the influences of epigallocatechin-3-gallate (EGCG) on BTG2 gene expressions have not been well evaluated. The objectives of this study were to examine the effect of EGCG-induced BTG2 expression and the potential signal pathways involved. The (3)H-thymidine incorporation and Western-blot assays revealed cell proliferation was attenuated by EGCG via upregulation of BTG2 expression causing cell cycle G1 phase arrest in OSCC cells. BTG2 overexpression decreased tumor cell growth, while BTG2 knockdown illuminated the opposite effect in xenograft animal studies. Overexpressed BTG2 arrested the cell cycle at the G1 phase and downregulated protein expressions of cyclin A, cyclin D, and cyclin E. Western-blot assays indicated that EGCG induced phosphorylation of p38, JNK, and ERK. However, pretreatments with selective mitogen-activated protein kinase (MAPK) inhibitors, SB203580 (p38 inhibitor) and PD0325901 (ERK1/2 inhibitor), significantly suppressed the activation of EGCG on BTG2 expression. Our results indicate that EGCG attenuates cell proliferation of OSCC cells by upregulating BTG2 expression via p38 and ERK pathways.

  14. Acemannan, an extracted product from Aloe vera, stimulates dental pulp cell proliferation, differentiation, mineralization, and dentin formation.

    PubMed

    Jittapiromsak, Nawaporn; Sahawat, Dusida; Banlunara, Wijit; Sangvanich, Polkit; Thunyakitpisal, Pasutha

    2010-06-01

    This study investigated the effect of acemannan (Aloe vera gel polysaccharide) on dentin formation. Primary human dental pulp cells were treated with acemannan. New DNA synthesis, bone morphogenetic protein-2, alkaline phosphatase activity, dentin sialoprotein expression, and mineralization were determined by [(3)H]-thymidine incorporation, enzyme-linked immunosorbent assay, biochemical assay, western blotting, and Alizarin Red staining, respectively. Then the upper first molars of 24 male Sprague Dawley rats were intentionally exposed and capped with either acemannan or calcium hydroxide. At day 28, the teeth were histopathologically examined and evaluated for the degree of inflammation, dentin bridge formation, and pulp tissue organization. The results revealed that acemannan significantly increased pulp cell proliferation, bone morphogenetic protein-2, alkaline phosphatase activity, dentin sialoprotein expression, and mineralization, compared with the untreated group. The acemannan-treated group also exhibited a complete homogeneous calcified dentin bridge and good pulp tissue organization, whereas neither was detected in the calcium hydroxide-treated and sham groups. In the acemannan-treated group, either mild or no inflammation was found, whereas the other groups had various degrees of inflammation. The data suggest that acemannan promotes dentin formation by stimulating primary human dental pulp cell proliferation, differentiation, extracellular matrix formation, and mineralization. Acemannan also has pulpal biocompatibility and promotes soft tissue organization.

  15. Myostatin inhibits cell proliferation and protein synthesis in C2C12 muscle cells.

    PubMed

    Taylor, W E; Bhasin, S; Artaza, J; Byhower, F; Azam, M; Willard, D H; Kull, F C; Gonzalez-Cadavid, N

    2001-02-01

    Myostatin mutations in mice and cattle are associated with increased muscularity, suggesting that myostatin is a negative regulator of skeletal muscle mass. To test the hypothesis that myostatin inhibits muscle cell growth, we examined the effects of recombinant myostatin in mouse skeletal muscle C2C12 cells. After verification of the expression of cDNA constructs in a cell-free system and in transfected Chinese hamster ovary cells, the human recombinant protein was expressed as the full-length (375-amino acid) myostatin in Drosophila cells (Mst375D), or the 110-amino acid carboxy-terminal protein in Escherichia coli (Mst110EC). These proteins were identified by immunoblotting and were purified. Both Mst375D and Mst110EC dose dependently inhibited cell proliferation (cell count and Formazan assay), DNA synthesis ([3H]thymidine incorporation), and protein synthesis ([1-14C]leucine incorporation) in C2C12 cells. The inhibitory effects of both proteins were greater in myotubes than in myoblasts. Neither protein had any significant effects on protein degradation or apoptosis. In conclusion, recombinant myostatin proteins inhibit cell proliferation, DNA synthesis, and protein synthesis in C2C12 muscle cells, suggesting that myostatin may control muscle mass by inhibiting muscle growth or regeneration.

  16. Face Recognition Incorporating Ancillary Information

    NASA Astrophysics Data System (ADS)

    Kim, Sang-Ki; Toh, Kar-Ann; Lee, Sangyoun

    2007-12-01

    Due to vast variations of extrinsic and intrinsic imaging conditions, face recognition remained to be a challenging computer vision problem even today. This is particularly true when the passive imaging approach is considered for robust applications. To advance existing recognition systems for face, numerous techniques and methods have been proposed to overcome the almost inevitable performance degradation due to external factors such as pose, expression, occlusion, and illumination. In particular, the recent part-based method has provided noticeable room for verification performance improvement based on the localized features which have good tolerance to variation of external conditions. The part-based method, however, does not really stretch the performance without incorporation of global information from the holistic method. In view of the need to fuse the local information and the global information in an adaptive manner for reliable recognition, in this paper we investigate whether such external factors can be explicitly estimated and be used to boost the verification performance during fusion of the holistic and part-based methods. Our empirical evaluations show noticeable performance improvement adopting the proposed method.

  17. Incorporating transgenerational testing and epigenetic ...

    EPA Pesticide Factsheets

    A number of environmental chemicals have been shown to alter markers of epigenetic change. Some published multi-generation rodent studies have identified effects on F2 and greater generations after chemical exposures solely to F0 dams, but were not focused on chemical safety. We were interested in how outcomes related to epigenetic changes could be identified and incorporated into chemical testing and risk assessment. To address this question, we conducted a systematic literature review to identify transgenerational (TG) epigenetic studies in rodents. These were analyzed to characterize the methods and observed outcomes, and to evaluate strengths, limitations, and biases. Our analysis found that test substances were administered to pregnant F0 dams; endpoints assessed in F1 to F4 generation offspring included growth, puberty timing, steroid hormone levels, abdominal adiposity, organ weights, histopathology, and epigenetic biomarkers. Biases were minimized through, e.g., randomization procedures, avoiding sibling or cousin matings, and independent multiple reviews of histopathology data. However, the numbers of litters assigned to control and test groups were not always transparently reported, nested statistical analyses of data was not always utilized to address litter effects, and “blind” testing was seldom performed. Many of these studies identified chemicals or combinations of chemicals that produced TG effects and/or adult-onset diseases, but there is a

  18. Phenotype expression of gingival fibroblasts cultured on membranes used in guided tissue regeneration.

    PubMed

    Locci, P; Calvitti, M; Belcastro, S; Pugliese, M; Guerra, M; Marinucci, L; Staffolani, N; Becchetti, E

    1997-09-01

    Human gingival fibroblasts were cultured in vitro using as substrates an extracellular matrix (matrix) and polytetrafluoride (PTFE) membranes, which are used in guided tissue regeneration. To test the degree of biocompatibility of these membranes, the cellular proliferation and the accumulation of extracellular matrix (ECM) macromolecules were considered as parameters. The fibroblasts were cultured in vitro for 24 and 48 hours without serum on plastic, matrix, and PTFE membranes in the presence of 3H-thymidine, 3H-glucosamine, and 3H-proline to study the neo-synthesis of DNA, glycosaminoglycans (GAG), and collagen proteins, respectively. Studies on cell proliferation showed that fibroblasts grown on matrix membrane significantly increased 3H-thymidine incorporation, while fibroblasts grown on PTFE membrane decreased 3H-thymidine incorporation, compared to plastic used as a control. Moreover, the PTFE membrane induced a marked decrease of collagen and GAG accumulation both in the cellular and extracellular pool, while the matrix membrane provoked a decrease of the two macromolecules in the cellular pool and an increase in the extracellular one, compared to the control. The data we obtained demonstrate that matrix membranes are the most suitable to stimulate both cellular proliferation and ECM macromolecule accumulation.

  19. Immunoregulation by low density lipoproteins in man. Inhibition of mitogen-induced T lymphocyte proliferation by interference with transferrin metabolism.

    PubMed Central

    Cuthbert, J A; Lipsky, P E

    1984-01-01

    Human low density lipoprotein (LDL, d = 1.020-1.050 g/ml) inhibits mitogen-stimulated T lymphocyte DNA synthesis. Because both LDL and transferrin bind to specific cell surface receptors and enter cells by the similar means of receptor-mediated endocytosis, and because transferrin is necessary for lymphocyte DNA synthesis, we investigated the possibility that LDL may inhibit mitogen-stimulated lymphocyte responses by interfering with transferrin metabolism. LDL inhibited mitogen-stimulated lymphocyte [3H]thymidine incorporation in a concentration-dependent manner. The degree of inhibition was most marked in serum-free cultures, but was also observed in serum-containing cultures. The addition of transferrin not only augmented mitogen-induced lymphocyte [3H]thymidine incorporation in serum-free medium but also completely reversed the inhibitory effect of LDL in both serum-free and serum-containing media. Similar results were obtained when lymphocyte proliferation was assayed by counting the number of cells in culture. Transferrin also reversed the inhibition of lymphocyte responses caused by very low density lipoproteins and by cholesterol. The ability of transferrin to reverse the inhibitory effect of lipoproteins was specific, in that native but not denatured transferrin was effective whereas a variety of other proteins were ineffective. These results indicate that LDL inhibits mitogen-stimulated lymphocyte responses by interfering with transferrin metabolism. LDL only inhibited lymphocyte responses after a 48-h incubation if present from the initiation of the culture. By contrast, transferrin reversed inhibition when added after 24 h of the 48-h incubation. LDL did not inhibit lymphocyte responses by nonspecifically associating with transferrin. In addition, the acquisition of specific lymphocyte transferrin receptors was not blocked by LDL. Moreover, transferrin did not prevent the binding and uptake of fluorescent-labeled LDL by activated lymphocytes

  20. Incorporating Population Variability and Susceptible Subpopulations into Dosimetry for High-Throughput Toxicity Testing

    EPA Science Inventory

    Momentum is growing worldwide to use in vitro high-throughput screening (HTS) to evaluate human health effects of chemicals. However, the integration of dosimetry into HTS assays and incorporation of population variability will be essential before its application in a risk assess...

  1. Protein tyrosine phosphatase: enzymatic assays.

    PubMed

    Montalibet, Jacqueline; Skorey, Kathryn I; Kennedy, Brian P

    2005-01-01

    Activity assays for tyrosine phosphatases are based on the hydrolysis of a arylphosphate moiety from a synthetic substrate yielding a spectroscopically active product. Many different substrates can be used for these assays with p-nitrophenyl phosphate (pNPP), fluorescein diphosphate (FDP), and 6,8-difluoro-4-methylumbellyferyl phosphate (DiFMUP) being the most efficient and versatile. Equally, larger molecules such as phosphotyrosyl peptides can also be used to mimic more natural substrates. Activity assays include the determinations of the rate of dephosphorylation and calculations of kinetic constants such as k(cat) and K(M). These assays are useful to identify and characterize tyrosine phosphatases and are commonly used to evaluate the efficiency of inhibitors.

  2. Oestradiol assays: fitness for purpose?

    PubMed

    Middle, Jonathan G; Kane, John W

    2009-11-01

    In this review we discuss the analytical inadequacies of oestradiol assays in relation to the clinical requirements for performing them, and make recommendations for their improvement. The measurement of oestradiol can be requested in a number of clinical scenarios (precocious puberty, infertility, assisted conception, hormone replacement therapy). The very wide dynamic range of oestradiol concentrations is a huge challenge for routine assays, which they are unlikely to meet on theoretical as well as practical grounds. The EQA performance of oestradiol assays in terms of trueness, comparability, recovery and analytical sensitivity leaves much to be desired and indicates that calibration is compromised by poor analytical specificity. To make oestradiol assays fit for purpose requires concerted action by all stakeholders to define analytical quality specifications for the various clinical scenarios involved, and then to encourage concerted action by the diagnostic industry to use the steroid reference measurement system to improve specificity, trueness and traceability.

  3. Functional Assays for Ricin Detection

    NASA Astrophysics Data System (ADS)

    Ezan, Eric; Duriez, Elodie; Fenaille, François; Becher, François

    In this review, we provide background information on ricin structure, present available functional assays for other toxins that are potential biothreat agents, and finish by describing the functional assay of ricin itself. Using appropriate sample preparation and optimized detection based on N-glycosidase activity, we demonstrate that specific detection of whole ricin at a level of around 0.1 ng/mL is possible and applicable to environmental samples.

  4. Microbiological assay using bioluminescent organism

    SciTech Connect

    Stiffey, A.V.

    1987-12-21

    This invention relates to testing processes for toxicity involving microorganisms and, more particularly, to testing processes for toxicity involving bioluminescent organisms. The present known method of testing oil-well drilling fluids for toxicity employs the mysid shrimp (Mysidopsis bahia) as the assay organism. The shrimp are difficult to raise and handle as laboratory assay organisms. This method is labor-intensive, because it requires a assay time of about 96 hours. Summary of the Invention: A microbiological assay in which the assay organism is the dinoflagellate, Pyrocystis lunula. A sample of a substance to be assayed is added to known numbers of the bioluminescent dinoflagellate and the mixture is agitated to subject the organisms to a shear stress causing them to emit light. The amount of light emitted is measured and compared with the amount of light emitted by a known non-toxic control mixture to determine if there is diminution or non-diminution of light emitted by the sample under test which is an indication of the presence or absence of toxicity, respectively. Accordingly, an object of the present invention is the provision of an improved method of testing substances for toxicity. A further object of the invention is the provision of an improved method of testing oil-well drilling fluids for toxicity using bioluminescent dinoflagellate (Pyrocystis lunula).

  5. 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

  6. A fluorogenic assay for methylglyoxal.

    PubMed

    Shaheen, Fozia; Shmygol, Anatoly; Rabbani, Naila; Thornalley, Paul J

    2014-04-01

    MG (methylglyoxal) is a potent glycating agent and an endogenous reactive dicarbonyl metabolite formed in all live cells and organisms. It is an important precursor of AGEs (advanced glycation end-products) and is implicated in aging and disease. MG is assayed by derivatization by 1,2-diaminobenzene derivatives in cell extracts. Such assays are not applicable to high sample throughput, subcellular, live-cell and in vivo estimations. The use of fluorogenic probes designed for NO (nitric oxide) detection in biological samples and living cells has inadvertently provided probes for the detection of dicarbonyls such as MG. We describe the application of DAF-2 (4,5-diaminofluorescein) and DAR-1 (4,5-diaminorhodamine) for the detection of MG in cell-free systems and application for high-throughput assay of glyoxalase activity and assay of glucose degradation products in peritoneal dialysis fluids. DAF-2 and DAR-1, as for related BODIPY probes, do not have sufficient sensitivity to detect MG in live cells. Care will also be required to control for NO and dehydroascorbate co-detection and interference from peroxidase catalysing the degradation of probes to MG and glyoxal. Fluorogenic detection of MG, however, has great potential to facilitate the assay of MG and to advance towards that capability of imaging this product in live cells in vitro and small animals in vivo.

  7. 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.

  8. HUMAN VASCULAR ENDOTHELIAL CELLS IN CULTURE

    PubMed Central

    Gimbrone, Michael A.; Cotran, Ramzi S.; Folkman, Judah

    1974-01-01

    Human endothelial cells, obtained by collagenase treatment of term umbilical cord veins, were cultured using Medium 199 supplemented with 20% fetal calf serum. Small clusters of cells initially spread on plastic or glass, coalesced and grew to form confluent monolayers of polygonal cells by 7 days. Cells in primary and subcultures were identified as endothelium by the presence of Weibel-Palade bodies by electron microscopy. A morphologically distinct subpopulation of cells contaminating some primary endothelial cultures was selectively subcultured, and identified by ultrastructural criteria as vascular smooth muscle. Autoradiography of endothelial cells after exposure to [3H]thymidine showed progressive increases in labeling in growing cultures beginning at 24 h. In recently confluent cultures, labeling indices were 2.4% in central closely packed regions, and 53.2% in peripheral growing regions. 3 days after confluence, labeling was uniform, being 3.5 and 3.9% in central and peripheral areas, respectively. When small areas of confluent cultures were experimentally "denuded," there were localized increases in [3H]thymidine labeling and eventual reconstitution of the monolayer. Liquid scintillation measurements of [3H]thymidine incorporation in primary and secondary endothelial cultures in microwell trays showed a similar correlation of DNA synthesis with cell density. These data indicate that endothelial cell cultures may provide a useful in vitro model for studying pathophysiologic factors in endothelial regeneration. PMID:4363161

  9. Barcoded microchips for biomolecular assays.

    PubMed

    Zhang, Yi; Sun, Jiashu; Zou, Yu; Chen, Wenwen; Zhang, Wei; Xi, Jianzhong Jeff; Jiang, Xingyu

    2015-01-20

    Multiplexed assay of analytes is of great importance for clinical diagnostics and other analytical applications. Barcode-based bioassays with the ability to encode and decode may realize this goal in a straightforward and consistent manner. We present here a microfluidic barcoded chip containing several sets of microchannels with different widths, imitating the commonly used barcode. A single barcoded microchip can carry out tens of individual protein/nucleic acid assays (encode) and immediately yield all assay results by a portable barcode reader or a smartphone (decode). The applicability of a barcoded microchip is demonstrated by human immunodeficiency virus (HIV) immunoassays for simultaneous detection of three targets (anti-gp41 antibody, anti-gp120 antibody, and anti-gp36 antibody) from six human serum samples. We can also determine seven pathogen-specific oligonucleotides by a single chip containing both positive and negative controls.

  10. Use of the spiral Salmonella assay to detect the mutagenicity of complex environmental mixtures

    SciTech Connect

    Houk, V.S.; Early, G.; Claxton, L.D. )

    1991-01-01

    The success demonstrated by the spiral Salmonella assay in a recent study of 20 pure prompted us to examine the effectiveness of this automated bacterial mutagenicity assay for testing complex environmental mixtures. Three sets of combustion emissions were selected for evaluation: automotive diesel exhaust, woodsmoke, and a coal combustion emission. Each sample was tested in the Salmonella mutagenicity assay according to standard protocol (plate incorporation) and spiral assay techniques. In the spiral assay, a specialized plating instrument dispenses the bacteria, test agent, and S9 mix in a spiral pattern onto a minimal agar plate supplemented with histidine and biotin. The components of the assay are administered in such a way that a uniform density of bacteria is exposed to a concentration gradient of the test agent on a single plate. When results are analyzed, a dose-response curve comprised of 13 data points is generated. A comparison of results from the two assays demonstrated the following: (1) Diesel exhaust was generally the most mutagenically potent sample in both assays, followed closely by the coal combustion emission. The woodsmoke sample was only weakly mutagenic in the standard assay but demonstrated higher mutagenic activity in the spiral assay. (2) Samples were more mutagenic on rev/microgram basis in the spiral assay, especially when metabolic activation was added. This disparity presumably was due to differences in the relative amounts of S9 administered across the dose range. (3) The spiral assay required 1/20 the sample mass of the standard assay to test equivalent doses; in addition, for some samples, 50 times more sample mass was required by the standard assay to generate a comparable dose response. (4) Dichloromethane extracts of the complex mixtures could be tested for mutagenicity in the spiral assay.

  11. Antioxidant assays for plant and food components.

    PubMed

    Moon, Joon-Kwan; Shibamoto, Takayuki

    2009-03-11

    Recently, research on natural antioxidants has become increasingly active in various fields. Accordingly, numerous articles on natural antioxidants, including polyphenols, flavonoids, vitamins, and volatile chemicals, have been published. Assays developed to evaluate the antioxidant activity of plants and food constituents vary. Therefore, to investigate the antioxidant activity of chemical(s), choosing an adequate assay based on the chemical(s) of interest is critical. There are two general types of assays widely used for different antioxidant studies. One is an assay associated with lipid peroxidations, including the thiobarbituric acid assay (TBA), malonaldehyde/high-performance liquid chromatography (MA/HPLC) assay, malonaldehyde/gas chromatography (MA/GC) assay, beta-carotene bleaching assay, and conjugated diene assay. Other assays are associated with electron or radical scavenging, including the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay, ferric reducing/antioxidant power (FRAP) assay, ferrous oxidation-xylenol orange (FOX) assay, ferric thiocyanate (FTC) assay, and aldehyde/carboxylic acid (ACA) assay. In this review, assays used recently were selected for extended discussion, including discussion of the mechanisms underlying each assay and its application to various plants and foods.

  12. Incorporating Sociology into Community Service Classes

    ERIC Educational Resources Information Center

    Hochschild, Thomas R., Jr.; Farley, Matthew; Chee, Vanessa

    2014-01-01

    Sociologists and instructors who teach about community service share an affinity for understanding and addressing social problems. While many studies have demonstrated the benefits of incorporating community service into sociology courses, we examine the benefits of incorporating sociological content into community service classes. The authors…

  13. 49 CFR 572.40 - Incorporated materials.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 7 2010-10-01 2010-10-01 false Incorporated materials. 572.40 Section 572.40... Percentile Male § 572.40 Incorporated materials. (a) The drawings, specifications, manual, and computer... by reference. These materials are thereby made part of this regulation. The Director of the...

  14. 49 CFR 572.40 - Incorporated materials.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 7 2011-10-01 2011-10-01 false Incorporated materials. 572.40 Section 572.40... Percentile Male § 572.40 Incorporated materials. (a) The drawings, specifications, manual, and computer... by reference. These materials are thereby made part of this regulation. The Director of the...

  15. 49 CFR 572.40 - Incorporated materials.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 7 2013-10-01 2013-10-01 false Incorporated materials. 572.40 Section 572.40... Percentile Male § 572.40 Incorporated materials. (a) The drawings, specifications, manual, and computer... by reference. These materials are thereby made part of this regulation. The Director of the...

  16. 49 CFR 572.40 - Incorporated materials.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 7 2012-10-01 2012-10-01 false Incorporated materials. 572.40 Section 572.40... Percentile Male § 572.40 Incorporated materials. (a) The drawings, specifications, manual, and computer... by reference. These materials are thereby made part of this regulation. The Director of the...

  17. Influence of tunicamycin, sialidase, and cholera toxin on gangliosides and T-lymphocyte responses to interleukin 2

    SciTech Connect

    Semmes, O.J.; Bailey, J.M.; Merritt, W.D.

    1986-05-01

    The authors have shown that gangliosides inhibit interleukin 2 (IL 2)-dependent proliferation of murine T cells. Tunicamycin (TM), sialidase, and cholera toxin-..beta.. subunit (..beta..-CT) are known modulators of cell surface glycoconjugates. To test the possible role of endogenous gangliosides in T cell responses to IL-2, the effect of these agents on ganglioside expression and cell proliferation was studied. Gangliosides were labelled for 24 hrs with /sup 3/H-glucosamine/galactose in the presence of IL-2 and purified sialidase, TM or ..beta..-CT. Gangliosides were isolated and the species separated by TLC. Alternatively, proliferation was assayed by /sup 3/H-thymidine uptake after 48 hrs culture. TM treatment at a concentration (10 ..mu..g/ml) that completely inhibited proliferation resulted in a 86% reduction of incorporation of saccharide precursors into gangliosides compared to a 50% reduction into proteins. Sialidase treatment (0.1 IU/ml) resulted in a 70% inhibition of proliferation and 30% reduction of radiolabel into gangliosides, of which 3 species were specifically reduced. ..beta..-CT, which binds to GM/sub 1/ and to a lesser extent GD/sub 1a/, caused a 50% reduction in proliferation response at 35 units/ml. The results support the hypothesis that gangliosides are involved in IL-2-dependent proliferation.

  18. Effect of microwaves (2450-MHz) on the immune system in mice: studies of nucleic acid and protein synthesis

    SciTech Connect

    Wiktor-Jedrzejczak, W.; Ahmed, A.; Czerski, P.; Leach, W.M.; Sell, K.W.

    1980-01-01

    CBA/J adult male mice were given single or triple exposures to 2450-mHz microwaves in an environmentally controlled wave guide facility. The average absorbed dose rate for a single exposure varied from 12 to 15 mW/g. Sham-exposed mice served as controls. Lymphoid cells were collected and tested for metabolic activity on days 3, 6, and 9 following a single exposure, and on days 9, 12, and 16 following triple exposures on days 0, 3, and 6. Cells were cultured in vitro for four hours to seven days before their metabolic rates were assayed. Under these conditions, microwaves failed to produce any detectable change in deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein synthesis, as measured by the incorporation of methyl(3H)-thymidine (3H-TDR) (DNA substrate), 3H-uridine (3H-UR) (RNA substrate), and 3H-leucine (protein substrate) by spleen, bone marrow, and peripheral blood lymphocytes (PBL) in vitro. These data suggest that microwave-induced increases in the frequency of complement-receptor (CR)- or surface-immunoglobulin (sIg)-bearing cells were not associated with a concomitant increase in cell proliferation and/or protein synthesis, and favor the concept that microwaves under these conditions stimulate already existing B-cell precursors for maturation.

  19. Morphologic and phenotypic changes of human neuroblastoma cells in culture induced by cytosine arabinoside

    SciTech Connect

    Ponzoni, M.; Lanciotti, M.; Melodia, A.; Casalaro, A.; Cornaglia-Ferraris, P. )

    1989-03-01

    The effects of cytosine-arabinoside (ARA-C) on the growth and phenotypic expression of a new human neuroblastoma (NB) cell line (GI-ME-N) have been extensively tested. Low doses of ARA-C allowing more than 90% cell viability induce morphological differentiation and growth inhibition. Differentiated cells were larger and flattened with elongated dendritic processes; such cells appeared within 48 hours after a dose of ARA-C as low as 0.1 {mu}g/ml. The new morphological aspect reached the maximum expression after 5-6 days of culture being independent from the addition of extra drug to the culture. A decrease in ({sup 3}H)thymidine incorporation was also observed within 24 hours and the cell growth was completely inhibited on the sixth day. Moreover, ARA-C strongly inhibited anchorage-independent growth in soft agar assay. Membrane immunofluorescence showed several dramatic changes in NB-specific antigen expression after 5 days of treatment with ARA-C. At the same time ARA-C also modulated cytoskeletal proteins and slightly increased catecholamine expression. These findings suggest that noncytotoxic doses of ARA-C do promote the differentiation of GI-ME-N neuroblastoma cells associated with reduced expression of the malignant phenotype.

  20. Experiment K-7-23: Effect of Spaceflight on Level and Function of Immune Cells. Part 2; Proliferation and Cytokines

    NASA Technical Reports Server (NTRS)

    Nash, P. V.; Konstantinova, I. V.; Fuchs, B. B.; Rakhmilevich, A. L.; Lesnyak, A. T.; Mastro, A. M.

    1994-01-01

    Lymphocytes from the superficial inguinal lymph nodes of rats flown on the Cosmos 2044 space mission were tested for proliferation in response to polyclonal activators. Cells were cultured with T or B cell mitogens, phorbol ester and calcium ionophore, or T cell mitogen and the lymphokines interleukin-1 (IL-1) or interleukin-2 (IL-2), and assayed for DNA synthesis by (3)H-thymidine incorporation. Lymphocytes also were incubated with concanavalin A (Con A), a T cell mitogen, and tested for IL-2 production. Mitogen-stimulated proliferation of lymphocytes from rats exposed to microgravity was not significantly different from synchronous or vivarium controls. Responses to Con A and IL-2, and Con A and IL-1 likewise were unaffected by space flight. Lymphocytes from all of these groups responded well to phorbol ester and calcium ionophore stimulation. Furthermore, lymph node cells (LNC) from control rats and rats flown on Cosmos 2044 produced similar amounts of IL-2. The results obtained using hindlimb suspended rats were notably different from those of flight and control animals. LNC from suspended rats generally had greater proliferative responses to T cell mitogens than did lymphocytes from other groups. Responsiveness to a B cell mitogen was not enhanced. Con A-stimulated LNC from hindlimb suspended rats also produced more IL-2 than did lymphocytes from the other groups. This difference was statistically significant at both IL-2 induction times tested.

  1. Correlation of histology and drug response of human tumors grown in native-state three-dimensional histoculture and in nude mice

    SciTech Connect

    Vescio, R.A.; Connors, K.M. ); Kubota, Tetsuro. ); Hoffman, R.M. Univ. of California, San Diego, La Jolla, CA )

    1991-06-15

    An in vitro histoculture system in which a native-state collagen-sponge gel supports the three-dimensional growth of tumor tissue has been recently developed that allows the culture and drug response assay for most every tumor type. Important features of the histoculture system include the maintenance of three-dimensional tissue architecture and the use of histological autoradiography to measure nuclear incorporation of ({sup 3}H)thymidine as an endpoint. The authors describe in this report in vitro-in vivo correlations for drug response and tumor histology by using human tumor xenografts grown in the native-state three-dimensional histoculture system and as xenografts in nude mice. This comparison eliminates many of the confounding variables seen in most correlative clinical trials. Results demonstrate (1) a very high preservation of in vivo tissue architecture in vitro, (2) an 86% accuracy in vitro of predicting drug resistance in vivo, and (3) an overall predictive frequency of drug resistance and sensitivity ranging from 53% for 5-fluorouracil to 78% for doxorubicin.

  2. Critical role of androgen receptor level in prostate cancer cell resistance to new generation antiandrogen enzalutamide

    PubMed Central

    Hoefer, Julia; Akbor, Mohammady; Handle, Florian; Ofer, Philipp; Puhr, Martin; Parson, Walther; Culig, Zoran; Klocker, Helmut; Heidegger, Isabel

    2016-01-01

    Enzalutamide is an androgen receptor (AR) inhibitor approved for therapy of metastatic castration resistant prostate cancer. However, clinical application revealed that 30 to 40% of patients acquire resistance after a short period of treatment. Currently, the molecular mechanisms underlying such resistances are not completely understood, partly due to a lack of model systems. In the present study we established three different cellular models of enzalutamide resistance including a cell line with wild type AR (LAPC4), DuCaP cells which overexpress wild-type AR, as well as a cell which has been adapted to long term androgen ablation (LNCaP Abl) and harbors the AR T878A mutation. After 10 months of cultivation, sustained growth in the presence of enzalutamide was achieved. When compared to controls, resistant cells exhibit significantly decreased sensitivity to enzalutamide as measured with 3[H]thymidine incorporation and WST assay. Moreover, these cell models exhibit partly re-activated AR signaling despite presence of enzalutamide. In addition, we show that enzalutamide resistant cells are insensitive to bicalutamide but retain considerable sensitivity to abiraterone. Mechanistically, enzalutamide resistance was accompanied by increased AR and AR-V7 mRNA and protein expression as well as AR gene amplification, while no additional AR mutations have been identified. PMID:27486973

  3. Antiproliferative activity of shark cartilage with and without tumor necrosis factor-alpha in human umbilical vein endothelium.

    PubMed

    McGuire, T R; Kazakoff, P W; Hoie, E B; Fienhold, M A

    1996-01-01

    We evaluated the antiangiogenic activity of shark cartilage, tumor necrosis factor-alpha (TNF-alpha), and a combination of the two using a human umbilical vein endothelial cell proliferation assay. Proliferation of endothelium is a hallmark of angiogenesis, and inhibition of endothelial cell proliferation indicates potential antiangiogenic activity. Shark cartilage produced a concentration-dependent decline in endothelial cell 3H-thymidine incorporation. This activity was heat stable and was found in molecular weight fractions of less than 10 kd. The antiproliferative effect of shark cartilage was specific for vascular endothelium and did not affect the proliferative rate of human astrocytoma cells or human skin fibroblasts. Shark cartilage at a concentration of 500 mu g/ml and TNF-alpha at a concentration of 10 ng/ml reduced endothelial cell proliferation by 32% and 29%, respectively. Treatment of endothelial cells with the combination of shark cartilage and TNF-alpha resulted in a 44% reduction in endothelial cell proliferation. The isolation and identification of the active components of shark cartilage is continuing.

  4. Cytotoxic activity of octahydropyrazin[2,1-a:5,4-a']diisoquinoline derivatives in human breast cancer cells.

    PubMed

    Lepiarczyk, Monika; Kałuża, Zbigniew; Bielawska, Anna; Czarnomysy, Robert; Gornowicz, Agnieszka; Bielawski, Krzysztof

    2015-01-01

    Evaluation of the cytotoxicity of novel octahydropyrazin[2,1-a:5,4-a']diisoquinoline derivatives (1a-2c) employing a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and inhibition of [(3)H]thymidine incorporation into DNA demonstrated that these compounds were more active than etoposide and camptothecin in both MDA-MB-231 and MCF-7 human breast cancer cells. Flow cytometric analysis after Annexin V-FITC and propidium iodide staining also confirmed that apoptosis was the main response of human breast cancer cells to 1a-2c treatment. Our results suggest that apoptosis of human breast cancer cells in the presence of 1a-2c follows the mitochondrial pathway, with the decrease in mitochondrial membrane potential and activation of caspase 9, as well as by the external pathway with the significant increase in caspase 8 expression. Cytotoxic properties of compounds 1a-2c in cultured human breast cancer cells correlate to their ability to inhibit topoisomerase I/II.

  5. Lymphocytic proliferative response to outer-membrane proteins isolated from Salmonella.

    PubMed

    González, C R; Isibasi, A; Ortiz-Navarrete, V; Paniagua, J; García, J A; Blanco, F; Kumate, J

    1993-01-01

    Porins isolated from Salmonella typhi have been demonstrated to protect against the challenge with this bacteria in mice. The mechanism has not been clarified, but could be associated with activation of both humoral and cellular immunity. In order to evaluate the induction of specific T cell responses, the lymphocytic proliferation to porins isolated from Salmonella typhimurium, Salmonella typhi and Escherichia coli was examined by 3H-thymidine incorporation assay in mice immunized with three different antigens: acetone-killed S. typhimurium, its porins, or outer-membrane proteins (OMPs) isolated from S. typhi. Higher proliferative responses were observed in mice immunized with porins and OMPs compared with those which received the acetone-killed bacteria. Although cross-reactivity was observed between porins, they were not mitogenic. Moreover, porins were able to activate T lymphocytes isolated from mice immunized with S. typhi OMPs. These results suggest that T cell activation, through the release of lymphokines, may play a role in the induction of protective immunity with porins.

  6. Acid phosphatase activity: a marker of androgen action in prostate explant cultures.

    PubMed

    Shao, T C; Kong, A Y; Cunningham, G R

    1987-01-01

    Acid phosphatase activity in rat ventral prostate explants has been assayed to determine if this parameter could serve as a specific and quantitative marker of androgen action in this in vitro model. Dihydrotestosterone (10 nM) caused an absolute increase in both total (42.5 +/- 2.9 vs control 27.1 +/- 4.0 nmoles p-nitrophenol generated in 30 min/micrograms DNA, P less than .01) and tartrate-resistant acid phosphatase activity (34.1 +/- 1.5 vs control 17.2 +/- 2.8 U/micrograms DNA, P less than .05), and this effect was maximal on the 4th day of culture. This was the time when explant weight and DNA content tended to fall or only to be maintained by androgen. Similar changes were observed with the potent synthetic androgen, mibolerone. The addition of either the antiandrogen cyproterone acetate or flutamide in a 100-fold excess to that of androgen caused significant inhibition in acid phosphatase activity. No significant change was observed at low concentrations of estradiol or progesterone, and only minimal and inconsistent increases in activity were noted at high concentrations. No increase was noted when cortisol, cyproterone acetate, or flutamide was added to the media. We conclude that measurement of acid phosphatase activity in cultured explants of rat ventral prostate provides a biochemical marker of androgenicity that is more specific than measurement of [3H]-thymidine incorporation.

  7. Critical role of androgen receptor level in prostate cancer cell resistance to new generation antiandrogen enzalutamide.

    PubMed

    Hoefer, Julia; Akbor, Mohammady; Handle, Florian; Ofer, Philipp; Puhr, Martin; Parson, Walther; Culig, Zoran; Klocker, Helmut; Heidegger, Isabel

    2016-09-13

    Enzalutamide is an androgen receptor (AR) inhibitor approved for therapy of metastatic castration resistant prostate cancer. However, clinical application revealed that 30 to 40% of patients acquire resistance after a short period of treatment. Currently, the molecular mechanisms underlying such resistances are not completely understood, partly due to a lack of model systems. In the present study we established three different cellular models of enzalutamide resistance including a cell line with wild type AR (LAPC4), DuCaP cells which overexpress wild-type AR, as well as a cell which has been adapted to long term androgen ablation (LNCaP Abl) and harbors the AR T878A mutation. After 10 months of cultivation, sustained growth in the presence of enzalutamide was achieved. When compared to controls, resistant cells exhibit significantly decreased sensitivity to enzalutamide as measured with 3[H]thymidine incorporation and WST assay. Moreover, these cell models exhibit partly re-activated AR signaling despite presence of enzalutamide. In addition, we show that enzalutamide resistant cells are insensitive to bicalutamide but retain considerable sensitivity to abiraterone. Mechanistically, enzalutamide resistance was accompanied by increased AR and AR-V7 mRNA and protein expression as well as AR gene amplification, while no additional AR mutations have been identified.

  8. Induction and disappearance of DNA strand breaks in human peripheral blood lymphocytes and fibroblasts treated with methyl methanesulfonate

    SciTech Connect

    Boerrigter, M.E.T.I.; Mullaart, E.; Vijg, J. )

    1991-01-01

    The induction and disappearance of DNA single-strand breaks (SSB) in human peripheral blood lymphocytes (PBL) and fibroblasts exposed to methyl methanesulfonate (MMS) were investigated by using the alkaline filter elution assay. In the two cell types, identical amounts of SSB were induced during a 45-minute treatment with a given dose of MMS. In quiescent PBL only 9{plus minus}4% (mean {plus minus} SD) of the induced SSB had disappeared at 1 hour after exposure, whereas in phytohemagglutinin-stimulated PBL, 23 {plus minus} 12% disappeared within the same repair period. The accumulation of SSB in PBL, but not in fibroblasts, during MMS exposure in the presence of the excision-repair inhibitor 1-{beta}-D-arabinofuranosylcytosine indicated the utilization of different repair pathways in these two cell types. The generally lower rate of disappearance of MMS-induced SSB in PBL as compared to fibroblasts correlated with an increased loss of cell viability, measured by determining the incorporation of ({sup 3}H)thymidine.

  9. Morphological and Functional Features of Hepatic Cyst Epithelium in Autosomal Dominant Polycystic Kidney Disease

    PubMed Central

    Alvaro, Domenico; Onori, Paolo; Alpini, Gianfranco; Franchitto, Antonio; Jefferson, Douglas M.; Torrice, Alessia; Cardinale, Vincenzo; Stefanelli, Fabrizio; Mancino, Maria Grazia; Strazzabosco, Mario; Angelico, Mario; Attili, Adolfo; Gaudio, Eugenio

    2008-01-01

    We evaluated the morphological and functional features of hepatic cyst epithelium in adult autosomal dominant polycystic kidney disease (ADPKD). In six ADPKD patients, we investigated the morphology of cyst epithelium apical surface by scanning electron microscopy and the expression of estrogen receptors (ERs), insulin-like growth factor 1 (IGF1), IGF1 receptors (IGF1-R), growth hormone receptor, the proliferation marker proliferating cell nuclear antigen, and pAKT by immunohistochemistry and immunofluorescence. Proliferation of liver cyst-derived epithelial cells was evaluated by both MTS proliferation assay and [3H]thymidine incorporation into DNA. The hepatic cyst epithelium displayed heterogeneous features, being normal in small cysts (<1 cm), characterized by rare or shortened cilia in 1- to 3-cm cysts, and exhibiting the absence of both primary cilia and microvilli in large cysts (>3 cm). Cyst epithelium showed marked immunohistochemical expression of ER, growth hormone receptor, IGF1, IGF1-R, proliferating cell nuclear antigen, and pAKT. IGF1 was 10-fold more enriched in the hepatic cyst fluid than in serum. Serum-deprived liver cyst-derived epithelial cells proliferated when exposed to 17β-estradiol and IGF1 and when exposed to human cyst fluid. ER or IGF1-R antagonists inhibited the proliferative effect of serum readmission, cyst fluid, 17β-estradiol, and IGF1. Our findings could explain the role of estrogens in accelerating the progression of ADPKD and may suggest a potential benefit of therapeutic strategies based on estrogen antagonism. PMID:18202196

  10. Protection against apoptosis in chicken bursa and thymus cells by phorbol ester in vitro

    SciTech Connect

    Asakawa, J.; Thorbecke, G.J. )

    1991-03-15

    Programmed suicide or apoptosis, due to activation of endogenous nucleases, occurs in immature CD4{sup {minus}}85{sup {minus}} mammalian thymus cells. Like the thymus, the bursa of Fabricius is a site of massive lymphopoiesis accompanied by cell death in vivo. In the present study the authors have, therefore, examined whether chicken bursa and thymus cells exhibit apoptosis. Bursa and thymus cells from SC chickens, 4-10 weeks of age, were incubated for 8-24 hrs with various reagents. Genomic DNA was isolated, electrophoresed in 3% Nusieve agarose gels, and examined for patterns of DNA fragmentation. A laddering of DNA in multiples of 200 base pairs, indicative of apoptosis, was observed with both bursa and thymus cells. These patterns of DNA fragmentation from bursa cells could be prevented by adding phorbol myristic acetate during culture and, more effectively, by PMA plus ionomycin, but not by ionomycin alone or by anti-{mu}. PMA did not affect the patterns of DNA fragmentation seen with spleen cells. Addition of the protein kinase C inhibitor staurosporin inhibited the preventive effect of PMA on apoptosis. PMA also greatly promoted the survival of bursa cells in culture, as assayed by percentage cell death and by {sup 3}H-thymidine incorporation. It is concluded that bursa and thymus cells from the chicken exhibit apoptosis. The data further suggest that protein kinase C activation protects apoptosis in cultured bursa cells.

  11. Modulation in vitro of human natural cytotoxicity, lymphocyte proliferative response to mitogens and cytokine production by essential fatty acids.

    PubMed Central

    Purasiri, P; Mckechnie, A; Heys, S D; Eremin, O

    1997-01-01

    Essential fatty acids (EFA) have been shown in animal studies to have a differential effect on various aspects of immune reactivity. However, there have been few studies in humans. Therefore, we elected to investigate the effects of a variety of EFA [gamma-linolenic acid (GLA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] in vitro on human blood lymphocyte reactivity, cytokine secretion and natural cytotoxicity. The proliferative response to polyclonal mitogens (phytohaemagglutinin, pokeweed mitogen, concanavalin A), as measured by [3H]thymidine incorporation into newly synthesized lymphocytes, was inhibited (P < 0.05) by all EFAs tested, in a dose-dependent manner (3-15 micrograms/ml). The greatest inhibition of proliferation was caused by EPA and DHA. Similarly, EPA, DHA and GLA significantly reduced cytotoxic activity [expressed as lytic units, using 51 chromium-release assays natural killer (NK) (K562 cells) and lymphokine-activated (LAK) (Daudi cells) cells] (P < 0.05) in a concentration-dependent manner (5-50 micrograms/ml), without affecting cell viability. EPA and DHA exhibited greater suppression than GLA. Furthermore, the inhibition of cell proliferation and suppression of natural cytotoxicity was associated with marked decrease in cytokine [interleukin-1 (IL-1), IL-2, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma)] production in vitro. Our findings demonstrate that EFAs (GLA, EPA, DHA) have the potential to inhibit significantly various aspects of human lymphocyte cell-mediated and humoral immune reactivities. PMID:9415022

  12. Effects of argon laser radiation on aortic endothelial cells: Early membrane changes and proliferative response

    SciTech Connect

    Franceschi, D.; Graham, D.; Alexander, J.J.; Koehler, K. )

    1989-06-01

    Membrane fluidity, transmembrane signaling responses, and proliferative characteristics of endothelial cells were studied to characterize biochemical and molecular changes after treatment with argon laser energy. Bovine aortic endothelial cells grown in monolayers were irradiated at 50, 100, and 200 J with an argon laser (wavelength, 488 and 514 nm). Proliferation, assayed by ({sup }3H)thymidine incorporation, was measured daily for 6 days. An initial lag phase was observed for irradiated cells when compared to nonirradiated controls (P less than 0.03), with eventual recovery by the third day. Membrane fluidity, determined by fluorescence anisotropy, was measured 1 hr after irradiation. A decrease in static rotational motion of 1,6-diphenyl-1,3,5-hexatriene (DPH) was noted in irradiated versus nonirradiated cells indicating a decrease in membrane fluidity (P less than 0.02). Dynamic studies of intracellular calcium and pH flux utilizing fluorescent probes demonstrated a preserved response to mitogenic stimulation. An increase in intracellular Ca2+ with a concomitant alkalinization of the intracellular milieu was observed in irradiated and non-irradiated cells in response to stimulation with endothelial cell growth factor (ECGF). These responses resemble those characterized for other mitogens. Argon laser energy applied to aortic endothelial cells decreases membrane fluidity early after irradiation. These alterations probably cause the initial lag observed in their proliferative response; however, the capacity to respond to exogenous mitogenic stimulation is maintained.

  13. Characterization and cytotoxicity of L-amino acid oxidase from the venom of king cobra (Ophiophagus hannah).

    PubMed

    Ahn, M Y; Lee, B M; Kim, Y S

    1997-06-01

    The aim of this project was to determine the cytotoxic components from the venom of king cobra, Ophiophagus hannah. Venom was purified by a combination of gel-filtration, ion-exchange and reversed-phase chromatographic steps. The biochemical properties of the cytotoxic component were consistent with those of L-amino acid oxidase. The molecular weight of the enzyme was estimated to be 150,000 by gel filtration and 70,000 under the denaturing conditions of SDS-PAGE, indicating a dimer. It has an isoelectric point of 4.5 and is a glycoprotein. The N-terminal sequence of L-amino acid oxidase from the king cobra venom was determined to be SVINLEESFQEPEYE. The cytotoxicity of L-amino acid oxidase was observed in stomach cancer, murine melanoma, fibrosarcoma, colorectal cancer and Chinese hamster ovary cell lines. Cytotoxicity resulted in the loss of ability in attachment and inhibition of cell proliferation. The cytotoxic protein decreased the level of cell proliferation by 74% according to [3H]thymidine uptake assay. The mechanism of enzyme action may be related to the inhibition of thymidine incorporation and an interaction with DNA.

  14. Dual Effect of Serum Amyloid A on the Invasiveness of Glioma Cells

    PubMed Central

    Knebel, Franciele Hinterholz; Albuquerque, Renata Chaves; Massaro, Renato Ramos; Maria-Engler, Silvya Stuchi; Campa, Ana

    2013-01-01

    Evidence sustains a role for the acute-phase protein serum amyloid A (SAA) in carcinogenesis and metastasis, and the protein has been suggested as a marker for tumor progression. Nevertheless, the demonstration of a direct activity of SAA on tumor cells is still incipient. We have investigated the effect of human recombinant SAA (rSAA) on two human glioma cell lines, A172 and T98G. rSAA stimulated the [3H]-thymidine incorporation of both lines, but had dual effects on migration and invasiveness which varied according to the cell line. In T98G, the rSAA increased migration and invasion behaviors whereas in A172 it decreased these behaviors. These findings agree with the effect triggered by rSAA on matrix metalloproteinases (MMPs) activities measured in a gelatinolytic assay. rSAA inhibited activity of both MMPs in A172 cells while increasing them in T98G cells. rSAA also affected the production of compounds present in the tumor microenvironment that orchestrate tumor progression, such as IL-8, the production of reactive oxygen species (ROS) and nitric oxide (NO). We also observed that both lines expressed all three of the isoforms of SAA: SAA1, SAA2, and SAA4. These data suggest that some tumor cells are responsive to SAA and, in these cases, SAA may have a role in cancer progression that varies according to the cell type. PMID:23533307

  15. Expression and functional activity of P-glycoprotein in cultured cerebral capillary endothelial cells.

    PubMed

    Hegmann, E J; Bauer, H C; Kerbel, R S

    1992-12-15

    Analysis of a panel of endothelial cells passaged between 5 and 25 times and derived from various organs and species demonstrated that murine and porcine cerebral capillary endothelial cells actively excluded the fluorescent dye rhodamine 123, a substrate of P-glycoprotein. In addition, rhodamine 123 accumulation could be enhanced by the multidrug resistance chemosensitizer verapamil, known to reduce P-glycoprotein-mediated drug efflux. Cloned murine and porcine cerebral capillary endothelial cells were immunoreactive with the C219 monoclonal antibody to P-glycoprotein, and a C219 epitope-specific blocking peptide could abolish staining. The antiproliferative and cytotoxic effects of vincristine, but not cis-platinum(II) diamminedichloride, were increased by the addition of either verapamil or cyclosporin A to brain endothelial cell cultures in a 72-h assay, as determined by [3H]thymidine incorporation and total protein measurement. Cyclosporin A was a more effective reversal agent than verapamil. Thus, a P-glycoprotein isoform may be constitutively expressed in brain endothelial cells in vitro and supports the available data on in situ immunohistochemical staining of P-glycoprotein at the blood-brain barrier. In addition, these findings may indicate that one function of P-glycoprotein in vivo at the blood-brain barrier is the exclusion of xenobiotics from central nervous system tissues.

  16. Inhibition of allogeneic T-cell response by Kupffer cells expressing indoleamine 2,3-dioxygenase

    PubMed Central

    Yan, Mao-Lin; Wang, Yao-Dong; Tian, Yi-Feng; Lai, Zhi-De; Yan, Lv-Nan

    2010-01-01

    AIM: To explore the possibility and mechanism of inhibiting allogeneic T-cell responses by Kupffer cells (KC) pretreated with interferon-γ (IFN-γ) in vitro. METHODS: The expressions of indoleamine 2,3-dioxygenase (IDO) mRNA and FasL mRNA in KC pretreated with IFN-γ were studied with real-time polymerase chain reaction (PCR). The catabolism of tryptophan by IDO from KC was analyzed by high performance liquid chromatography. Allogeneic T-cell response was used to confirm the inhibition of KC in vitro. The proliferation of lymphocytes was detected using [3H] thymidine incorporation. Cell cycle and lymphocyte apoptosis were evaluated by flow cytometric assay. RESULTS: Real-time PCR revealed IDO mRNA and FasL mRNA expressions in KC pretreated with IFN-γ, and IDO catabolic effect was confirmed by a decrease in tryptophan and increase in kynurenine concentration. KC expressing IDO and FasL in BABL/c mice acquired the ability to suppress the proliferation of T-cells from C57BL/6, which could be blocked by addition of 1-methyl-tryptophan and anti-FasL antibody. KC expressing IDO could induce allogeneic T-cell apoptosis. CONCLUSION: In addition to Fas/FasL pathway, IDO may be another mechanism for KC to induce immune tolerance. PMID:20128035

  17. Modulation of breast cancer cell survival by aromatase inhibiting hop (Humulus lupulus L.) flavonoids.

    PubMed

    Monteiro, Rosário; Faria, Ana; Azevedo, Isabel; Calhau, Conceição

    2007-01-01

    Hop flavonoids are being regarded as attractive molecules to prevent or treat certain forms of cancer. Studies have focused mainly on xanthohumol, the most abundant prenylated chalcone existing in hops extract. However, during the production of beer, or after its ingestion, xanthohumol originates different metabolites, among which isoxanthohumol and 8-prenylnaringenin. The aim of this work was to study the effect of the prenylflavonoids xanthohumol, isoxanthohumol and 8-prenylnaringenin on the breast cancer Sk-Br-3 cell line proliferation, apoptosis and activity of the enzyme aromatase (estrogen synthase). Aromatase activity was determined by a tritiated water assay, cell proliferation was assessed by [(3)H]thymidine incorporation, sulforhodamine B protein measurement and Ki-67 immunostaining and apoptosis was determined by TUNEL. Our results show that all tested prenylflavonoids were able to inhibit aromatase activity and thus, estrogen formation. Additionally, breast cancer cell line proliferation was decreased and apoptosis induced by all three compounds. The presence of 17beta-estradiol in treatment medium was able to revert the effect of the prenylflavonoids on cellular proliferation. These observations strengthen the idea that hop flavonoids may have anti-breast cancer effects and shed new light on a possible mechanism of action by which these effects occur, namely through their ability to decrease estrogen synthesis.

  18. Apoptosis and cell cycle disturbances induced by coumarin and 7-hydroxycoumarin on human lung carcinoma cell lines.

    PubMed

    Lopez-Gonzalez, Jose Sullivan; Prado-Garcia, Heriberto; Aguilar-Cazares, Dolores; Molina-Guarneros, Juan A; Morales-Fuentes, Jorge; Mandoki, Juan Jose

    2004-03-01

    Coumarin and 7-hydroxycoumarin have anti-tumour actions in vitro and in vivo. There are no previous reports on the cytostatic and apoptotic actions of coumarin and 7-hydroxycoumarin in non-small cell lung carcinoma (NSCLC) cell lines. Here we report on: (1) the inhibition of cell proliferation, (2) the phase in which cell cycle arrest occurs, and (3) the induction of apoptosis. Inhibition of cell proliferation was determined by 3H-thymidine incorporation. The effects on cell cycle phases were determined at 100 microg/ml of coumarin or 7-hydroxycoumarin using propidium iodide and flow cytometry. Higher concentrations were used to study apoptosis, detected by: (1) morphological cell changes, (2) subG1 peak detection and (3) Annexin-V assay. Peripheral blood mononuclear cells (PBMC) stimulated with phytohemagglutinin were used as controls. The actions of these compounds depended on drug concentrations and on histological cell type. Coumarin and 7-hydroxycoumarin inhibited cell growth by inducing cell cycle arrest in the G1 phase in all the lung carcinoma cell lines. Apoptosis required large concentrations of the coumarin compounds and was observed in adenocarcinomas. Apoptosis was not associated with intra-nucleosomal DNA fragmentation. Apoptosis was not observed in squamous lung carcinoma cell lines, but an increase in G1 cell cycle arrest was detected. In PBMC, only large concentrations of the coumarin compounds elicited a cystostatic action. Coumarins in combination with other anti-neoplastic drugs might increase the effectiveness of NSCLC treatments.

  19. Immune sensitization against epidermal antigens in polymorphous light eruption

    SciTech Connect

    Gonzalez-Amaro, R.; Baranda, L.; Salazar-Gonzalez, J.F.; Abud-Mendoza, C.; Moncada, B. )

    1991-01-01

    To get further insight into the pathogenesis of polymorphous light eruption, we studied nine patients with polymorphous light eruption and six healthy persons. Two skin biopsy specimens were obtained from each person, one from previously ultraviolet light-irradiated skin and another one from unirradiated skin. An epidermal cell suspension, skin homogenate, or both were prepared from each specimen. Autologous cultures were made with peripheral blood mononuclear cells combined with irradiated or unirradiated skin homogenate and peripheral blood mononuclear cells combined with irradiated or unirradiated epidermal cell suspension. Cell proliferation was assessed by 3H-thymidine incorporation assay. The response of peripheral blood mononuclear cells to unirradiated epidermal cells or unirradiated skin homogenate was similar in both patients and controls. However, peripheral blood mononuclear cells from patients with polymorphous light eruption showed a significantly increased proliferative response to both irradiated epidermal cells and irradiated skin homogenate. Our results indicate that ultraviolet light increases the stimulatory capability of polymorphous light eruption epidermal cells in a unidirectional mixed culture with autologous peripheral blood mononuclear cells. This suggests that an immune sensitization against autologous ultraviolet light-modified skin antigens occurs in polymorphous light eruption.

  20. Acemannan stimulates gingival fibroblast proliferation; expressions of keratinocyte growth factor-1, vascular endothelial growth factor, and type I collagen; and wound healing.

    PubMed

    Jettanacheawchankit, Suwimon; Sasithanasate, Siriruk; Sangvanich, Polkit; Banlunara, Wijit; Thunyakitpisal, Pasutha

    2009-04-01

    Aloe vera has long been used as a traditional medicine for inducing wound healing. Gingival fibroblasts (GFs) play an important role in oral wound healing. In this study, we investigated the effects of acemannan, a polysaccharide extracted from Aloe vera gel, on GF proliferation; keratinocyte growth factor-1 (KGF-1), vascular endothelial growth factor (VEGF), and type I collagen production; and oral wound healing in rats. [(3)H]-Thymidine incorporation assay and ELISA were used. Punch biopsy wounds were created at the hard palate of male Sprague Dawley rats. All treatments (normal saline; 0.1% triamcinolone acetonide; plain 1% Carbopol; and Carbopol containing 0.5%, 1%, and 2% acemannan (w/w)) were applied daily. Wounded areas and histological features were observed at day 7 after treatment. From our studies, acemannan at concentrations of 2, 4, 8, and 16 mg/ml significantly induced cell proliferation (P<0.05). Acemannan concentrations between 2 - 16 mg/ml significantly stimulated KGF-1, VEGF, and type I collagen expressions (P<0.05). Wound healing of animals receiving Carbopol containing 0.5% acemannan (w/w) was significantly better than that of the other groups (P<0.05). These findings suggest that acemannan plays a significant role in the oral wound healing process via the induction of fibroblast proliferation and stimulation of KGF-1, VEGF, and type I collagen expressions.

  1. Fenugreek Incorporated Silk Fibroin Nanofibers-A Potential Antioxidant Scaffold for Enhanced Wound Healing.

    PubMed

    Selvaraj, Sowmya; Fathima, Nishter Nishad

    2017-02-22

    Free radicals are generated by various biochemical pathways in the living system, causing severe oxidative damage to the biomolecules leading to adverse disease conditions. Hence, there is an increasing interest in antioxidant studies for preventing the effects of these free radicals. Herein, we propose a novel electrospun scaffold with antioxidant properties that can be used as wound healing material. Fenugreek, a natural antioxidant incorporated silk fibroin nanofiber, was prepared in four different ratios by the co-electrospinning method. The biocompatibility of the nanofibers and its antioxidant activity were evaluated through 3-(4, 5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT) assay and 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging assay, respectively. The experimental observations indicate that the incorporation of fenugreek increases the thermal and mechanical properties of silk fibroin nanofibers. DPPH assay proves that the antioxidant property is enhanced with increasing concentration of fenugreek in nanofiber mats, and the Swiss albino 3T6 fibroblasts show better proliferation on the nanofibrous scaffolds. Further, the wound healing efficiency of fenugreek incorporated silk fibroin nanofibrous scaffolds was evaluated using full thickness excisional wounds in rat model. Wound healing was accelerated in silk fibroin-fenugreek nanofibers treated wounds with complete re-epithelialization and enhanced collagen deposition. The present study validates the use of fenugreek incorporated silk fibroin nanofiber mats as antioxidant scaffolds in wound healing applications.

  2. Biochemical Assays of Cultured Cells

    NASA Technical Reports Server (NTRS)

    Barlow, G. H.

    1985-01-01

    Subpopulations of human embryonic kidney cells isolated from continuous flow electrophoresis experiments performed at McDonnell Douglas and on STS-8 have been analyzed. These analyses have included plasminogen activator assays involving indirect methodology on fibrin plated and direct methodology using chromogenic substrates. Immunological studies were performed and the conditioned media for erythropoietin activity and human granulocyte colony stimulating (HGCSF) activity was analyzed.

  3. Turbidimetric Assay of Staphylococcal Nuclease

    PubMed Central

    Erickson, Alan; Deibel, R. H.

    1973-01-01

    A simplified turbidimetric procedure was developed to assay staphylococcal nuclease activity. The ease of performance and sensitivity to nanogram quantities enhance the utilization of the method for the quantitative or qualitative estimation of the enzyme. Unlike plating methods, the turbidimetric procedure affords the differentiation between heat-stable and heat-labile nuclease activity. PMID:4735446

  4. Three dimensional colorimetric assay assemblies

    SciTech Connect

    Charych, D.; Reichart, A.

    2000-06-27

    A direct assay is described using novel three-dimensional polymeric assemblies which change from a blue to red color when exposed to an analyte, in one case a flu virus. The assemblies are typically in the form of liposomes which can be maintained in a suspension, and show great intensity in their color changes. Their method of production is also described.

  5. Three dimensional colorimetric assay assemblies

    DOEpatents

    Charych, Deborah; Reichart, Anke

    2000-01-01

    A direct assay is described using novel three-dimensional polymeric assemblies which change from a blue to red color when exposed to an analyte, in one case a flu virus. The assemblies are typically in the form of liposomes which can be maintained in a suspension, and show great intensity in their color changes. Their method of production is also described.

  6. An improved choline monooxygenase assay

    SciTech Connect

    Lafontaine, P.J.; Hanson, A.D. )

    1991-05-01

    Glycine betaine accumulates in leaves of plants from several angiosperm families in response to drought or salinization. Its synthesis, from the oxidation of choline, is mediated by a two step pathway. In spinach the first enzyme of this pathway is a ferredoxin-dependent choline monooxygenase (CMO). In order to purify this enzyme a sensitive and reliable assay is necessary. Two types of modifications were explored to improve the existing assay. (1) Ferredoxin reduction - one way of providing reduced Fd to CMO is by the addition of isolated spinach thylakoids in the assay mixture. In order to optimize the reduction of Fd two different systems were compared: (a) where only PS is active, by adding DCMU to inhibit electron transport from PS II and DAD as electron donor for PS I; (b) where both PS II and PS I are active. (2) Betaine aldehyde estimation - to simplify this, it is possible to couple the CMO reaction with betaine aldehyde dehydrogenase (BADH) from E. coli. BADH converts betaine aldehyde to betaine as it is formed in the assay, eliminating the need for a chemical oxidation step.

  7. Broad base biological assay using liquid based detection assays

    SciTech Connect

    Milanovich, F; Albala, J; Colston, B; Langlois, R; Venkateswaren, K

    2000-10-31

    The release of a biological agent by terrorists represents a serious threat to the safety of US citizens. At present there are over 50 pathogens and toxins on various agency threat lists. Most of these pathogens are rarely seen by public health personnel so the ability to rapidly identify their infection is limited. Since many pathogenic infections have symptomatic delays as long as several days, effective treatment is often compromised. This translates into two major deficiencies in our ability to counter biological terrorism (1) the lack of any credible technology to rapidly detect and identify all the pathogens or toxins on current threat lists and (2) the lack of a credible means to rapidly diagnose thousands of potential victims. In this SI we are developing a rapid, flexible, inexpensive, high throughput, and deeply multiplex-capable biological assay technology. The technology, which we call the Liquid Array (LA), utilizes optical encoding of small diameter beads which serve as the templates for biological capture assays. Once exposed to a fluid sample these beads can be identified and probed for target pathogens at rates of several thousand beads per second. Since each bead can be separately identified, one can perform parallel assays by assigning a different assay to each bead in the encoded set. The goal for this development is a detection technology capable of simultaneously identifying 100s of different bioagents and/or of rapidly diagnosing several thousand individuals. We are pursuing this research in three thrusts. In the first we are exploring the fundamental interactions of the beads with proteins and nucleic acids in complex mixtures. This will provide us with a complete understanding of the limits of the technology with respect to throughput and complex environment. A major spin-off of this activity is in the rapidly emerging field of proteomics where we may be able to rapidly assess the interactions responsible for cell metabolism, structural

  8. Assay strategies and methods for phospholipases

    SciTech Connect

    Reynolds, L.J.; Washburn, W.N.; Deems, R.A.; Dennis, E.A.

    1991-01-01

    Of the general considerations discussed, the two issues which are most important in choosing an assay are (1) what sensitivity is required to assay a particular enzyme and (2) whether the assay must be continuous. One can narrow the options further by considering substrate availability, enzyme specificity, assay convenience, or the presence of incompatible side reactions. In addition, the specific preference of a particular phospholipase for polar head group, micellar versus vesicular substrates, and anionic versus nonionic detergents may further restrict the options. Of the many assays described in this chapter, several have limited applicability or serious drawbacks and are not commonly employed. The most commonly used phospholipase assays are the radioactive TLC assay and the pH-stat assay. The TLC assay is probably the most accurate, sensitive assay available. These aspects often outweigh the disadvantages of being discontinuous, tedious, and expensive. The radioactive E. coli assay has become popular recently as an alternative to the TLC assay for the purification of the mammalian nonpancreatic phospholipases. The assay is less time consuming and less expensive than the TLC assay, but it is not appropriate when careful kinetics are required. Where less sensitivity is needed, or when a continuous assay is necessary, the pH-stat assay is often employed. With purified enzymes, when free thiol groups are not present, a spectrophotometric thiol assay can be used. This assay is {approximately} as sensitive as the pH-stat assay but is more convenient and more reproducible, although the substrate is not available commercially. Despite the many assay choices available, the search continues for a convenient, generally applicable assay that is both sensitive and continuous.

  9. 21 CFR 225.158 - Laboratory assays.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 4 2012-04-01 2012-04-01 false Laboratory assays. 225.158 Section 225.158 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS... Laboratory assays. Where the results of laboratory assays of drug components, including assays by State...

  10. 21 CFR 225.158 - Laboratory assays.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 4 2013-04-01 2013-04-01 false Laboratory assays. 225.158 Section 225.158 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS... Laboratory assays. Where the results of laboratory assays of drug components, including assays by State...

  11. 21 CFR 225.158 - Laboratory assays.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 4 2014-04-01 2014-04-01 false Laboratory assays. 225.158 Section 225.158 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS... Laboratory assays. Where the results of laboratory assays of drug components, including assays by State...

  12. 21 CFR 225.158 - Laboratory assays.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 4 2011-04-01 2011-04-01 false Laboratory assays. 225.158 Section 225.158 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS... Laboratory assays. Where the results of laboratory assays of drug components, including assays by State...

  13. Toxin activity assays, devices, methods and systems therefor

    DOEpatents

    Koh, Chung-Yan; Schaff, Ulrich Y.; Sommer, Gregory Jon

    2016-04-05

    Embodiments of the present invention are directed toward devices, system and method for conducting toxin activity assay using sedimentation. The toxin activity assay may include generating complexes which bind to a plurality of beads in a fluid sample. The complexes may include a target toxin and a labeling agent, or may be generated due to presence of active target toxin and/or labeling agent designed to be incorporated into complexes responsive to the presence of target active toxin. The plurality of beads including the complexes may be transported through a density media, wherein the density media has a lower density than a density of the beads and higher than a density of the fluid sample, and wherein the transporting occurs, at least in part, by sedimentation. Signal may be detected from the labeling agents of the complexes.

  14. Microbiologic assay of space hardware.

    NASA Technical Reports Server (NTRS)

    Favero, M. S.

    1971-01-01

    Review of the procedures used in the microbiological examination of space hardware. The general procedure for enumerating aerobic and anaerobic microorganisms and spores is outlined. Culture media and temperature-time cycles used for incubation are reviewed, along with assay systems designed for the enumeration of aerobic and anaerobic spores. The special problems which are discussed are involved in the precise and accurate enumeration of microorganisms on surfaces and in the neutralization of viable organisms buried inside solid materials that could be released to a planet's surface if the solid should be fractured. Special attention is given to sampling procedures including also the indirect techniques of surface assays of space hardware such as those using detachable or fallout strips. Some data on comparative levels of microbial contamination on lunar and planetary spacecraft are presented.

  15. Important Norwegian crude assays updated

    SciTech Connect

    Corbett, R.A

    1990-03-12

    New assays on two important Norwegian North Sea crude oils, Statfjord and Gullfaks, are presented. Both are high-quality, low-sulfur crudes that will yield a full range of good-quality products. All assay data came from industry-standard test procedures. The Statfjord field is the largest in the North Sea. Production started in 1979. Statfjord is a typical North Sea crude, produced from three separate platforms and three separate loading buoys with interconnecting lines. Current production is about 700,000 b/d. Gullfaks is produced from a large field in Block 34/10 of the Norwegian sector of the North Sea production area. Gullfaks crude oil is more biodegraded than other crudes from the region. Biodegradation has removed most of the waxy normal paraffins, resulting in a heavier, more naphthenic and aromatic crude.

  16. Comet Assay in Cancer Chemoprevention.

    PubMed

    Santoro, Raffaela; Ferraiuolo, Maria; Morgano, Gian Paolo; Muti, Paola; Strano, Sabrina

    2016-01-01

    The comet assay can be useful in monitoring DNA damage in single cells caused by exposure to genotoxic agents, such as those causing air, water, and soil pollution (e.g., pesticides, dioxins, electromagnetic fields) and chemo- and radiotherapy in cancer patients, or in the assessment of genoprotective effects of chemopreventive molecules. Therefore, it has particular importance in the fields of pharmacology and toxicology, and in both environmental and human biomonitoring. It allows the detection of single strand breaks as well as double-strand breaks and can be used in both normal and cancer cells. Here we describe the alkali method for comet assay, which allows to detect both single- and double-strand DNA breaks.

  17. Protein binding assay for hyaluronate

    SciTech Connect

    Lacy, B.E.; Underhill, C.B.

    1986-11-01

    A relatively quick and simple assay for hyaluronate was developed using the specific binding protein, hyaluronectin. The hyaluronectin was obtained by homogenizing the brains of Sprague-Dawley rats, and then centrifuging the homogenate. The resulting supernatant was used as a source of crude hyaluronectin. In the binding assay, the hyaluronectin was mixed with (/sup 3/H)hyaluronate, followed by an equal volume of saturated (NH/sub 4/)/sub 2/SO/sub 4/, which precipitated the hyaluronectin and any (/sup 3/H)hyaluronate associated with it, but left free (/sup 3/H)hyaluronate in solution. The mixture was then centrifuged, and the amount of bound (/sup 3/H)hyaluronate in the precipitate was determined. Using this assay, the authors found that hyaluronectin specifically bound hyaluronate, since other glycosaminoglycans failed to compete for the binding protein. In addition, the interaction between hyaluronectin and hyaluronate was of relatively high affinity, and the size of the hyaluronate did not appear to substantially alter the amount of binding. To determine the amount of hyaluronate in an unknown sample, they used a competition assay in which the binding of a set amount of (/sup 3/H)hyaluronate was blocked by the addition of unlabeled hyaluronate. By comparing the degree of competition of the unknown samples with that of known amounts of hyaluronate, it was possible to determine the amount of hyaluronate in the unknowns. They have found that this method is sensitive to 1 ..mu..g or less of hyaluronate, and is unaffected by the presence of proteins.

  18. [Visible spectrophotometric assay of ranitidine].

    PubMed

    Apostu, M; Dorneanu, V; Bibire, Nela

    2003-01-01

    Ranitidine, belonging to H2-antagonist group, is a compound containing a furanic moiety and is used in peptic ulcer therapy. This paper debates the possibility of developing a new visible spectrophotometric assessment by using the reaction between ranitidine and eosine. We carried out our determinations at 505 nm, where the absorbency of ranitidine-eosine complex is maximal, and we have established the optimal reaction conditions. This method was successfully applied for ranitidine assay from pharmaceutical dosage forms.

  19. Two offshore Australian crudes assayed

    SciTech Connect

    Rhodes, A.K.

    1994-05-09

    Two light, sweet crudes from offshore Australia have been assayed. Gippsland crude, also called Bass Strait, is produced off the coast of Victoria, in southeastern Australia. The 47 API, 0.09% sulfur crude was analyzed in mid-1993. Skua, a 42 API, 0.06 wt % sulfur crude, is produced in the Timor Sea. Data are given on the whole crude and fractions for both deposits. Both chemical and physical properties are listed.

  20. Bioluminescence assay for cell viability.

    PubMed

    Lomakina, G Yu; Modestova, Yu A; Ugarova, N N

    2015-06-01

    Theoretical aspects of the adenosine triphosphate bioluminescence assay based on the use of the firefly luciferin-luciferase system are considered, as well as its application for assessing cell viability in microbiology, sanitation, medicine, and ecology. Various approaches for the analysis of individual or mixed cultures of microorganisms are presented, and capabilities of the method for investigation of biological processes in live cells including necrosis, apoptosis, as well as for investigation of the dynamics of metabolism are described.

  1. Optimization of a UDP-glucuronosyltransferase assay for trout ...

    EPA Pesticide Factsheets

    An existing assay for hepatic UDP-glucuronosyltransferase (UGT) activity was optimized for use with trout liver S9 fractions. Individual experiments were conducted to determine the time dependence of UGT activity as well as optimal levels of S9 protein, uridine 5’-diphosphoglucuronic acid (UDPGA; a necessary cofactor), alamethicin (a pore-forming agent added to eliminate latency), and substrate (p-nitrophenol). Addition of Mg2+ (to 1 mM) or bovine serum albumin (BSA; to 2% w/v) had variable effects on activity, but these effects were minor. Eliminating alamethicin from the system resulted in very low levels of activity. A portion of this activity could be recovered by adding Triton X-100 or Brij 58; however, the optimal concentration range for either detergent was very narrow. All studies were performed under physiological conditions (pH 7.8, 11 °C) to support ongoing development of methods for extrapolating in vitro rates of biotransformation to the intact animal. When expressed on a pmol/min/g liver basis, UGT activities determined using this updated assay were substantially higher than those reported previously for uninduced trout. The purpose of the present study was to optimize an existing in vitro assay for hepatic UGT activity in rainbow trout. The original assay, adapted here for use with trout S9 fractions, was updated by incorporating a membrane disrupting agent (alamethicin) to reduce latency. Additional experiments were conducted to evaluate

  2. Arabinogalactan proteins are incorporated in negatively charged coffee brew melanoidins.

    PubMed

    Bekedam, E Koen; De Laat, Marieke P F C; Schols, Henk A; Van Boekel, Martinus A J S; Smit, Gerrit

    2007-02-07

    The charge properties of melanoidins in high molecular weight (HMw) coffee brew fractions, isolated by diafiltration and membrane dialysis, were studied. Ion exchange chromatography experiments with the HMw fractions showed that coffee brew melanoidins were negatively charged whereas these molecules did not expose any positive charge at the pH of coffee brew. Fractions with different ionic charges were isolated and subsequently characterized by means of the specific extinction coefficient (K(mix 405nm)), sugar composition, phenolic group content, nitrogen content, and the arabinogalactan protein (AGP) specific Yariv gel-diffusion assay. The isolated fractions were different in composition and AGP was found to be present in one of the HMw fractions. The AGP accounted for 6% of the coffee brew dry matter and had a moderate negative charge, probably caused by the presence of uronic acids. As the fraction that precipitated with Yariv was brown (K(mix 405nm) = 1.2), compared to a white color in the green bean, it was concluded that these AGPs had undergone Maillard reaction resulting in an AGP-melanoidin complex. The presence of mannose (presumably from galactomannan) indicates the incorporation of galactomannans in the AGP-melanoidin complex. As the uronic acid content in the more negatively charged melanoidin-rich, AGP-poor HMw fractions decreased, it was hypothesized that acidic groups are formed or incorporated during melanoidin formation.

  3. Effects of MMP inhibitors incorporated within dental adhesives.

    PubMed

    Almahdy, A; Koller, G; Sauro, S; Bartsch, J W; Sherriff, M; Watson, T F; Banerjee, A

    2012-06-01

    Matrix metalloproteinase (MMP) inhibition has been shown to reduce adhesive bond degradation when applied as a pre-conditioner, adding to clinical steps in the placement of adhesives, but their incorporation within dental adhesives has not been fully explored. This study examined the effect of including 2 MMP inhibitors (BB94 and GM6001) within the primers of 3 commercially available adhesives. Fluorometric assay and zymography showed that adhesives with MMP inhibitors had high affinity toward both synthetic fluorogenic FRET peptides (95%) and dentin powder substrates, respectively. The immediate microtensile bond strength was enhanced for 2 types of adhesives following the addition of both inhibitors. However, no changes were detected between the control and the inhibitor groups following 3-month storage. The modified two-step etch-and-rinse and single-step systems showed less Rhodamine B penetration to the "hybrid layer" and to the "adhesive", respectively. The incorporation of BB94 and GM6001 within the primers resulted in the inhibition of dentin MMPs with improved initial bond strength and enhanced sealing ability.

  4. Effects of MMP Inhibitors Incorporated within Dental Adhesives

    PubMed Central

    Almahdy, A.; Koller, G.; Sauro, S.; Bartsch, J.W.; Sherriff, M.; Watson, T.F.; Banerjee, A.

    2012-01-01

    Matrix metalloproteinase (MMP) inhibition has been shown to reduce adhesive bond degradation when applied as a pre-conditioner, adding to clinical steps in the placement of adhesives, but their incorporation within dental adhesives has not been fully explored. This study examined the effect of including 2 MMP inhibitors (BB94 and GM6001) within the primers of 3 commercially available adhesives. Fluorometric assay and zymography showed that adhesives with MMP inhibitors had high affinity toward both synthetic fluorogenic FRET peptides (95%) and dentin powder substrates, respectively. The immediate microtensile bond strength was enhanced for 2 types of adhesives following the addition of both inhibitors. However, no changes were detected between the control and the inhibitor groups following 3-month storage. The modified two-step etch-and-rinse and single-step systems showed less Rhodamine B penetration to the “hybrid layer” and to the “adhesive”, respectively. The incorporation of BB94 and GM6001 within the primers resulted in the inhibition of dentin MMPs with improved initial bond strength and enhanced sealing ability. PMID:22518030

  5. The Rabbit Corneal Pocket Assay.

    PubMed

    Morbidelli, Lucia; Ziche, Marina

    2016-01-01

    The rabbit corneal micropocket angiogenesis assay uses the avascular cornea as a substrate canvas to study angiogenesis in vivo. Through the use of standardized slow-release pellets, a predictable angiogenic response is generated over the course of 1-2 weeks and then quantified. Uniform slow-release pellets are prepared by mixing purified angiogenic growth factors such as basic fibroblast growth factor or vascular endothelial growth factor and a synthetic polymer to allow slow release. A micropocket is surgically created in the rabbit cornea under anesthesia and a pellet implanted. On the days later, the angiogenic response is measured and qualified using a slit lamp, as well as the concomitant vascular phenotype or inflammatory features. The results of the assay are used to assess the ability of potential therapeutic molecules to modulate angiogenesis in vivo, both when released locally or given by ocular formulations or through systemic treatment. In this chapter, the experimental details of the rabbit cornea assay and technical implementations to the original protocol are described.

  6. Assay of ribulose bisphosphate carboxylase

    SciTech Connect

    Pike, C.; Berry, J.

    1987-04-01

    Assays of ribulose bisphosphate carboxylase (rubisco) can be used to illustrate many properties of photosynthetic systems. Many different leaves have been assayed with this standard procedure. The tissue is ground with a mortar and pestle in extraction buffer. The supernatant after centrifugation is used as the source of enzyme. Buffer, RuBP, (/sup 14/C)-NaHCO/sub 3/, and enzyme are combined in a scintillation vial; the reaction is run for 1 min at 30/sup 0/. The acid-stable products are counted. Reproducibility in student experiments has been excellent. The assay data can be combined with analyses of leaf properties such as fresh and dry weight, chlorophyll and protein content, etc. Students have done projects such as the response of enzyme to temperature and to various inhibitors. They also report on the use of a transition state analog, carboxyarabinitol bisphosphate, to titrate the molar concentration of rubisco molecules (active sites) in an enzyme sample. Thus, using crude extracts the catalytic activity of a sample can be compared to the absolute quantity of enzyme or to the turnover number.

  7. Optical fiber hybridization assay fluorosensor

    NASA Astrophysics Data System (ADS)

    Pilevar, Saeed; Davis, Christopher C.; Hodzic, Vildana; Portugal, Frank

    1999-04-01

    The present work describes an all-fiber hybridization assay sensor that relies on the evanescent field excitation of fluorescence from surface-bound fluorophores. The evanescent field is made accessible through the use of a long adiabatically tapered single-mode fiber probe. A semiconductor laser operating at 785 nm wavelength is used in a pulsed mode to excite fluorescence in the tapered region of a fiber probe using the near-infrared fluorophore IRD 41. We have carried out real-time hybridization tests for IRD 41-labeled oligonucleotide at various probe concentrations binding to complementary oligonucleotide cross-linked to the tapered fiber surface. Short oligonucleotides (20-mer) bound to the fiber surface have been used to detect near-infrared dye labeled complementary sequences at sub-nanomolar levels. Sandwich assays with total RNA were conducted to examine the capability of the biosensor for detecting bacterial cells using rRNA as the target. The results indicate that this fluorosensor is capable of detecting H. pylori in a sandwich assay at picomolar concentrations.

  8. Optimized PCR assay for detection of white spot syndrome virus (WSSV).

    PubMed

    Nunan, Linda M; Lightner, Donald V

    2011-01-01

    A rapid PCR assay for detection of white spot syndrome virus (WSSV) was developed based on the nested PCR procedure described by Lo et al. (1996) and outlined as the recommended PCR diagnostic assay in the Manual of Diagnostic Tests for Aquatic Animals published by the Office of International Epizootics (OIE, 2009). The optimized procedure incorporated the second step primers used in the nested WSSV PCR. By adjusting the annealing temperature and shortening the cycling times, this modified assay is substantially faster and as sensitive as the recommended OIE protocol. The modified PCR test was compared directly to the two-step nested PCR protocol and a modified nested procedure. The sensitivity of the published assay was determined by template dilutions of semi-purified WSSV virions that had been quantitated using real-time PCR for detection of WSSV. Various isolates were tested using the modified procedure, to ensure that the assay was able to detect WSSV from different geographical locations.

  9. Noncompetitive affinity assays of glucagon and amylin using mirror-image aptamers as affinity probes.

    PubMed

    Yi, Lian; Wang, Xue; Bethge, Lucas; Klussmann, Sven; Roper, Michael G

    2016-03-21

    The ability to detect picomolar concentrations of glucagon and amylin using fluorescently labeled mirror-image aptamers, so-called Spiegelmers, is demonstrated. Spiegelmers rival the specificity of antibodies and overcome the problem of biostability of natural aptamers in a biological matrix. Using Spiegelmers as affinity probes, noncompetitive capillary electrophoresis affinity assays of glucagon and murine amylin were developed and optimized. The detection limit for glucagon was 6 pM and for amylin was 40 pM. Glucagon-like peptide-1 and -2 did not interfere with the glucagon assay, while the amylin assay showed cross-reactivity to calcitonin gene related peptide. The developed assays were combined with a competitive immunoassay for insulin to measure glucagon, amylin, and insulin secretion from batches of islets after incubation with different glucose concentrations. The development of these assays is an important step towards incorporation into an online measurement system for monitoring dynamic secretion from single islets.

  10. Assays for determination of protein concentration.

    PubMed

    Olson, Bradley J S C; Markwell, John

    2007-05-01

    Biochemical analysis of proteins relies on accurate quantitation of protein concentration. This unit describes how to perform commonly used protein assays, e.g., Lowry, Bradford, BCA, and UV spectroscopic protein assays. The primary focus of the unit is assay selection, emphasizing sample and buffer compatibility. Protein assay standard curves and data processing fundamentals are discussed in detail. This unit also details high-throughput adaptations of the commonly used protein assays, and also contains a protocol for BCA assay of total protein in SDS-PAGE sample buffer that is used for equal loading of SDS-PAGE gels, which is reliable, inexpensive, and quick.

  11. Assays for determination of protein concentration.

    PubMed

    Olson, Bradley J S C; Markwell, John

    2007-09-01

    Biochemical analysis of proteins relies on accurate quantitation of protein concentration. This appendix describes how to perform commonly used protein assays, e.g., Lowry, Bradford, BCA, and UV spectroscopic protein assays. The primary focus of the appendix is assay selection, emphasizing sample and buffer compatibility. Protein assay standard curves and data processing fundamentals are discussed in detail. This appendix also details high-throughput adaptations of the commonly used protein assays, and also contains a protocol for BCA assay of total protein in SDS-PAGE sample buffer that is used for equal loading of SDS-PAGE gels, which is reliable, inexpensive, and quick.

  12. Radioenzymatic assay for quinolinic acid

    SciTech Connect

    Foster, A.C.; Okuno, E.; Brougher, D.S.; Schwarcz, R.

    1986-10-01

    A new and rapid method for the determination of the excitotoxic tryptophan metabolite quinolinic acid is based on its enzymatic conversion to nicotinic acid mononucleotide and, in a second step utilizing (/sup 3/H)ATP, further to (/sup 3/H) deamido-NAD. Specificity of the assay is assured by using a highly purified preparation of the specific quinolinic acid-catabolizing enzyme, quinolinic acid phosphoribosyltransferase, in the initial step. The limit of sensitivity was found to be 2.5 pmol of quinolinic acid, sufficient to conveniently determine quinolinic acid levels in small volumes of human urine and blood plasma.

  13. Rotor assembly and assay method

    DOEpatents

    Burtis, Carl A.; Johnson, Wayne F.; Walker, William A.

    1993-01-01

    A rotor assembly for carrying out an assay includes a rotor body which is rotatable about an axis of rotation, and has a central chamber and first, second, third, fourth, fifth, and sixth chambers which are in communication with and radiate from the central chamber. The rotor assembly further includes a shuttle which is movable through the central chamber and insertable into any of the chambers, the shuttle including a reaction cup carrying an immobilized antigen or an antibody for transport among the chambers. A method for carrying out an assay using the rotor assembly includes moving the reaction cup among the six chambers by passing the cup through the central chamber between centrifugation steps in order to perform the steps of: separating plasma from blood cells, binding plasma antibody or antigen, washing, drying, binding enzyme conjugate, reacting with enzyme substrate and optically comparing the resulting reaction product with unreacted enzyme substrate solution. The movement of the reaction cup can be provided by attaching a magnet to the reaction cup and supplying a moving magnetic field to the rotor.

  14. SNO+ Scintillator Purification and Assay

    SciTech Connect

    Ford, R.; Vazquez-Jauregui, E.; Chen, M.; Chkvorets, O.; Hallman, D.

    2011-04-27

    We describe the R and D on the scintillator purification and assay methods and technology for the SNO+ neutrino and double-beta decay experiment. The SNO+ experiment is a replacement of the SNO heavy water with liquid scintillator comprised of 2 g/L PPO in linear alkylbenzene (LAB). During filling the LAB will be transported underground by rail car and purified by multi-stage distillation and steam stripping at a flow rate of 19 LPM. While the detector is operational the scintillator can be recirculated at 150 LPM (full detector volume in 4 days) to provide repurification as necessary by either water extraction (for Ra, K, Bi) or by functional metal scavenger columns (for Pb, Ra, Bi, Ac, Th) followed by steam stripping to remove noble gases and oxygen (Rn, O{sub 2}, Kr, Ar). The metal scavenger columns also provide a method for scintillator assay for ex-situ measurement of the U and Th chain radioactivity. We have developed ''natural'' radioactive spikes of Pb and Ra in LAB and use these for purification testing. Lastly, we present the planned operating modes and purification strategies and the plant specifications and design.

  15. Assay of potentially contaminated propellant

    SciTech Connect

    Koster, J.E.; Williams, H.E. III; Scott, W.S.

    1995-02-01

    One of the decontamination and decommissioning projects within DOD is demilitarization of an aging stockpile of munitions. A large portion of the stockpile contains depleted uranium (DU) as an armor piercing core and so these munitions must be assayed for the presence of uranium in other components. The assay method must be fast and preferably easy to implement. Presence of DU is indicated by its alpha decay. The alpha particles in turn produce ions in the ambient air. If a significant fraction of these ions can escape the quantity of propellant, the ions can be detected instead of the alpha particles. As a test of the feasibility of detecting alpha emissions from DU somewhere within a cartridge of propellant, the transmission of ions through layers of real propellant was measured. The propellant is in the form of graphite-coated cylindrical pellets. A 105nun cartridge was modified for use as a pellet chamber. A check source served as an ion source. The ion detector consisted of a grid held at 300V coupled to an ammeter. Results confirm that this is a promising technique for testing the propellant for the presence of DU quickly yet with sensitivity.

  16. SNO+ Scintillator Purification and Assay

    NASA Astrophysics Data System (ADS)

    Ford, R.; Chen, M.; Chkvorets, O.; Hallman, D.; Vázquez-Jáuregui, E.

    2011-04-01

    We describe the R&D on the scintillator purification and assay methods and technology for the SNO+ neutrino and double-beta decay experiment. The SNO+ experiment is a replacement of the SNO heavy water with liquid scintillator comprised of 2 g/L PPO in linear alkylbenzene (LAB). During filling the LAB will be transported underground by rail car and purified by multi-stage distillation and steam stripping at a flow rate of 19 LPM. While the detector is operational the scintillator can be recirculated at 150 LPM (full detector volume in 4 days) to provide repurification as necessary by either water extraction (for Ra, K, Bi) or by functional metal scavenger columns (for Pb, Ra, Bi, Ac, Th) followed by steam stripping to remove noble gases and oxygen (Rn, O2, Kr, Ar). The metal scavenger columns also provide a method for scintillator assay for ex-situ measurement of the U and Th chain radioactivity. We have developed "natural" radioactive spikes of Pb and Ra in LAB and use these for purification testing. Lastly, we present the planned operating modes and purification strategies and the plant specifications and design.

  17. Rotor assembly and assay method

    DOEpatents

    Burtis, C.A.; Johnson, W.F.; Walker, W.A.

    1993-09-07

    A rotor assembly for carrying out an assay includes a rotor body which is rotatable about an axis of rotation, and has a central chamber and first, second, third, fourth, fifth, and sixth chambers which are in communication with and radiate from the central chamber. The rotor assembly further includes a shuttle which is movable through the central chamber and insertable into any of the chambers, the shuttle including a reaction cup carrying an immobilized antigen or an antibody for transport among the chambers. A method for carrying out an assay using the rotor assembly includes moving the reaction cup among the six chambers by passing the cup through the central chamber between centrifugation steps in order to perform the steps of: separating plasma from blood cells, binding plasma antibody or antigen, washing, drying, binding enzyme conjugate, reacting with enzyme substrate and optically comparing the resulting reaction product with unreacted enzyme substrate solution. The movement of the reaction cup can be provided by attaching a magnet to the reaction cup and supplying a moving magnetic field to the rotor. 34 figures.

  18. Therapy for incorporated radionuclides: scope and need

    SciTech Connect

    Smith, V.H.

    1981-03-01

    In the United States the recent termination of funding for research on therapy for incorporated radionuclides has virtually halted progress on improved or new agents and procedures for removing radioactivity from the body. Research was eliminated, but is still needed on new removal agents, improved delivery system, in vitro test systems, and the toxicology of treatments. For many radionuclides, no adequate therapy exists. The relationship between radionuclide removal and reduction in cancer risk is still unanswered. Without proper research support, needed improvements in the treatment for incorporated radionuclides in the US are uncertain.

  19. Erythropoietin assay: present status of methods, pitfalls, and results in polycythemic disorders.

    PubMed

    Popovic, W J; Adamson, J W

    1978-01-01

    Mammalian erythropoiesis is regulated primarily by the hormone erythropoietin (ESP). Studies of ESF have provided information about its biochemistry and its role in regulating hemoglobin synthesis. Such studies rely on assays for erythropoietic activity in biological fluid. The assay which has proven most valuable and is used most widely is based upon the incorporation of radioactive iron into newly-formed red cells of polycythemic mice. While this assay has gained wide acceptance, it is expensive, cumbersome, imprecise, and insensitive, capable of reliably detecting no less than 50 milliunits of erythropoietin. Improvements in assay techniques will require new methodology relying primarily on immunologic recognition for the determination of hormone activity. Currently under development and in experimental use are radioimmunoassays and a hemagglutination inhibition assay. While work has progressed in these areas, these assays are not of proven value at present and meaningful physiological correlations have not emerged from their use. Alternatively, assays for hormone activity using suspensions of hematopoietic cells and the measurement of incorporation of radioactive isotopes into hemoglobin have provided both improvement in sensitivity and precision. The disadvantage of these types of assays is that they are sensitive to factors other than ESF and may give misleading information, depending on whether the factors present stimulate or inhibit cellular proliferation and hemoglobin synthesis. While such techniques may provide a temporary solution to some problems associated with assaying ESF for purification or physiological studies, they are not the best answer to the overall problem of hormone detection and characterization. The most important contribution to this field will be the availability of large amounts of highly purified and well-characterized ESF.

  20. 21 CFR 864.7425 - Carboxyhemoglobin assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Carboxyhemoglobin assay. 864.7425 Section 864.7425...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7425 Carboxyhemoglobin assay. (a) Identification. A carboxyhemoglobin assay is a device used to determine the...

  1. 21 CFR 864.7425 - Carboxyhemoglobin assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Carboxyhemoglobin assay. 864.7425 Section 864.7425...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7425 Carboxyhemoglobin assay. (a) Identification. A carboxyhemoglobin assay is a device used to determine the...

  2. 21 CFR 864.7425 - Carboxyhemoglobin assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Carboxyhemoglobin assay. 864.7425 Section 864.7425...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7425 Carboxyhemoglobin assay. (a) Identification. A carboxyhemoglobin assay is a device used to determine the...

  3. 21 CFR 864.7425 - Carboxyhemoglobin assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Carboxyhemoglobin assay. 864.7425 Section 864.7425...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7425 Carboxyhemoglobin assay. (a) Identification. A carboxyhemoglobin assay is a device used to determine the...

  4. 21 CFR 864.7425 - Carboxyhemoglobin assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Carboxyhemoglobin assay. 864.7425 Section 864.7425...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7425 Carboxyhemoglobin assay. (a) Identification. A carboxyhemoglobin assay is a device used to determine the...

  5. A colorimetric assay for cytokinin oxidase.

    PubMed

    Libreros-Minotta, C A; Tipton, P A

    1995-11-01

    A simple and rapid colorimetric assay for cytokinin oxidase is described. The assay is based on the formation of a Schiff base between the enzymatic reaction product 3-methyl-2-butenal and p-aminophenol. The assay is effective in the submicromolar concentration range and can be used in crude plant extracts as well as in more highly purified preparations.

  6. Data transformation methods for multiplexed assays

    DOEpatents

    Tammero, Lance F. Bentley; Dzenitis, John M; Hindson, Benjamin J

    2013-07-23

    Methods to improve the performance of an array assay are described. A correlation between fluorescence intensity-related parameters and negative control values of the assay is determined. The parameters are then adjusted as a function of the correlation. As a result, sensitivity of the assay is improved without changes in its specificity.

  7. 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.

  8. Sperm chromatin structure assay (SCSA®).

    PubMed

    Evenson, Donald P

    2013-01-01

    The SCSA(®) is the pioneering assay for the detection of damaged sperm DNA and altered proteins in sperm nuclei via flow cytometry of acridine orange (AO) stained sperm. The SCSA(®) is considered to be the most precise and repeatable test providing very unique, dual parameter data (red vs. green fluorescence) on a 1,024 × 1,024 channel scale, not only on DNA fragmentation but also on abnormal sperm characterized by lack of normal exchange of histones to protamines. Raw semen/sperm aliquots or purified sperm can be flash frozen, placed in a box with dry ice and shipped by overnight courier to an experienced SCSA(®) lab. The samples are individually thawed, prepared, and analyzed in ∼10 min. Of significance, data on 5,000 individual sperm are recorded on a 1,024 × 1,024 dot plot of green (native DNA) and red (broken DNA) fluorescence. Repeat measurements have virtually identical dot plot patterns demonstrating that the low pH treatment that opens up the DNA strands at the sites of breaks and staining by acridine orange (AO) are highly precise and repeatable (CVs of 1-3%) and the same between fresh and frozen samples. SCSAsoft(®) software transforms the X-Y data to total DNA stainability versus red/red + green fluoresence (DFI) providing a more accurate determination of % DFI as well as the more sensitive value of standard deviation of DFI (SD DFI) as demonstrated by animal fertility and dose-response toxicology studies. The current established clinical threshold is 25% DFI for placing a man into a statistical probability of the following: (a) longer time to natural pregnancy, (b) low odds of IUI pregnancy, (c) more miscarriages, or (d) no pregnancy. Changes in lifestyle as well as medical intervention can lower the %DFI to increase the probability of natural pregnancy. Couples of men with >25% DFI are counseled to try ICSI and when in the >50% range may consider TESE/ICSI. The SCSA(®) simultaneously determines the % of sperm with high DNA stainability (%HDS

  9. Forensically relevant SNaPshot(®) assays for human DNA SNP analysis: a review.

    PubMed

    Mehta, Bhavik; Daniel, Runa; Phillips, Chris; McNevin, Dennis

    2017-01-01

    Short tandem repeats are the gold standard for human identification but are not informative for forensic DNA phenotyping (FDP). Single-nucleotide polymorphisms (SNPs) as genetic markers can be applied to both identification and FDP. The concept of DNA intelligence emerged with the potential for SNPs to infer biogeographical ancestry (BGA) and externally visible characteristics (EVCs), which together enable the FDP process. For more than a decade, the SNaPshot(®) technique has been utilised to analyse identity and FDP-associated SNPs in forensic DNA analysis. SNaPshot is a single-base extension (SBE) assay with capillary electrophoresis as its detection system. This multiplexing technique offers the advantage of easy integration into operational forensic laboratories without the requirement for any additional equipment. Further, the SNP panels from SNaPshot(®) assays can be incorporated into customised panels for massively parallel sequencing (MPS). Many SNaPshot(®) assays are available for identity, BGA and EVC profiling with examples including the well-known SNPforID 52-plex identity assay, the SNPforID 34-plex BGA assay and the HIrisPlex EVC assay. This review lists the major forensically relevant SNaPshot(®) assays for human DNA SNP analysis and can be used as a guide for selecting the appropriate assay for specific identity and FDP applications.

  10. Incorporating the National Standards in Performance Classes.

    ERIC Educational Resources Information Center

    Kerchner, Jody L.

    2001-01-01

    Addresses how the National Standards can be incorporated into band rehearsals using the music piece, "Balladair" (Frank Erickson), as an example when teaching techniques and musical concepts. Provides ideas such as using material from "Balladair" to create warm-ups or relating the piece to other arts. (CMK)

  11. Incorporating Engineering Design Challenges into STEM Courses

    ERIC Educational Resources Information Center

    Householder, Daniel L., Ed.; Hailey, Christine E., Ed.

    2012-01-01

    Successful strategies for incorporating engineering design challenges into science, technology, engineering, and mathematics (STEM) courses in American high schools are presented in this paper. The developers have taken the position that engineering design experiences should be an important component of the high school education of all American…

  12. 49 CFR 572.190 - Incorporated materials.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... Impact Crash Test Dummy, July 1, 2008,” incorporated by reference in § 572.191; (4) SAE Recommended...) SAE J1733 of 1994-12, “Sign Convention for Vehicle Crash Testing.” (b) The Director of the Federal... 20879, (301) 670-0090. (2) The SAE materials referred to in paragraphs (a)(4) and (a)(5) of this...

  13. 49 CFR 572.190 - Incorporated materials.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Impact Crash Test Dummy, July 1, 2008,” incorporated by reference in § 572.191; (4) SAE Recommended...) SAE J1733 of 1994-12, “Sign Convention for Vehicle Crash Testing.” (b) The Director of the Federal... 20879, (301) 670-0090. (2) The SAE materials referred to in paragraphs (a)(4) and (a)(5) of this...

  14. 49 CFR 572.40 - Incorporated materials.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 7 2014-10-01 2014-10-01 false Incorporated materials. 572.40 Section 572.40 Transportation Other Regulations Relating to Transportation (Continued) NATIONAL HIGHWAY TRAFFIC SAFETY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) ANTHROPOMORPHIC TEST DEVICES Side Impact Dummy...

  15. Combined cycle power plant incorporating coal gasification

    DOEpatents

    Liljedahl, Gregory N.; Moffat, Bruce K.

    1981-01-01

    A combined cycle power plant incorporating a coal gasifier as the energy source. The gases leaving the coal gasifier pass through a liquid couplant heat exchanger before being used to drive a gas turbine. The exhaust gases of the gas turbine are used to generate both high pressure and low pressure steam for driving a steam turbine, before being exhausted to the atmosphere.

  16. The Incorporation and Abjection of Official Knowledge

    ERIC Educational Resources Information Center

    Kearl, Benjamin Kelsey

    2012-01-01

    In this essay, the author analyzes two theoretical perspectives--incorporation and abjection--that inform official knowledge generally and high school American history textbooks specifically. While contemporary textbooks increasingly depict the experiences of historically marginalized groups such as women, African Americans, Latinos, American…

  17. 77 FR 16761 - Incorporation by Reference

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-03-22

    ...; ] OFFICE OF THE FEDERAL REGISTER 1 CFR Part 51 Incorporation by Reference AGENCY: Office of the Federal... Register (OFR or we) received a petition to revise our regulations at 1 CFR part 51 on February 13, 2012... 11414. You can view the petition and its suggested revisions to the regulations 1 CFR part 51 on...

  18. 49 CFR 587.5 - Incorporated materials.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 7 2014-10-01 2014-10-01 false Incorporated materials. 587.5 Section 587.5 Transportation Other Regulations Relating to Transportation (Continued) NATIONAL HIGHWAY TRAFFIC SAFETY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) DEFORMABLE BARRIERS Side Impact Moving...

  19. Incorporating Technology into a Hawaiian Language Curriculum.

    ERIC Educational Resources Information Center

    Ka'awa, Makalapua; Hawkins, Emily

    This paper describes Hawaiian language courses that incorporate computer technology at the University of Hawaii at Manoa. In the past decade, enrollments in all types of Hawaiian language programs have increased rapidly. The University of Hawaii is committed to extending Hawaiian language education, especially the full development of Hawaiian…

  20. 49 CFR 572.180 - Incorporated materials.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ...,” incorporated by reference in §§ 572.180(a)(2), and 572.181(a); (4) Society of Automotive Engineers (SAE) Recommended Practice J211, Rev. Mar 95 “Instrumentation for Impact Tests—Part 1—Electronic Instrumentation... the National Archives and Records Administration (NARA), and in electronic format through...

  1. Semiconducting compounds and devices incorporating same

    DOEpatents

    Marks, Tobin J.; Facchetti, Antonio; Boudreault, Pierre-Luc; Miyauchi, Hiroyuki

    2016-01-19

    Disclosed are molecular and polymeric compounds having desirable properties as semiconducting materials. Such compounds can exhibit desirable electronic properties and possess processing advantages including solution-processability and/or good stability. Organic transistor and photovoltaic devices incorporating the present compounds as the active layer exhibit good device performance.

  2. Semiconducting compounds and devices incorporating same

    DOEpatents

    Marks, Tobin J; Facchetti, Antonio; Boudreault, Pierre-Luc; Miyauchi, Hiroyuki

    2014-06-17

    Disclosed are molecular and polymeric compounds having desirable properties as semiconducting materials. Such compounds can exhibit desirable electronic properties and possess processing advantages including solution-processability and/or good stability. Organic transistor and photovoltaic devices incorporating the present compounds as the active layer exhibit good device performance.

  3. Design of Schools to Incorporate Fallout Protection.

    ERIC Educational Resources Information Center

    Folley, Milo D.

    Means are suggested by which a school district may incorporate low-cost fallout protection in a school construction program, through construction of an underground shelter beneath the concrete slab foundation. Ways of controlling distribution and filtering air are discussed. The author also suggests consideration of a completely underground…

  4. Incorporating Mobile Learning into Athletic Training Education

    ERIC Educational Resources Information Center

    Davie, Emily

    2009-01-01

    Objective: To introduce and present techniques for incorporating mobile learning into athletic training education. Background: The matriculation of digital natives into college has stimulated the identification and development of new teaching and learning strategies. Electronic learning (e-learning), including the use of learning management…

  5. Incorporating the Aesthetic Dimension into Pedagogy

    ERIC Educational Resources Information Center

    Webster, R. Scott; Wolfe, Melissa

    2013-01-01

    This paper reports on a case study that was undertaken to discover not only the belief and intent behind the everyday opportunities that four exemplary teachers offered their high performing students but what activities they incorporated into their everyday lessons in an attempt to make sense of how aesthetic experiences may enhance learning. The…

  6. Incorporation of labeled small molecules into rubratoxin.

    PubMed

    Emeh, C O; Marth, E H

    1978-07-01

    A sterile glucose-mineral salts broth was inoculated with conidia of Penicillium rubrum P-13 and P-3290. Radiolabeled compounds were added to some cultures, these being incubated quiescently at 28 degrees C for 14 days. Other stationary cultures were grown for 21 days, received labeled compounds, and were then grown for 5 more days. The remaining cultures were inoculated with 72-h-old mycelial pellets, received labeled materials and were incubated with shaking for 60 h. Rubratoxin was resolved by thin-layer chromatography. Labeled [1(14)C]acetate, [1,5(14)C]citrate, [2(14)C]malonate, [1(14)C]glucose, [U14C]glucose or [1(14)C]hexanoate were incorporated into rubratoxins A and B by P. rubrum 3290 and into rubratoxin B by P. rubrum 13. Incorporation of [1(14)C]acetate and [2(14)C]malonate increased when exogenous unlabeled acetate, malonate, pyruvate, or phosphoenol-pyruvate was added. Acetate incorporation was influenced by cultural conditions, attaining maximum amounts in quiescent cultures which received labeled acetate after 21 days of incubation. Acetate incorporation in shake cultures was enhanced by reduced nicotinamide adenine dinucleotide phosphate (NADPH) and by unlabeled exogenous citrate.

  7. 49 CFR 572.30 - Incorporated materials.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 7 2014-10-01 2014-10-01 false Incorporated materials. 572.30 Section 572.30 Transportation Other Regulations Relating to Transportation (Continued) NATIONAL HIGHWAY TRAFFIC SAFETY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) ANTHROPOMORPHIC TEST DEVICES Hybrid III Test Dummy §...

  8. Incorporating Social Media in the Classroom

    ERIC Educational Resources Information Center

    McMeans, April

    2015-01-01

    Incorporating social media into the classroom will provide a positive, upbeat learning environment that students are engaged in on a regular basis. In doing this, educators will be ensuring discussion, collaboration, critical thinking, and creativity amongst their students. Social media is a knowledgeable topic for our students, and it is an…

  9. Identification, Introjection, Incorporation, Internalization: A Bibliography.

    ERIC Educational Resources Information Center

    Pedrini, D. T.; Pedrini, Bonnie C.

    Identification, introjection, incorporation, and internalization dealt with the same basic process according to S. Freud. The process has been discussed and researched in many and varied personal-social contexts. There have been two general areas of concern: identification for positive reasons (often called developmental) and identification for…

  10. Incorporating Feminism into Rehabilitation Counselor Education

    ERIC Educational Resources Information Center

    Jeon, Mookyong

    2015-01-01

    Purpose: The author describes how rehabilitation counselor educators can incorporate the feminist perspective in teaching rehabilitation counselors-in-training by exploring history, core values, and training methods of feminism. Method: Based on a literature review, the author compares philosophy and concepts of rehabilitation counseling and…

  11. Steroid assays in paediatric endocrinology.

    PubMed

    Honour, John W

    2010-01-01

    Most steroid disorders of the adrenal cortex come to clinical attention in childhood and in order to investigate these problems, there are many challenges to the laboratory which need to be appreciated to a certain extent by clinicians. The analysis of sex steroids in biological fluids from neonates, over adrenarche and puberty present challenges of specificities and concentrations often in small sample sizes. Different reference ranges are also needed for interpretations. For around 40 years, quantitative assays for the steroids and their regulatory peptide hormones have been possible using immunoassay techniques. Problems are recognised and this review aims to summarise the benefits and failings of immunoassays and introduce where tandem mass spectrometry is anticipated to meet the clinical needs for steroid analysis in paediatric endocrine investigations. It is important to keep a dialogue between clinicians and the laboratory, especially when any laboratory result does not make sense in the clinical investigation.

  12. Steroid Assays in Paediatric Endocrinology

    PubMed Central

    2010-01-01

    Most steroid disorders of the adrenal cortex come to clinical attention in childhood and in order to investigate these problems, there are many challenges to the laboratory which need to be appreciated to a certain extent by clinicians. The analysis of sex steroids in biological fluids from neonates, over adrenarche and puberty present challenges of specificities and concentrations often in small sample sizes. Different reference ranges are also needed for interpretations. For around 40 years, quantitative assays for the steroids and their regulatory peptide hormones have been possible using immunoassay techniques. Problems are recognised and this review aims to summarise the benefits and failings of immunoassays and introduce where tandem mass spectrometry is anticipated to meet the clinical needs for steroid analysis in paediatric endocrine investigations. It is important to keep a dialogue between clinicians and the laboratory, especially when any laboratory result does not make sense in the clinical investigation. Conflict of interest:None declared. PMID:21274330

  13. Optimized microturbidimetric assay for fibrinogen.

    PubMed

    Macart, M; Koffi, A; Henocque, G; Mathieu, J F; Guilbaud, J C

    1989-02-01

    In this assay we measure the turbidity produced by precipitation of plasma fibrinogen with a reagent composed of ammonium sulfate, EDTA, and guanidine hydrochloride. The two-step reagent addition, and use of fixed reaction times, eliminates interference from bilirubin, hemoglobin, and chylomicrons. We checked 135 monoclonal proteins for interference, finding the probability of encountering major interference in samples from adults to be very low, P = 0.0002. The method is calibrated with purified fibrinogen and the response is linear over the range 0-10 g/L. Within-run precision (CV) is less than 2% from 1 to 10 g/L. Correlations with the immunoturbidimetric (r = 0.99), chronometric (r = 0.99), and clotting (r = 0.97) methods were extremely high.

  14. Human Arterial Ring Angiogenesis Assay.

    PubMed

    Seano, Giorgio; Primo, Luca

    2016-01-01

    In this chapter we describe a model of human angiogenesis where artery explants from umbilical cords are embedded in gel matrices and subsequently produce capillary-like structures. The human arterial ring (hAR) assay is an innovative system that enables three-dimensional (3D) and live studies of human angiogenesis. This ex vivo model has the advantage of recapitulating several steps of angiogenesis, including endothelial sprouting, migration, and differentiation into capillaries. Furthermore, it can be exploited for (1) identification of new genes regulating sprouting angiogenesis, (2) screening for pro- or anti-angiogenic drugs, (3) identification of biomarkers to monitor the efficacy of anti-angiogenic regimens, and (4) dynamic analysis of tumor microenvironmental effects on vessel formation.

  15. Proteasomes: Isolation and Activity Assays

    PubMed Central

    Li, Yanjie; Tomko, Robert J.; Hochstrasser, Mark

    2015-01-01

    In eukaryotes, damaged or unneeded proteins are typically degraded by the ubiquitin-proteasome system. In this system, the protein substrate is often first covalently modified with a chain of ubiquitin polypeptides. This chain serves as a signal for delivery to the 26S proteasome, a 2.5 MDa, ATP-dependent multisubunit protease complex. The proteasome consists of a barrel-shaped 20S core particle (CP) that is capped on one or both of its ends by a 19S regulatory particle (RP). The RP is responsible for recognizing the substrate, unfolding it, and translocating it into the CP for destruction. Here we describe simple, one-step purifications scheme for isolating the 26S proteasome and its 19S RP and 20S CP subcomplexes from the yeast Saccharomyces cerevisiae, as well as assays for measuring ubiquitin-dependent and ubiquitin-independent proteolytic activity in vitro. PMID:26061243

  16. Fluorogenic assays for immediate confirmation of Escherichia coli.

    PubMed Central

    Feng, P C; Hartman, P A

    1982-01-01

    Rapid assays for Escherichia coli were developed by using the compound 4-methylumbelliferone glucuronide (MUG), which is hydrolyzed by glucuronidase to yield a fluorogenic product. The production of glucuronidase was limited to strains of E. coli and some Salmonella and Shigella strains in the family Enterobacteriaceae. For immediate confirmation of the presence of E. coli in most-probable-number tubes, MUG was incorporated into lauryl tryptose broth at a final concentration of 100 micrograms/ml. Results of both the presumptive test (gas production) and the confirmed test (fluorescence) for E. coli were obtained from a variety of food, water, and milk samples after incubation for only 24 h at 35 degrees C. Approximately 90% of the tubes showing both gas production and fluorescence contained fecal coliforms (they were positive in EC broth incubated at 45 degrees C). Few false-positive reactions were observed. The lauryl tryptose broth-MUG-most-probable-number assay was superior to violet red bile agar for the detection of heat- and chlorine-injured E. coli cells. Anaerogenic strains produced positive reactions, and small numbers of E. coli could be detected in the presence of large numbers of competing bacteria. The fluorogenic assay was sensitive and rapid; the presence of one viable cell was detected within 20 h. E. coli colonies could be distinguished from other coliforms on membrane filters and plates of violet red bile agar if MUG was incorporated into the culture media. A rapid confirmatory test for E. coli that is amenable to automation was developed by using microtitration plates filled with a nonselective medium containing MUG. Pure or mixed cultures containing E. coli produced fluorescence within 4 h (most strains) to 24 h (a few weakly positive strains). Images PMID:7049088

  17. Predictive assay for cancer targets

    NASA Astrophysics Data System (ADS)

    Suess, Amanda; Nguyen, Christine; Sorensen, Karen; Montgomery, Jennifer; Souza, Brian; Kulp, Kris; Dugan, Larry; Christian, Allen

    2005-11-01

    Early detection of cancer is a key element in successful treatment of the disease. Understanding the particular type of cancer involved, its origins and probable course, is also important. PhIP (2-amino-1- methyl-6 phenylimidazo [4,5-b]pyridine), a heterocyclic amine produced during the cooking of meat at elevated temperatures, has been shown to induce mammary cancer in female, Sprague-Dawley rats. Tumors induced by PhIP have been shown to contain discreet cytogenetic signature patterns of gains and losses using comparative genomic hybridization (CGH). To determine if a protein signature exists for these tumors, we are analyzing expression levels of the protein products of the above-mentioned tumors in combination with a new bulk protein subtractive assay. This assay produces a panel of antibodies against proteins that are either on or off in the tumor. Hybridization of the antibody panel onto a 2-D gel of tumor or control protein will allow for identification of a distinct protein signature in the tumor. Analysis of several gene databases has identified a number of rat homologs of human cancer genes located in these regions of gain and loss. These genes include the oncogenes c-MYK, ERBB2/NEU, THRA and tumor suppressor genes EGR1 and HDAC3. The listed genes have been shown to be estrogen-responsive, suggesting a possible link between delivery of bio-activated PhIP to the cell nucleus via estrogen receptors and gene-specific PhIP-induced DNA damage, leading to cell transformation. All three tumors showed similar silver staining patterns compared to each other, while they all were different than the control tissue. Subsequent screening of these genes against those from tumors know to be caused by other agents may produce a protein signature unique to PhIP, which can be used as a diagnostic to augment optical and radiation-based detection schemes.

  18. Predictive Assay For Cancer Targets

    SciTech Connect

    Suess, A; Nguyen, C; Sorensen, K; Montgomery, J; Souza, B; Kulp, K; Dugan, L; Christian, A

    2005-09-19

    Early detection of cancer is a key element in successful treatment of the disease. Understanding the particular type of cancer involved, its origins and probable course, is also important. PhIP (2-amino-1-methyl-6 phenylimidazo [4,5-b]pyridine), a heterocyclic amine produced during the cooking of meat at elevated temperatures, has been shown to induce mammary cancer in female, Sprague-Dawley rats. Tumors induced by PhIP have been shown to contain discreet cytogenetic signature patterns of gains and losses using comparative genomic hybridization (CGH). To determine if a protein signature exists for these tumors, we are analyzing expression levels of the protein products of the above-mentioned tumors in combination with a new bulk protein subtractive assay. This assay produces a panel of antibodies against proteins that are either on or off in the tumor. Hybridization of the antibody panel onto a 2-D gel of tumor or control protein will allow for identification of a distinct protein signature in the tumor. Analysis of several gene databases has identified a number of rat homologs of human cancer genes located in these regions of gain and loss. These genes include the oncogenes c-MYK, ERBB2/NEU, THRA and tumor suppressor genes EGR1 and HDAC3. The listed genes have been shown to be estrogen-responsive, suggesting a possible link between delivery of bio-activated PhIP to the cell nucleus via estrogen receptors and gene-specific PhIP-induced DNA damage, leading to cell transformation. All three tumors showed similar silver staining patterns compared to each other, while they all were different than the control tissue. Subsequent screening of these genes against those from tumors know to be caused by other agents may produce a protein signature unique to PhIP, which can be used as a diagnostic to augment optical and radiation-based detection schemes.

  19. Microdroplet chain array for cell migration assays.

    PubMed

    Ma, Yan; Pan, Jian-Zhang; Zhao, Shi-Ping; Lou, Qi; Zhu, Ying; Fang, Qun

    2016-11-29

    Establishing cell migration assays in multiple different microenvironments is important in the study of tissue repair and regeneration, cancer progression, atherosclerosis, and arthritis. In this work, we developed a miniaturized and massive parallel microfluidic platform for multiple cell migration assays combining the traditional membrane-based cell migration technique and the droplet-based microfluidic technique. Nanoliter-scale droplets are flexibly assembled as building blocks based on a porous membrane to form microdroplet chains with diverse configurations for different assay modes. Multiple operations including in-droplet 2D/3D cell culture, cell co-culture and cell migration induced by a chemoattractant concentration gradient in droplet chains could be flexibly performed with reagent consumption in the nanoliter range for each assay and an assay scale-up to 81 assays in parallel in one microchip. We have applied the present platform to multiple modes of cell migration assays including the accurate cell migration assay, competitive cell migration assay, biomimetic chemotaxis assay, and multifactor cell migration assay based on the organ-on-a-chip concept, for demonstrating its versatility, applicability, and potential in cell migration-related research.

  20. Development of forensic assay signatures for ebolaviruses.

    PubMed

    Song, Jian; Doggett, Norman; Wren, Melinda; Burr, Tom; Fenimore, P W; Hatcher, Eneida L; Bruno, William J; Li, Po-E; Stubben, Chris; Wolinsky, Murray

    2015-03-01

    Ebolaviruses are a diverse group of RNA viruses comprising five different species, four of which cause fatal hemorrhagic fever in humans. Because of their high infectivity and lethality, ebolaviruses are considered major biothreat agents. Although detection assays exist, no forensic assays are currently available. Here, we report the development of forensic assays that differentiate ebolaviruses. We performed phylogenetic analyses and identified canonical SNPs for all species, major clades and isolates. TaqMan-MGB allelic discrimination assays based on these SNPs were designed, screened against synthetic RNA templates, and validated against ebolavirus genomic RNAs. A total of 45 assays were validated to provide 100% coverage of the species and variants with additional resolution at the isolate level. These assays enabled accurate forensic analysis on 4 "unknown" ebolaviruses. Unknowns were correctly classified to species and variant. A goal of providing resolution below the isolate level was not successful. These high-resolution forensic assays allow rapid and accurate genotyping of ebolaviruses for forensic investigations.

  1. Evaluating 6 ricin field detection assays.

    PubMed

    Slotved, Hans-Christian; Sparding, Nadja; Tanassi, Julia Tanas; Steenhard, Nina R; Heegaard, Niels H H

    2014-01-01

    This study presents data showing the performance of 6 commercial detection assays against ricin around concentrations specified as detection limits by the producers. A 2-fold dilution series of 20 ng/ml ricin was prepared and used for testing the lateral-flow kits: BADD, Pro Strips™, ENVI, RAID DX, Ricin BioThreat Alert, and IMASS™ device. Three of the 6 tested field assays (IMASS™ device, ENVI assay, and the BioThreat Alert assay) were able to detect ricin, although differences in the measured detection limits compared to the official detection limits and false-negative results were observed. We were not able to get the BADD, Pro Strips™, and RAID assays to function in our laboratory. We conclude that when purchasing a field responder assay, there is large variation in the specificity of the assays, and a number of in-house tests must be performed to ensure functionality.

  2. Investigation of the repair of single-strand breaks in human DNA using alkaline gel electrophoresis

    SciTech Connect

    Kovacs, E.; Langemann, H. )

    1990-11-01

    Unstimulated lymphocytes from eight healthy persons were exposed to 10-, 30-, and 100-Gy doses of 60Co gamma radiation. The repair of damaged DNA was measured by (1) alkaline gel electrophoresis (extracted DNA loaded on 0.25% agarose gel, run at 1 V/cm for 39-44 h) at 0, 1, and 2 h after exposure and (2) incorporation of (3H)thymidine into unstimulated lymphocytes in the presence of 2 mM hydroxyurea 1 and 2 h after exposure. Both methods--alkaline gel electrophoresis and thymidine incorporation--showed that repair was completed within 2 h.

  3. 46 CFR 105.01-3 - Incorporation by reference.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... COMMERCIAL FISHING VESSELS DISPENSING PETROLEUM PRODUCTS Administration § 105.01-3 Incorporation by reference..., Standard Test Method for Vapor Pressure of Petroleum Products (Reid Method), incorporation by...

  4. Towards incorporating epigenetic mechanisms into carcinogen identification and evaluation

    PubMed Central

    Herceg, Zdenko

    2013-01-01

    Remarkable progress in the field of epigenetics has turned academic, medical and public attention to the potential applications of these new advances in medicine and various fields of biomedical research. The result is a broader appreciation of epigenetic phenomena in the a etiology of common human diseases, most notably cancer. These advances also represent an exciting opportunity to incorporate epigenetics and epigenomics into carcinogen identification and safety assessment. Current epigenetic studies, including major international sequencing projects, are expected to generate information for establishing the ‘normal’ epigenome of tissues and cell types as well as the physiological variability of the epigenome against which carcinogen exposure can be assessed. Recently, epigenetic events have emerged as key mechanisms in cancer development, and while our search of the Monograph Volume 100 revealed that epigenetics have played a modest role in evaluating human carcinogens by the International Agency for Research on Cancer (IARC) Monographs so far, epigenetic data might play a pivotal role in the future. Here, we review (i) the current status of incorporation of epigenetics in carcinogen evaluation in the IARC Monographs Programme, (ii) potential modes of action for epigenetic carcinogens, (iii) current in vivo and in vitro technologies to detect epigenetic carcinogens, (iv) genomic regions and epigenetic modifications and their biological consequences and (v) critical technological and biological issues in assessment of epigenetic carcinogens. We also discuss the issues related to opportunities and challenges in the application of epigenetic testing in carcinogen identification and evaluation. Although the application of epigenetic assays in carcinogen evaluation is still in its infancy, important data are being generated and valuable scientific resources are being established that should catalyse future applications of epigenetic testing. PMID:23749751

  5. Towards incorporating epigenetic mechanisms into carcinogen identification and evaluation.

    PubMed

    Herceg, Zdenko; Lambert, Marie-Pierre; van Veldhoven, Karin; Demetriou, Christiana; Vineis, Paolo; Smith, Martyn T; Straif, Kurt; Wild, Christopher P

    2013-09-01

    Remarkable progress in the field of epigenetics has turned academic, medical and public attention to the potential applications of these new advances in medicine and various fields of biomedical research. The result is a broader appreciation of epigenetic phenomena in the a etiology of common human diseases, most notably cancer. These advances also represent an exciting opportunity to incorporate epigenetics and epigenomics into carcinogen identification and safety assessment. Current epigenetic studies, including major international sequencing projects, are expected to generate information for establishing the 'normal' epigenome of tissues and cell types as well as the physiological variability of the epigenome against which carcinogen exposure can be assessed. Recently, epigenetic events have emerged as key mechanisms in cancer development, and while our search of the Monograph Volume 100 revealed that epigenetics have played a modest role in evaluating human carcinogens by the International Agency for Research on Cancer (IARC) Monographs so far, epigenetic data might play a pivotal role in the future. Here, we review (i) the current status of incorporation of epigenetics in carcinogen evaluation in the IARC Monographs Programme, (ii) potential modes of action for epigenetic carcinogens, (iii) current in vivo and in vitro technologies to detect epigenetic carcinogens, (iv) genomic regions and epigenetic modifications and their biological consequences and (v) critical technological and biological issues in assessment of epigenetic carcinogens. We also discuss the issues related to opportunities and challenges in the application of epigenetic testing in carcinogen identification and evaluation. Although the application of epigenetic assays in carcinogen evaluation is still in its infancy, important data are being generated and valuable scientific resources are being established that should catalyse future applications of epigenetic testing.

  6. An improved 96-well turbidity assay for T4 lysozyme activity

    PubMed Central

    Toro, Tasha B.; Nguyen, Thao P.; Watt, Terry J.

    2015-01-01

    T4 lysozyme (T4L) is an important model system for investigating the relationship between protein structure and function. Despite being extensively studied, a reliable, quantitative activity assay for T4L has not been developed. Here, we present an improved T4L turbidity assay as well as an affinity-based T4L expression and purification protocol. This assay is designed for 96-well format and utilizes conditions amenable for both T4L and other lysozymes. This protocol enables easy, efficient, and quantitative characterization of T4L variants and allows comparison between different lysozymes. Our method: • Is applicable for all lysozymes, with enhanced sensitivity for T4 lysozyme compared to other 96-well plate turbidity assays; • Utilizes standardized conditions for comparing T4 lysozyme variants and other lysozymes; and • Incorporates a simplified expression and purification protocol for T4 lysozyme. PMID:26150996

  7. Rocking adhesion assay system to study adhesion and transendothelial migration of cancer cells.

    PubMed

    Bapu, Deepashree; Khadim, Munira; Brooks, Susan A

    2014-01-01

    Adhesion of metastatic cancer cells to the vascular endothelium of the target organs and their subsequent transendothelial migration is one of the critical, yet poorly understood, steps of the metastatic cascade. Conventionally, the mechanisms of this complex process have been studied using static adhesion systems or flow assay systems. Static assay systems are easy to set up and perform but do not mimic the physiological conditions of blood flow. Flow assays closely mimic physiological conditions of flow but are time consuming and require specialist equipment. In this chapter we describe the rocking adhesion system which incorporates the key advantages of both the static and flow assay systems and not only is easy to set up and perform but also mimics conditions of blood flow.

  8. Nick translation - a new assay for monitoring DNA damage and repair in cultured human fibroblasts

    SciTech Connect

    Snyder, R.D.; Matheson, D.W.

    1985-01-01

    An in vitro assay has been developed to detect DNA damage and repair following chemical treatment of human diploid fibroblasts. DNA damage is measured by following the Escherichia coli DNA polymerase I-catalyzed incorporation of radiolabeled deoxycytidine triphosphate (dCTP) into the DNA of lysolecithin-permeabilized cells. DNA strand breaks with free 3' OH termini serve as template sites for incorporation, and decrease of this incorporation with time, following removal of the test chemical, indicates loss (repair) of initial damage. Inhibition of the DNA excision repair process by the addition of the repair inhibitors arabinofuranosyl cytosine (ara-C) and hydroxyurea (HU) during the incubation period gives rise to an increased number of template sites, manifesting itself in increased incorporation and indicating the induction of long-patch excision repair. Results presented demonstrate that all 14 direct-acting carcinogens tested and 8 of 14 carcinogens requiring metabolic activation give positive indication of DNA damage, repair, or both. Eleven of 14 noncarcinogens tested were scored as negative, the other 3 having previously been shown to interact with cellular DNA. This assay is shown to have predictive capability at least equal to that of UDS assays but to allow a broader spectrum of genotoxic effects to be analyzed.

  9. Emissive sensors and devices incorporating these sensors

    SciTech Connect

    Swager, Timothy M; Zhang, Shi-Wei

    2013-02-05

    The present invention generally relates to luminescent and/or optically absorbing compositions and/or precursors to those compositions, including solid films incorporating these compositions/precursors, exhibiting increased luminescent lifetimes, quantum yields, enhanced stabilities and/or amplified emissions. The present invention also relates to sensors and methods for sensing analytes through luminescent and/or optically absorbing properties of these compositions and/or precursors. Examples of analytes detectable by the invention include electrophiles, alkylating agents, thionyl halides, and phosphate ester groups including phosphoryl halides, cyanides and thioates such as those found in certain chemical warfare agents. The present invention additionally relates to devices and methods for amplifying emissions, such as those produced using the above-described compositions and/or precursors, by incorporating the composition and/or precursor within a polymer having an energy migration pathway. In some cases, the compositions and/or precursors thereof include a compound capable of undergoing a cyclization reaction.

  10. Toxicity of sediment-incorporated drilling fluids

    SciTech Connect

    Clark, J.R.; Patrick, J.M.

    1987-01-01

    The 24, 96, or 168-h LC50s of four used drilling fluids or barite incorporated into sediment were determined in toxicity tests with lancelets (Branchiostoma caribaeum), a benthic chordate. The number of lancelets that did not burrow into contaminated sediments was used to calculate EC50s at the same times that LC50s were determined. Observations of the burrowing behavior allowed quantitation of effects after 24-h exposures to each of the drilling fluids whereas lancelet mortality was sufficient to calculate 24-h LC50s for only one drilling fluid. Drilling fluids were less toxic to lancelets when incorporated into sediments than to mysids (Mysidopsis bahia) or benthic invertebrate communities in water-column exposures.

  11. 49 CFR 572.180 - Incorporated materials.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ...,” incorporated by reference in §§ 572.180(a)(2), and 572.181(a); (4) Society of Automotive Engineers (SAE...”; and, (5) SAE J1733 of 1994-12 “Sign Convention for Vehicle Crash Testing.” (b) The Director of the... Woodfield Road, Gaithersburg, MD 20879, telephone (301) 670-0090. (2) The SAE materials referred to...

  12. Improving techniques for clonogenic assays.

    PubMed

    Eliason, J F; Fekete, A; Odartchenko, N

    1984-01-01

    A serum-free medium has been developed which supports colony formation by cells from several human tumor cell lines, one colon adenocarcinoma (WiDr) and four melanoma (Me43, Me85, MP6, MeIuso). This medium consists of a 1:1 mixture of an enriched Dulbecco's modified Eagle's medium (EMED) and a modified Ham's F-12 nutrient mixture (FMED) supplemented with 0.9% methylcellulose, 1% bovine serum albumin, 80 micrograms/ml human transferrin, 3 micrograms/ml insulin, 2.8 micrograms/ml linoleic acid, 2.6 micrograms/ml cholesterol, 20 microM ethanolamine, and trace elements. Colony formation by WiDr cells is linear with the numbers of cells plated, having a plating efficiency (PE) of 34%, as compared to 26% in serum-containing medium. Two of the melanoma cell lines. MP6 and MeIuso, exhibit linear relationships between colony numbers and cell concentration with PEs of 21% and 70% respectively. Colony formation by the other two melanoma cell lines appears to be nonlinear. This work represents a step toward standardizing culture conditions for human tumor clonogenic cell assays.

  13. Benefits of oxygen incorporation in atomic laminates

    NASA Astrophysics Data System (ADS)

    Dahlqvist, Martin

    2016-04-01

    Atomic laminates such as MAX phases benefit from the addition of oxygen in many ways, from the formation of a protective oxide surface layer with self-healing capabilities when cracks form to the tuning of anisotropic conductivity. In this paper oxygen incorporation and vacancy formation in M 2AlC (M  =  Ti, V, Cr) MAX phases have been studied using first-principles calculations where the focus is on phase stability and electronic structure for different oxygen and/or vacancy configurations. Oxygen prefers different lattice sites depending on M-element and this can be correlated to the number of available non-bonding M d-electrons. In Ti2AlC, oxygen substitutes carbon while in Cr2AlC it is located interstitially within the Al-layer. I predict that oxygen incorporation in Ti2AlC stabilizes the material, which explains the experimentally observed 12.5 at% oxygen (x  =  0.5) in Ti2Al(C1-x O x ). In addition, it is also possible to use oxygen to stabilize the hypothetical Zr2AlC and Hf2AlC. Hence, oxygen incorporation may be beneficial in many ways. Not only can it make a material more stable, but it also can act as a reservoir for internal self-healing with shorter diffusion paths.

  14. Structural incorporation of As5+ into hematite.

    PubMed

    Bolanz, Ralph M; Wierzbicka-Wieczorek, Mária; Čaplovičová, Mária; Uhlík, Peter; Göttlicher, Jörg; Steininger, Ralph; Majzlan, Juraj

    2013-08-20

    Hematite (α-Fe2O3) is one of the most common iron oxides and a sink for the toxic metalloid arsenic. Arsenic can be immobilized by adsorption to the hematite surface; however, the incorporation of As in hematite was never seriously considered. In our study we present evidence that, besides adsorption, the incorporation of As into the hematite crystals can be of great relevance for As immobilization. With the coupling of nanoresolution techniques and X-ray absorption spectroscopy the presence of As (up to 1.9 wt %) within the hematite crystals could be demonstrated. The incorporated As(5+) displays a short-range order similar to angelellite-like clusters, epitaxially intergrown with hematite. Angelellite (Fe4As2O11), a triclinic iron arsenate with structural relations to hematite, can epitaxially intergrow along the (210) plane with the (0001) plane of hematite. This structural composite of hematite and angelellite-like clusters represents a new immobilization mechanism and potentially long-lasting storage facility for As(5+) by iron oxides.

  15. Use of luminescent Leptospira interrogans for enumeration in biological assays.

    PubMed

    Murray, Gerald L; King, Amy M; Srikram, Amporn; Sermswan, Rasana W; Adler, Ben

    2010-06-01

    Rapid and reliable in vitro methods for the detection of pathogenic leptospires, such as Leptospira interrogans, are lacking. The present study investigated the use of luminescence to replace the existing enumeration techniques. Transposon TnSC189 was modified to incorporate the luxCDABE cassette from Photorhabdus luminescens and was used to construct luminescent Leptospira spp. There was a linear relationship between luminescence and cell number, with the theoretical detection limit being less than 10(4) leptospires. A comparison of enumeration by a standard method (counting by dark-field microscopy) and enumeration by luminescence was conducted with luminescent L. interrogans. There was a good correlation between the two methods of enumeration (R(2) = 0.766), although variation in the luminescence early and late in growth phase reduced the degree of correlation. To demonstrate the utility of luminescence as a viability and cell number reporter, in vitro assays, including MIC determination, an extracellular matrix binding experiment, and a complement killing experiment, were conducted. In each case, the results obtained by luminescence matched those obtained by traditional means with high correlations (binding assay R(2) = 0.916, complement killing assay R(2) = 0.988). A strain expressing the luxCDABE transposon retained virulence in the hamster model of infection. Despite some variation in luminescence as a result of the growth phase or the particular assay conditions, enumeration by luminescence was found to be a quick, reliable, and highly sensitive method for the in vitro detection of leptospires that has the potential to replace more time-consuming methods of enumeration.

  16. Assay development status report for total cyanide

    SciTech Connect

    Simpson, B.C.; Jones, T.E.; Pool, K.H.

    1993-02-01

    A validated cyanide assay that is applicable to a variety of tank waste matrices is necessary to resolve certain waste tank safety issues and for purposes of overall waste characterization. The target for this effort is an assay with an applicable range of greater than 1,000 ppM (0.10 wt%) total cyanide and a confidence level greater than 80%. Figure 1 illustrates the operating regime of the proposed cyanide assay method. The Assay Development Status Report for Total Cyanide will summarize the past experience with cyanide analyses on-tank waste matrices and will rate the status of the analytical methods used to assay total cyanide (CN{sup {minus}} ion) in the tank waste matrices as acceptable or unacceptable. This paper will also briefly describe the current efforts for improving analytical resolution of the assays and the attempts at speciation.

  17. Multiplexed Molecular Assays for Rapid Rule-Out of Foot-and-Mouth Disease

    SciTech Connect

    Lenhoff, R; Naraghi-Arani, P; Thissen, J; Olivas, J; Carillo, C; Chinn, C; Rasmussen, M; Messenger, S; Suer, L; Smith, S M; Tammero, L; Vitalis, E; Slezak, T R; Hullinger, P J; Hindson, B J; Hietala, S; Crossley, B; Mcbride, M

    2007-06-26

    A nucleic acid-based multiplexed assay was developed that combines detection of foot-and-mouth disease virus (FMDV) with rule-out assays for two other foreign animal diseases and four domestic animal diseases that cause vesicular or ulcerative lesions indistinguishable from FMDV infection in cattle, sheep and swine. The FMDV 'look-alike' diagnostic assay panel contains five PCR and twelve reverse transcriptase PCR (RT-PCR) signatures for a total of seventeen simultaneous PCR amplifications for seven diseases plus incorporating four internal assay controls. It was developed and optimized to amplify both DNA and RNA viruses simultaneously in a single tube and employs Luminex{trademark} liquid array technology. Assay development including selection of appropriate controls, a comparison of signature performance in single and multiplex testing against target nucleic acids, as well of limits of detection for each of the individual signatures is presented. While this assay is a prototype and by no means a comprehensive test for FMDV 'look-alike' viruses, an assay of this type is envisioned to have benefit to a laboratory network in routine surveillance and possibly for post-outbreak proof of freedom from foot-and-mouth disease.

  18. Sensitive field assays for water analysis

    SciTech Connect

    Douglas, W.L.

    1984-08-01

    The goal of the project is to develop a rapid, simple, and inexpensive dry-film assay device for detection of environmental contaminants using the compound geosmin as a model. Phase I activities centered upon the immunochemical reagents necessary for the assay, development of an enzyme-cycling system that makes possible detection of substances in the parts per billion (PPB) range or lower, and demonstration of how the Immuno-Replacement-Assay can be used to detect geosmin.

  19. Using the CPTAC Assay Portal to Identify and Implement Highly Characterized Targeted Proteomics Assays.

    PubMed

    Whiteaker, Jeffrey R; Halusa, Goran N; Hoofnagle, Andrew N; Sharma, Vagisha; MacLean, Brendan; Yan, Ping; Wrobel, John A; Kennedy, Jacob; Mani, D R; Zimmerman, Lisa J; Meyer, Matthew R; Mesri, Mehdi; Boja, Emily; Carr, Steven A; Chan, Daniel W; Chen, Xian; Chen, Jing; Davies, Sherri R; Ellis, Matthew J C; Fenyö, David; Hiltke, Tara; Ketchum, Karen A; Kinsinger, Chris; Kuhn, Eric; Liebler, Daniel C; Liu, Tao; Loss, Michael; MacCoss, Michael J; Qian, Wei-Jun; Rivers, Robert; Rodland, Karin D; Ruggles, Kelly V; Scott, Mitchell G; Smith, Richard D; Thomas, Stefani; Townsend, R Reid; Whiteley, Gordon; Wu, Chaochao; Zhang, Hui; Zhang, Zhen; Rodriguez, Henry; Paulovich, Amanda G

    2016-01-01

    The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as an open-source repository of well-characterized targeted proteomic assays. The portal is designed to curate and disseminate highly characterized, targeted mass spectrometry (MS)-based assays by providing detailed assay performance characterization data, standard operating procedures, and access to reagents. Assay content is accessed via the portal through queries to find assays targeting proteins associated with specific cellular pathways, protein complexes, or specific chromosomal regions. The position of the peptide analytes for which there are available assays are mapped relative to other features of interest in the protein, such as sequence domains, isoforms, single nucleotide polymorphisms, and posttranslational modifications. The overarching goals are to enable robust quantification of all human proteins and to standardize the quantification of targeted MS-based assays to ultimately enable harmonization of results over time and across laboratories.

  20. Celastrol blocks interleukin-6 gene expression via downregulation of NF-κB in prostate carcinoma cells.

    PubMed

    Chiang, Kun-Chun; Tsui, Ke-Hung; Chung, Li-Chuan; Yeh, Chun-Nan; Chen, Wen-Tsung; Chang, Phei-Lang; Juang, Horng-Heng

    2014-01-01

    Interleukin-6 (IL-6), a multifunctional cytokine, contributes to proliferation or differentiation of prostate carcinoma cells in a highly cell type-specific manner. Celastrol (3-hydroxy-24-nor-2oxo-1(10),3,5,7-friedelatetrane-29-oic acid), also named as tripterine, is extracted from root of Chinese traditional herb Tripterygiumwilfordii Hook f with potent anti-inflammatory and anti-cancer activities. In this study, we evaluated the molecular mechanisms of celastrol on cell proliferation and IL-6 gene expression in prostate carcinoma cells. 3H-thymidine incorporation and flow cytometric analysis indicated that celastrol treatments arrested the cell cycle at the G0/G1 phase, thus attenuating cell proliferation in prostate carcinoma PC-3 cells; moreover, celastrol induced cell apoptosis at higher dosage. Knockdown of IL-6 attenuated the anti-proliferative effect of celastrol on PC-3 cells. Results from ELISA and 5'-deletion transient gene expression assays indicated that celastrol treatment decreased IL-6 secretion and gene expression, and this effect is dependent on the NF-κB response element within IL-6 promoter area since mutation of the NF-κB response element from AAATGTCCCATTTTCCC to AAATGTTACATTTTCCC by site-directed mutagenesis abolished the inhibition of celastrol on the IL-6 promoter activity. Celastrol also attenuated the activation of PMA and TNFα on the gene expression and secretion of IL-6 in PC-3 cells. Immunoblot assays revealed that celastrol treatment downregulated the expressions of IKKα, p50 and p65, supporting the 5'-deletion transient gene expression assay result that celastrol blocked IL-6 expression through the NF-κB pathway in PC-3 cells. For the first time, our results concluded that celastrol attenuates PC-3 cell proliferation via downregulation of IL-6 gene expression through the NF-κB-dependent pathway.

  1. Magnetoresistive Sensors in Biological Assays

    NASA Astrophysics Data System (ADS)

    Tondra, Mark

    2010-03-01

    Magnetic beads or nanoparticles can be used as ``labels'' in biochemical assays by attaching the beads to the biospecies of interest using a bio-specific attachment. Once the labels are attached, they can be used to manipulate, capture, and detect the species to be analyzed. Magnetoresistive (MR) sensors may be used to detect and count these labels, and thus make an inference about the concentration of the species of interest. MR technology is especially promising for biosensor applications where making the detector small and integrated with related sample handling tools to form a ``lab-on-a-chip'' miniaturized system. The function of the MR sensors is to detect stray magnetic fields from the beads while they are exposed to a magnetic excitation field. Generally, the stray fields from beads and clusters of beads are complicated functions of geometry, so some care is required to relate the detected magnetic signal to the number and location of the bead labels. This presentation will begin with a broad overview of results from many groups working in this area. For convenience, the applications are divided into three categories, detection of: flowing magnetic beads, immobilized beads, and scanned samples. Next will be some discussion of how the choice of spintronic sensor technology might affect detection capabilities (AMR, GMR, TMR, Hall effect, etc). Then, challenges relating to integration of MR sensors into microfluidic products will be discussed. This is the focus of the presenter's current day-to-day work on developing and producing MR-based biosensors. And finally, a description of possible future avenues of study and development will be presented.

  2. Measurement of DNA damage in individual cells using the Single Cell Gel Electrophoresis (Comet) assay.

    PubMed

    Hartley, Janet M; Spanswick, Victoria J; Hartley, John A

    2011-01-01

    The Single Cell Gel Electrophoresis (Comet) assay is a simple, versatile and sensitive method for measuring DNA damage in individual cells, allowing the determination of heterogeneity of response within a cell population. The basic alkaline technique described is for the determination of DNA strand break damage and its repair at a single cell level. Specific modifications to the method use a lower pH ('neutral' assay), or allow the measurement of DNA interstrand cross-links. It can be further adapted to, for example, study specific DNA repair mechanisms, be combined with fluorescent in situ hybridisation, or incorporate lesion specific enzymes.

  3. Manganese incorporation into ferroelectric lead titanate

    NASA Astrophysics Data System (ADS)

    Stoupin, Stanislav

    Substitution with 3d magnetic transition elements in ABO 3 ferroelectric perovskite host media is widely utilized to produce relaxor ferroelectrics. Many resulting solid solutions exhibit magnetoelectric properties affected by concentration levels of the introduced magnetic ions. For conventional material preparation techniques such as firing of mechanically mixed oxides, incorporation is often limited by 5 mol% concentration level. Doping at higher concentrations requires introduction of other substituents to compensate charge within the unit cell to promote formation of the perovskite phase. In contrast, molecular mixing of precursor materials at the initial phase of preparation procedure offers an advantage of achieving higher incorporation levels of the 3d elements without additional charge-compensating ions. Presented in this thesis is a new sol-gel procedure utilized for high level incorporation of 3d magnetic ions into ferroelectric lead titanate. The technique was applied to produce PbTi1-xMnxO 3 solid solution, a perovskite system promising for high degree of magnetoelectric coupling. Concentration dependent studies were performed to characterize structural, thermal, ferroelectric and magnetic properties of the material. The solubility limit of Mn has been found to be 20 mol% and the material remains tetragonally distorted. X-ray Absorption Spectroscopy confirms that local structural environment of Mn, Ti, and Pb is consistent with tetragonal symmetry of the unit cell. Increase in Mn concentration leads to reduction in melting point, broadening of the ferroelectric transition, reduction of the transition temperature and increase in dielectric constant of the material. At the solubility limit the system was found to be ferromagnetic below 50 K.

  4. Staphylococcus aureus Sortase A-Mediated Incorporation of Peptides: Effect of Peptide Modification on Incorporation

    PubMed Central

    Hansenová Maňásková, Silvie; Nazmi, Kamran; van ‘t Hof, Wim; van Belkum, Alex; Martin, Nathaniel I.; Bikker, Floris J.; van Wamel, Willem J. B.; Veerman, Enno C. I.

    2016-01-01

    The endogenous Staphylococcus aureus sortase A (SrtA) transpeptidase covalently anchors cell wall-anchored (CWA) proteins equipped with a specific recognition motif (LPXTG) into the peptidoglycan layer of the staphylococcal cell wall. Previous in situ experiments have shown that SrtA is also able to incorporate exogenous, fluorescently labelled, synthetic substrates equipped with the LPXTG motif (K(FITC)LPETG-amide) into the bacterial cell wall, albeit at high concentrations of 500 μM to 1 mM. In the present study, we have evaluated the effect of substrate modification on the incorporation efficiency. This revealed that (i) by elongation of LPETG-amide with a sequence of positively charged amino acids, derived from the C-terminal domain of physiological SrtA substrates, the incorporation efficiency was increased by 20-fold at 10 μM, 100 μM and 250 μM; (ii) Substituting aspartic acid (E) for methionine increased the incorporation of the resulting K(FITC)LPMTG-amide approximately three times at all concentrations tested; (iii) conjugation of the lipid II binding antibiotic vancomycin to K(FITC)LPMTG-amide resulted in the same incorporation levels as K(FITC)LPETG-amide, but much more efficient at an impressive 500-fold lower substrate concentration. These newly developed synthetic substrates can potentially find broad applications in for example the in situ imaging of bacteria; the incorporation of antibody recruiting moieties; the targeted delivery and covalent incorporation of antimicrobial compounds into the bacterial cell wall. PMID:26799839

  5. Parameters influencing zeolite incorporation in PDMS membranes

    SciTech Connect

    Vankelecom, I.F.J.; Scheppers, E.; Heus, R.; Uytterhoeven, J.B. )

    1994-11-24

    The incorporation of several types of zeolite in PDMS membranes is studied, by measuring the tensile strength, xylene sorption, and density of the membranes. The zeolite is shown to be involved in the cross-linking of the membrane. The interaction between the PDMS matrix and the zeolites results in reinforced membranes in the case of zeolite Y. The parameters influencing the dispersion of the zeolite in the membrane are investigated, as well as several aspects of the preparation method. Finally, the idea of cross-linking is applied to explain the results of water/ethanol pervaporation. 25 refs., 9 figs., 4 tabs.

  6. Incorporating social concerns in environmental impact assessments

    SciTech Connect

    Wolfe, A.K.

    1990-03-01

    Social impact assessments most often focus on the population-driven impacts of projects. Such impacts may be insignificant when compared with social structural impacts of complex, controversial projects. This set of impacts includes social disruption, social group formation, and stigma effects. The National Environmental Policy Act does not explicitly call for assessment of, and assessors often are reluctant to address, these complex issues. This paper discusses why such impacts are critical to assess and gives examples of how they have been incorporated into environmental assessment documents. 6 refs.

  7. Incorporation of noble metals into aerogels

    DOEpatents

    Hair, L.M.; Sanner, R.D.; Coronado, P.R.

    1998-12-22

    Aerogels or xerogels containing atomically dispersed noble metals for applications such as environmental remediation are disclosed. New noble metal precursors, such as Pt--Si or Pd(Si--P){sub 2}, have been created to bridge the incompatibility between noble metals and oxygen, followed by their incorporation into the aerogel or xerogel through sol-gel chemistry and processing. Applications include oxidation of hydrocarbons and reduction of nitrogen oxide species, complete oxidation of volatile organic carbon species, oxidative membranes for photocatalysis and partial oxidation for synthetic applications.

  8. Incorporation of noble metals into aerogels

    DOEpatents

    Hair, Lucy M.; Sanner, Robert D.; Coronado, Paul R.

    1998-01-01

    Aerogels or xerogels containing atomically dispersed noble metals for applications such environmental remediation. New noble metal precursors, such as Pt--Si or Pd(Si--P).sub.2, have been created to bridge the incompatibility between noble metals and oxygen, followed by their incorporation into the aerogel or xerogel through sol-gel chemistry and processing. Applications include oxidation of hydrocarbons and reduction of nitrogen oxide species, complete oxidation of volatile organic carbon species, oxidative membranes for photocatalysis and partial oxidation for synthetic applications.

  9. Radiation collimator and systems incorporating same

    DOEpatents

    Norman, Daren R.; Yoon, Woo Y.; Jones, James L.; Haskell, Kevin J.; Bennett, Brion D.; Tschaggeny, Charles W.; Jones, Warren F.

    2011-09-13

    A collimator including a housing having disposed therein a shield element surrounding a converter core in which a photon beam is generated from electrons emanating from a linear accelerator. A beam channeler longitudinally adjacent the shield element has a beam aperture therethrough coaxially aligned with, and of the same diameter as, an exit bore of the converter core. A larger entry bore in the converter core is coaxial with, and longitudinally separated from, the exit bore thereof. Systems incorporating the collimator are also disclosed.

  10. Incorporation of Nanosensors into Aerospace Vehicles

    NASA Technical Reports Server (NTRS)

    Medelius, Pedro J.

    2007-01-01

    Traditional sensors are too big and heavy for installation in space vehicles, including the Space Shuttle Orbiter as well as future manned and unmanned vehicles currently in the early design phase. Advances in nanotechnology have led to the availability of smaller and more accurate sensors. Multiple and redundant nanosensors can be used to conduct more accurate and comprehensive measurements in a space vehicle. Early planning can lead to the relatively easy incorporation of miniature sensors sharing power and communication lines, thus reducing the requirement for large amount of electrical and/or optical cabling.

  11. Incorporating opponent models into adversary search

    SciTech Connect

    Carmel, D.; Markovitch, S.

    1996-12-31

    This work presents a generalized theoretical framework that allows incorporation of opponent models into adversary search. We present the M* algorithm, a generalization of minimax that uses an arbitrary opponent model to simulate the opponent`s search. The opponent model is a recursive structure consisting of the opponent`s evaluation function and its model of the player. We demonstrate experimentally the potential benefit of using an opponent model. Pruning in M* is impossible in the general case. We prove a sufficient condition for pruning and present the {alpha}{beta}* algorithm which returns the M* value of a tree while searching only necessary branches.

  12. Valve system incorporating single failure protection logic

    DOEpatents

    Ryan, Rodger; Timmerman, Walter J. H.

    1980-01-01

    A valve system incorporating single failure protective logic. The system consists of a valve combination or composite valve which allows actuation or de-actuation of a device such as a hydraulic cylinder or other mechanism, integral with or separate from the valve assembly, by means of three independent input signals combined in a function commonly known as two-out-of-three logic. Using the input signals as independent and redundant actuation/de-actuation signals, a single signal failure, or failure of the corresponding valve or valve set, will neither prevent the desired action, nor cause the undesired action of the mechanism.

  13. A homogeneous fluorometric assay platform based on novel synthetic proteins

    SciTech Connect

    Vardar-Schara, Goenuel; Krab, Ivo M.; Yi, Guohua; Su, Wei Wen . E-mail: wsu@hawaii.edu

    2007-09-14

    Novel synthetic recombinant sensor proteins have been created to detect analytes in solution, in a rapid single-step 'mix and read' noncompetitive homogeneous assay process, based on modulating the Foerster resonance energy transfer (FRET) property of the sensor proteins upon binding to their targets. The sensor proteins comprise a protein scaffold that incorporates a specific target-capturing element, sandwiched by genetic fusion between two molecules that form a FRET pair. The utility of the sensor proteins was demonstrated via three examples, for detecting an anti-biotin Fab antibody, a His-tagged recombinant protein, and an anti-FLAG peptide antibody, respectively, all done directly in solution. The diversity of sensor-target interactions that we have demonstrated in this study points to a potentially universal applicability of the biosensing concept. The possibilities for integrating a variety of target-capturing elements with a common sensor scaffold predict a broad range of practical applications.

  14. 21 CFR 866.3210 - Endotoxin assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Endotoxin assay. 866.3210 Section 866.3210 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3210 Endotoxin assay....

  15. 21 CFR 866.3210 - Endotoxin assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Endotoxin assay. 866.3210 Section 866.3210 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3210 Endotoxin assay....

  16. 21 CFR 866.3210 - Endotoxin assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Endotoxin assay. 866.3210 Section 866.3210 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3210 Endotoxin assay....

  17. 21 CFR 866.3210 - Endotoxin assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Endotoxin assay. 866.3210 Section 866.3210 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3210 Endotoxin assay....

  18. 21 CFR 866.3210 - Endotoxin assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Endotoxin assay. 866.3210 Section 866.3210 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3210 Endotoxin assay....

  19. 21 CFR 864.7525 - Heparin assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification....

  20. Statistical inference for serial dilution assay data.

    PubMed

    Lee, M L; Whitmore, G A

    1999-12-01

    Serial dilution assays are widely employed for estimating substance concentrations and minimum inhibitory concentrations. The Poisson-Bernoulli model for such assays is appropriate for count data but not for continuous measurements that are encountered in applications involving substance concentrations. This paper presents practical inference methods based on a log-normal model and illustrates these methods using a case application involving bacterial toxins.

  1. 21 CFR 864.7525 - Heparin assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification....

  2. 21 CFR 864.7525 - Heparin assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification....

  3. 21 CFR 864.7525 - Heparin assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES... circulation. These assays are quantitative clotting time procedures using the effect of heparin on...

  4. 21 CFR 864.7525 - Heparin assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES... circulation. These assays are quantitative clotting time procedures using the effect of heparin on...

  5. Differential regulation of the mitogen-activated protein and stress-activated protein kinase cascades by adrenergic agonists in quiescent and regenerating adult rat hepatocytes.

    PubMed Central

    Spector, M S; Auer, K L; Jarvis, W D; Ishac, E J; Gao, B; Kunos, G; Dent, P

    1997-01-01

    To study the mechanisms by which catecholamines regulate hepatocyte proliferation after partial hepatectomy (PHX), hepatocytes were isolated from adult male rats 24 h after sham operation or two-thirds PHX and treated with catecholamines and other agonists. In freshly isolated sham cells, p42 mitogen-activated protein (MAP) kinase activity was stimulated by the alpha1-adrenergic agonist phenylephrine (PHE). Activation of p42 MAP kinase by growth factors was blunted by pretreatment of sham hepatocytes with glucagon but not by that with the beta2-adrenergic agonist isoproterenol (ISO). In PHX cells, the ability of PHE to activate p42 MAP kinase was dramatically reduced, whereas ISO became competent to inhibit p42 MAP kinase activation. PHE treatment of sham but not PHX and ISO treatment of PHX but not sham hepatocytes also activated the stress-activated protein (SAP) kinases p46/54 SAP kinase and p38 SAP kinase. These data demonstrate that an alpha1- to beta2-adrenergic receptor switch occurs upon PHX and results in an increase in SAP kinase versus MAP kinase signaling by catecholamines. In primary cultures of hepatocytes, ISO treatment of PHX but not sham cells inhibited [3H]thymidine incorporation. In contrast, PHE treatment of sham but not PHX cells stimulated [3H]thymidine incorporation, which was reduced by approximately 25 and approximately 95% with specific inhibitors of p42 MAP kinase and p38 SAP kinase function, respectively. Inhibition of the p38 SAP kinase also dramatically reduced basal [3H]thymidine incorporation. These data suggest that p38 SAP kinase plays a permissive role in liver regeneration. Alterations in the abilities of catecholamines to modulate the activities of protein kinase A and the MAP and SAP kinase pathways may represent one physiological mechanism by which these agonists can regulate hepatocyte proliferation after PHX. PMID:9199291

  6. Aerobic glycolysis and lymphocyte transformation

    PubMed Central

    Hume, David A.; Radik, Judith L.; Ferber, Ernst; Weidemann, Maurice J.

    1978-01-01

    1. The role of enhanced aerobic glycolysis in the transformation of rat thymocytes by concanavalin A has been investigated. Concanavalin A addition doubled [U-14C]glucose uptake by rat thymocytes over 3h and caused an equivalent increased incorporation into protein, lipids and RNA. A disproportionately large percentage of the extra glucose taken up was converted into lactate, but concanavalin A also caused a specific increase in pyruvate oxidation, leading to an increase in the percentage contribution of glucose to the respiratory fuel. 2. Acetoacetate metabolism, which was not affected by concanavalin A, strongly suppressed pyruvate oxidation in the presence of [U-14C]glucose, but did not prevent the concanavalin A-induced stimulation of this process. Glucose uptake was not affected by acetoacetate in the presence or absence of concanavalin A, but in each case acetoacetate increased the percentage of glucose uptake accounted for by lactate production. 3. [3H]Thymidine incorporation into DNA in concanavalin A-treated thymocyte cultures was sensitive to the glucose concentration in the medium in a biphasic manner. Very low concentrations of glucose (25μm) stimulated DNA synthesis half-maximally, but maximum [3H]thymidine incorporation was observed only when the glucose concentration was raised to 1mm. Lactate addition did not alter the sensitivity of [3H]-thymidine uptake to glucose, but inosine blocked the effect of added glucose and strongly inhibited DNA synthesis. 4. It is suggested that the major function of enhanced aerobic glycolysis in transforming lymphocytes is to maintain higher steady-state amounts of glycolytic intermediates to act as precursors for macromolecule synthesis. PMID:310305

  7. Modulation of adipocyte biology by δ(9)-tetrahydrocannabinol.

    PubMed

    Teixeira, Diana; Pestana, Diogo; Faria, Ana; Calhau, Conceição; Azevedo, Isabel; Monteiro, Rosário

    2010-11-01

    It is recognized that the endocannabinoid system (ECS) plays a crucial role in the modulation of food intake and other aspects of energy metabolism. In this study, we aimed to investigate the effects of Δ(9)-tetrahydrocannabinol (THC) on adipocyte biology. 3T3-L1 cells were used to evaluate proliferation by sulforhodamine B (SRB) staining and methyl-(3)H-thymidine incorporation after 48 or 72 h of treatment with THC (1-500 nmol/l). Cells were differentiated in the presence or absence of the cannabinoid, and adipogenesis was determined by measuring lipid accumulation and peroxisome proliferator-activated receptor γ (PPARγ) transcription through reverse transcriptase-PCR (RT-PCR). Lipolysis was quantified under basal conditions or after isoproterenol (IP, 100 nmol/l) or insulin (INS, 100 nmol/l) treatment. Transforming growth factor β (TGFβ), diacylglycerol lipase α, and N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD) transcriptions were determined by RT-PCR in preadipocytes and adipocytes and adiponectin only in adipocytes. THC treatment increased culture protein content and reduced methyl-(3)H-thymidine incorporation. Cells treated with THC underwent adipogenesis shown by the expression of PPARγ and had increased lipid accumulation. Basal and IP-stimulated lipolyses were inhibited by THC and there was no effect on lipolysis of INS-treated adipocytes. The effects on methyl-(3)H-thymidine incorporation and lipolysis seem to be mediated through CB1- and CB2-dependent pathways. THC decreased NAPE-PLD in preadipocytes and increased adiponectin and TGFβ transcription in adipocytes. These results show that the ECS interferes with adipocyte biology and may contribute to adipose tissue (AT) remodeling. Although these observations point toward increased AT deposition, the stimulation of adiponectin production and inhibition of lipolysis may be in favor of improved INS sensitivity under cannabinoid influence.

  8. Dissimilar trophic effects of cerulein and xenopsin on the rat pancreas.

    PubMed

    Feurle, G E; Ohnheiser, G; Löser, C

    1990-03-01

    Cholecystokinin (CCK), gastrin, cerulein, and other analogs are known to stimulate the growth of the rat pancreas. In the present study, we compared the trophic action of a member of this gastrin/CCK family, the amphibian peptide cerulein, with a member of the structurally unrelated neurotensin/xenopsin group, the amphibian peptide xenopsin. For this purpose, 0.56 nmols/kg cerulein, 1.0 nmols/kg xenopsin, or normal saline were injected intraperitoneally three times a day in 28 rats for 3 d. Pancreatic weight, DNA, and incorporation of 3H-thymidine into DNA were determined. In another study, pancreatic weight, DNA, and the polyamines, putrescine, spermidine, and spermine, were determined after a single dose of 2.7 nmol/kg cerulein, 4.5 nmol/kg xenopsin, or saline. The polyamines were measured by reverse-phase HPLC and post-column derivatization. Cerulein increased pancreatic weight, stimulated 3H-thymidine incorporation into DNA, and raised putrescine concentrations significantly, but led to a significant reduction of pancreatic DNA concentration. Xenopsin also stimulated 3H-thymidine incorporation into DNA, but did not affect pancreatic weight, DNA concentration, or the polyamines during the 4 h of the experiment. These findings suggest that cerulein, in the dose and intervals applied, initiated hyperplasia and induced hypertrophy of the pancreas, whereas xenopsin only initiated hyperplasia. These results, together with the dissimilar secretory effects of the two peptide families, may be the expression of a dissimilar mode of action. However, it cannot be excluded that, since cerulein is more potent than xenopsin, the differences also are owing to dosage. We conclude that cerulein and xenopsin, which both have trophic effects on the pancreas, may act by different mechanisms.

  9. Inhibitor of DNA synthesis is present in normal chicken serum

    SciTech Connect

    Franklin, R.A.; Davila, D.R.; Westly, H.J.; Kelley, K.W.

    1986-03-05

    The authors have found that heat-inactivated serum (57/sup 0/C for 1 hour) from normal chickens reduces the proliferation of mitogen-stimulated chicken and murine splenocytes as well as some transformed mammalian lymphoblastoid cell lines. Greater than a 50% reduction in /sup 3/H-thymidine incorporation was observed when concanavalin A (Con A)-activated chicken splenocytes that were cultured in the presence of 10% autologous or heterologous serum were compared to mitogen-stimulated cells cultured in the absence of serum. Normal chicken serum (10%) also caused greater than 95% suppression of /sup 3/H-thymidine incorporation by bovine (EBL-1 and BL-3) and gibbon ape (MLA 144) transformed lymphoblastoid cell lines. The only cell line tested that was not inhibited by chicken serum was an IL-2-dependent, murine cell line. Chicken serum also inhibited both /sup 3/H-thymidine incorporation and IL-2 synthesis by Con A-activated murine splenocytes. Suppression was caused by actions other than cytotoxicity because viability of chicken splenocytes was unaffected by increasing levels of chicken serum. Furthermore, dialyzed serum retained its activity, which suggested that thymidine in the serum was not inhibiting uptake of radiolabeled thymidine. Suppressive activity was not due to adrenal glucocorticoids circulating in plasma because neither physiologic nor pharmacologic doses of corticosterone had inhibitory effects on mitogen-stimulated chicken splenocytes. These data demonstrate that an endogenous factor that is found in normal chicken serum inhibits proliferation of T-cells from chickens and mice as well as some transformed mammalian lymphoblastoid cell lines.

  10. 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.

  11. Optoelectronic devices incorporating fluoropolymer compositions for protection

    DOEpatents

    Chen, Xuming; Chum, Pak-Wing S.; Howard, Kevin E.; Lopez, Leonardo C.; Sumner, William C.; Wu, Shaofu

    2015-12-22

    The fluoropolymer compositions of the present invention generally incorporate ingredients comprising one or more fluoropolymers, an ultraviolet light protection component (hereinafter UV protection component), and optionally one or more additional ingredients if desired. The UV protection component includes a combination of at least one hindered tertiary amine (HTA) compound having a certain structure and a weight average molecular weight of at least 1000. This tertiary amine is used in combination with at least one organic, UV light absorbing compound (UVLA compound) having a weight average molecular weight greater than 500. When the HTA compound and the UVLA compound are selected according to principles of the present invention, the UV protection component provides fluoropolymer compositions with significantly improved weatherability characteristics for protecting underlying materials, features, structures, components, and/or the like. In particular, fluoropolymer compositions incorporating the UV protection component of the present invention have unexpectedly improved ability to resist blackening, coloration, or other de gradation that may be caused by UV exposure. As a consequence, devices protected by these compositions would be expected to have dramatically improved service life. The compositions have a wide range of uses but are particularly useful for forming protective layers in optoelectronic devices.

  12. Incorporating Geospatial Technology into Teacher Professional Development

    NASA Astrophysics Data System (ADS)

    Sproles, E. A.; Songer, L.

    2009-12-01

    The need for students to think spatially and use geospatial technologies is becoming more critical as these tools and concepts are increasingly incorporated into a broad range of occupations and academic disciplines. Geospatial Teaching Across the Curriculum (Geo-STAC) is a collaborative program that provides high school teachers with mentored professional development workshops in geospatial thought and technology. The seminars, led by community college faculty, give high school teachers the ability to incorporate geospatial technology into coursework across the curriculum — in Science, Technology, Engineering, and Math (STEM) and non-STEM disciplines. Students participating in the hands-on lessons gain experience in web-based and desktop Geographic Information Systems (GIS). The goals of the workshop are for teachers to: (1) understand the importance of geospatial thinking; (2) learn how to employ geospatial thinking in each discipline; (3) learn about geospatial technologies; (4) develop a Web-based GIS lesson; and, (5) implement a Web-based GIS lesson. Additionally, Geo-STAC works with high school students so that they: (1) understand the importance of geospatial technologies and careers in future job markets; (2) learn how to use Web-based GIS to solve problems; and, (3) visit the community college GIS lab and experience using desktop GIS. Geo-STAC actively disseminates this collaborative model to colleges to community colleges and high schools across the country.

  13. Incorporating intelligence into structured radiology reports

    NASA Astrophysics Data System (ADS)

    Kahn, Charles E.

    2014-03-01

    The new standard for radiology reporting templates being developed through the Integrating the Healthcare Enterprise (IHE) and DICOM organizations defines the storage and exchange of reporting templates as Hypertext Markup Language version 5 (HTML5) documents. The use of HTML5 enables the incorporation of "dynamic HTML," in which documents can be altered in response to their content. HTML5 documents can employ JavaScript, the HTML Document Object Model (DOM), and external web services to create intelligent reporting templates. Several reporting templates were created to demonstrate the use of scripts to perform in-template calculations and decision support. For example, a template for adrenal CT was created to compute contrast washout percentage from input values of precontrast, dynamic postcontrast, and delayed adrenal nodule attenuation values; the washout value can used to classify an adrenal nodule as a benign cortical adenoma. Dynamic templates were developed to compute volumes and apply diagnostic criteria, such as those for determination of internal carotid artery stenosis. Although reporting systems need not use a web browser to render the templates or their contents, the use of JavaScript creates innumerable opportunities to construct highly sophisticated HTML5 reporting templates. This report demonstrates the ability to incorporate dynamic content to enhance the use of radiology reporting templates.

  14. Self-Incorporation of Coenzymes by Ribozymes

    NASA Technical Reports Server (NTRS)

    Breaker, Ronald R.; Joyce, Gerald F.

    1995-01-01

    RNA molecules that are assembled from the four standard nucleotides contain a limited number of chemical functional groups, a characteristic that is generally thought to restrict the potential for catalysis by ribozymes. Although polypeptides carry a wider range of functional groups, many contemporary protein-based enzymes employ coenzymes to augment their capabilities. The coenzymes possess additional chemical moieties that can participate directly in catalysis and thereby enhance catalytic function. In this work, we demonstrate a mechanism by which ribozymes can supplement their limited repertoire of functional groups through RNAcatalyzed incorporation of various coenzymes and coenzyme analogues. The group I ribozyme of Tetrahymena thermophila normally mediates a phosphoester transfer reaction that results in the covalent attachment of guanosine to the ribozyme. Here, a shortened version of the ribozyme is shown to catalyze the self-incorporation of coenzymes and coenzyme analogues, such as NAD+ and dephosphorylated CoA-SH. Similar ribozyme activities may have played an important role in the "RNA world," when RNA enzymes are thought to have maintained a complex metabolism in the absence of proteins and would have benefited from the inclusion of additional functional groups.

  15. Incorporating Experience Curves in Appliance Standards Analysis

    SciTech Connect

    Garbesi, Karina; Chan, Peter; Greenblatt, Jeffery; Kantner, Colleen; Lekov, Alex; Meyers, Stephen; Rosenquist, Gregory; Buskirk, Robert Van; Yang, Hung-Chia; Desroches, Louis-Benoit

    2011-10-31

    The technical analyses in support of U.S. energy conservation standards for residential appliances and commercial equipment have typically assumed that manufacturing costs and retail prices remain constant during the projected 30-year analysis period. There is, however, considerable evidence that this assumption does not reflect real market prices. Costs and prices generally fall in relation to cumulative production, a phenomenon known as experience and modeled by a fairly robust empirical experience curve. Using price data from the Bureau of Labor Statistics, and shipment data obtained as part of the standards analysis process, we present U.S. experience curves for room air conditioners, clothes dryers, central air conditioners, furnaces, and refrigerators and freezers. These allow us to develop more representative appliance price projections than the assumption-based approach of constant prices. These experience curves were incorporated into recent energy conservation standards for these products. The impact on the national modeling can be significant, often increasing the net present value of potential standard levels in the analysis. In some cases a previously cost-negative potential standard level demonstrates a benefit when incorporating experience. These results imply that past energy conservation standards analyses may have undervalued the economic benefits of potential standard levels.

  16. Acellular comet assay: a tool for assessing variables influencing the alkaline comet assay.

    PubMed

    Kennedy, Erin K; McNamee, James P; Prud'homme Lalonde, Louise; Jones, Trevor; Wilkinson, Diana

    2012-01-01

    In this study, an acellular modification to the alkaline comet assay to further evaluate key variables within the assay that may influence the outcome of genotoxicity studies is described. This acellular comet assay can detect differences of 0.2 Gy of (60)Co gamma-ray radiation between 0 and 1 Gy and differences of 1 Gy between 0 and 8 Gy; thus, this assay is applicable for a wide range of DNA damage levels. It is also shown that DNA damage from different radiation energies was not significantly different from (60)Co gamma-ray. This assay displayed a statistical increase in DNA damage due to uncontrolled exposure to natural light; however, the slope of the dose-response curve for light-exposed samples was similar to that for samples protected from light. A comparison of the alkaline comet assay with the acellular comet assay allowed for the intrinsic repair capacity of the alkaline comet assay to be quantified.

  17. Paper disk assay for glycosaminoglycan sulfotransferases

    SciTech Connect

    Sugahara, K.; Ishii, T.; Yamashina, I.

    1987-11-01

    A method is described for the assay of sulfotransferases, which transfer sulfate from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to glycosaminoglycan acceptors. Following the sulfation reactions, the (/sup 35/S)sulfate-labeled products are precipitated and then separated from a sulfate donor ((/sup 35/S)PAPS) and its degradation products by a paper disk method, and then the radioactivity remaining on the paper disk is subsequently determined by liquid scintillation counting. The rapidity and simplicity of the method are advantageous for multiple assays and have allowed us to establish assay conditions for serum sulfotransferases which introduce sulfate at position 6 of the internal N-acetylgalactosamine units of chondroitin, position 2 (amino group) of the glucosamine units of heparan sulfate and sugar units of keratan sulfate, respectively. The assay method will be applicable with modification to the assay of other glycosaminoglycan sulfotransferases and glycoprotein sulfotransferases.

  18. Fluorometric assay for red blood cell antibodies

    SciTech Connect

    Schreiber, A.B.; Lambermont, M.; Strosberg, A.D.; Wybran, J.

    1981-03-01

    A fluorometric assay is described for the detection of red blood cell antibodies. The assay reveals as little as 600 molecules of bound, fluoroesceinated rabbit anti-human IgG antibodies per erythrocyte. Eleven patients with possible autoimmune erythrocyte disorder and negative direct antiglobulin test were studied by the fluorometric assay. The outcome of the fluorometric assay was compared with that of the human allogeneic rosette test. Results obtained by the two methods were in complete agreement. Five of the patients were shown to possess unexpectedly high levels of erythrocyte-bound IgG in spite of a negative, direct antiglobulin test. These findings and the validity of the fluorometric assay are discussed.

  19. Rapid fluorescence-based assay for radiosensitivity and chemosensitivity testing in mammalian cells in vitro

    SciTech Connect

    Begg, A.C.; Mooren, E.

    1989-02-01

    An efficient and rapid cytotoxicity assay has been developed, particularly for radiobiological studies, utilizing 96-well microtiter plates. Several days after treatment, cell numbers per well were measured by fluorescent intensity using an automatic reader after staining with the DNA specific dye Hoechst 33258. For radiobiological applications, a microtiter plate irradiation box was designed and built which allowed a variable number of wells (minimum 4, maximum 16) to be irradiated at one time. In this manner, complete dose-response curves could be obtained from one plate. The assay depends on the growth of surviving and untreated cells, and by appropriate choice of conditions (cell numbers plated, time of assay), cell survival curves for this quick fluorescence assay were in reasonable agreement with those from a clonogenic assay for cisplatin and X-ray-induced cell killing. The assay can span 1.5-2 decades of cell survival and is suitable for any cell line which grows as a monolayer. Radiobiological applications were tested using agents or conditions which modified radiation damage. Firstly, sublethal damage repair could be demonstrated in RIF1 mouse tumor cells by comparing the survival curve for a single X-ray dose with that for two fractions separated by 4 h. Secondly, incorporation of 5-iodo-2'-deoxyuridine into cellular DNA was shown to radiosensitize Chinese Hamster cells, with similar enhancement ratios obtained from the fluorescence and clonogenic assays. Thirdly, radiosensitization by cisplatin and radioprotection by cysteamine could be readily measured using the quick fluorescence assay. The ability to have multiple dose groups per plate makes it an efficient assay for both radiosensitivity and chemosensitivity testing.

  20. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of radiosensitivity

    SciTech Connect

    Carmichael, J.; DeGraff, W.G.; Gazdar, A.F.; Minna, J.D.; Mitchell, J.B.

    1987-02-15

    Radiation survival curves were generated for V79 Chinese hamster and two human lung cancer cell lines (NCI-H460 and NCI-H249) with doubling times of 10, 20, and 85 h, respectively, using a standard clonogenic assay, a dye exclusion assay, and a semiautomated colorimetric assay utilizing a tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylformazan bromide. Comparable results for D0 and extrapolation number (n) were observed for all assays in the lines with doubling times of 10 and 20 h. In these instances the tumor cell lines had undergone seven or more doublings after radiation. For the tumor line (H249) with an 80-h doubling time the D0S were comparable between the assays while the extrapolation number was increased in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylformazan bromide assay, a result probably related to the lower number of doublings (less than 4) after radiation. We then tested the ability of the assays to detect radiation protection and sensitization using known agents. We found that cysteamine treatment resulted in radioprotection (by a factor of 8 at 8 Gy) while 5-bromo-2-deoxyuridine incorporation caused enhancement of radiation sensitivity in all three assays. We conclude that, while optimal conditions for each cell line (cell number plated and doubling time) must be established, using characterized tumor cell lines, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylformazan bromide assay could be automated and thus be of great value in screening large numbers of potential radiosensitizers or protectors.

  1. Use of Cause-and-Effect Analysis to Design a High-Quality Nanocytotoxicology Assay.

    PubMed

    Rösslein, Matthias; Elliott, John T; Salit, Marc; Petersen, Elijah J; Hirsch, Cordula; Krug, Harald F; Wick, Peter

    2015-01-20

    An important consideration in developing standards and regulations that govern the production and use of commercial nanoscale materials is the development of robust and reliable measurements to monitor the potential adverse biological effects of such products. These measurements typically require cell-based and other biological assays that provide an assessment of the risks associated with the nanomaterial of interest. In this perspective, we describe the use of cause-and-effect (C&E) analysis to design robust, high quality cell-based assays to test nanoparticle-related cytotoxicity. C&E analysis of an assay system identifies the sources of variability that influence the test result. These sources can then be used to design control experiments that aid in establishing the validity of a test result. We demonstrate the application of C&E analysis to the commonly used 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell-viability assay. This is the first time to our knowledge that C&E analysis has been used to characterize a cell-based toxicity assay. We propose the use of a 96-well plate layout which incorporates a range of control experiments to assess multiple factors such as nanomaterial interference, pipetting accuracy, cell seeding density, and instrument performance, and demonstrate the performance of the assay using the plate layout in a case study. While the plate layout was formulated specifically for the MTS assay, it is applicable to other cytotoxicity, ecotoxicity (i.e., bacteria toxicity), and nanotoxicity assays after assay-specific modifications.

  2. Optimized plasmid systems for the incorporation of multiple different unnatural amino acids by evolved orthogonal ribosomes.

    PubMed

    Lammers, Christoph; Hahn, Liljan E; Neumann, Heinz

    2014-08-18

    Incorporation of multiple different unnatural amino acids into the same polypeptide remains a significant challenge. Orthogonal ribosomes, which are evolvable as they direct the translation of a single dedicated orthogonal mRNA, can provide an avenue to produce such polypeptides routinely. Recent advances in engineering orthogonal ribosomes have created a prototype system to enable genetically encoded introduction of two different functional groups, albeit with limited efficiency. Here, we systematically investigated the limiting factors of this system by using assays to measure the levels and activities of individual components; we identified Methanosarcina barkeri PylRS as a limiting factor for protein yield. Balancing the expression levels of individual components significantly improved growth rate and protein yield. This optimization of the system is likely to increase the scope of evolved orthogonal ribosome-mediated incorporation of multiple different unnatural amino acids.

  3. Persistence of Free Plasmid DNA in Soil Monitored by Various Methods, Including a Transformation Assay

    PubMed Central

    Romanowski, Gerd; Lorenz, Michael G.; Sayler, Gary; Wackernagel, Wilfried

    1992-01-01

    The persistence and stability of free plasmid pUC8-ISP DNA introduced into 10-g samples of various soils and kept at 23°C were monitored over a period of 60 days. The soils were sampled at a plant science farm and included a loamy sand soil (no. 1), a clay soil (no. 2), and a silty clay soil (no. 3). Four different methods allowed monitoring of (i) the production of acid-soluble radioactive material from [3H]thymidine-labeled plasmid DNA, (ii) the decrease of hybridizing nucleotide sequences in slot blot analysis, (iii) the loss of plasmid integrity measured by Southern hybridization, and (iv) the decay of the biological activity as determined by transformation of Ca2+-treated Escherichia coli cells with the DNA extracted from soil. Acid-soluble material was not produced within the first 24 h but then increased to 45% (soil no. 1), 27% (soil no. 2), and 77% (soil no. 3) until the end of incubation. A quite parallel loss of material giving a slot blot hybridization signal was observed. Southern hybridization indicated that after 1 h in the soils, plasmid DNA was mostly in the form of circular and full-length linear molecules but that, depending on the soil type, after 2 to 5 days full-length plasmid molecules were hardly detectable. The transforming activity of plasmid DNA reextracted from the soils followed inactivation curves over 2 to 4 orders of magnitude and dropped below the detection limit after 10 days. The inactivation was slower in soil no. 2 (28.2-h half-life time of the transforming activity of a plasmid molecule) than in soils no. 3 (15.1 h) and no. 1 (9.1 h). The studies provide data on the persistence of free DNA molecules in natural bacterial soil habitats. The data suggest that plasmid DNA may persist long enough to be available for uptake by competent recipient cells in situ. Images PMID:16348772

  4. Laboratory Assays for Epstein-Barr Virus-Related Disease

    PubMed Central

    Gulley, Margaret L.; Tang, Weihua

    2008-01-01

    Epstein-Barr virus (EBV) infects various cell types in a wide spectrum of benign and malignant diseases. Laboratory tests for EBV have improved and are increasingly used in diagnosis, prognosis, prediction, and prevention of diseases ranging from infectious mononucleosis to selected subtypes of lymphoma, sarcoma, and carcinoma. Indeed, the presence of EBV is among the most effective tumor markers supporting clinical management of cancer patients. In biopsies, localization of EBER transcripts by in situ hybridization remains the gold standard for identifying latent infection. Other RNA- and protein-based assays detect lytic viral replication and can distinguish carcinoma-derived from lymphocyte-derived EBV in saliva or nasopharyngeal brushings. Analysis of blood using EBV viral load and serology reflects disease status and risk of progression. This review summarizes prior research in the context of basic virologic principles to provide a rational strategy for applying and interpreting EBV tests in various clinical settings. Such assays have been incorporated into standard clinical practice in selected settings such as diagnosis of primary infection and management of patients with immune dysfunction or nasopharyngeal carcinoma. As novel therapies are developed that target virus-infected cells or overcome the adverse effects of infection, laboratory testing becomes even more critical for determining when intervention is appropriate and the extent to which it has succeeded. PMID:18556771

  5. INCORPORATING DYNAMIC 3D SIMULATION INTO PRA

    SciTech Connect

    Steven R Prescott; Curtis Smith

    2011-07-01

    Through continued advancement in computational resources, development that was previously done by trial and error production is now performed through computer simulation. These virtual physical representations have the potential to provide accurate and valid modeling results and are being used in many different technical fields. Risk assessment now has the opportunity to use 3D simulation to improve analysis results and insights, especially for external event analysis. By using simulations, the modeler only has to determine the likelihood of an event without having to also predict the results of that event. The 3D simulation automatically determines not only the outcome of the event, but when those failures occur. How can we effectively incorporate 3D simulation into traditional PRA? Most PRA plant modeling is made up of components with different failure modes, probabilities, and rates. Typically, these components are grouped into various systems and then are modeled together (in different combinations) as a “system” with logic structures to form fault trees. Applicable fault trees are combined through scenarios, typically represented by event tree models. Though this method gives us failure results for a given model, it has limitations when it comes to time-based dependencies or dependencies that are coupled to physical processes which may themselves be space- or time-dependent. Since, failures from a 3D simulation are naturally time related, they should be used in that manner. In our simulation approach, traditional static models are converted into an equivalent state diagram representation with start states, probabilistic driven movements between states and terminal states. As the state model is run repeatedly, it converges to the same results as the PRA model in cases where time-related factors are not important. In cases where timing considerations are important (e.g., when events are dependent upon each other), then the simulation approach will typically

  6. Development and Utilization of an Ex Vivo Bromodeoxyuridine Local Lymph Node Assay (LLNA) Protocol for Assessing Potential Chemical Sensitizers

    EPA Science Inventory

    The murine local lymph node assay (LLNA) is widely used to identify chemicals that may cause allergic contact dermatitis. Exposure to a dermal sensitizer results in proliferation of local lymph node T cells, which has traditionally been measured by in vivo incorporation of [3H]m...

  7. Choline incorporation by Schistosoma mansoni: distribution of choline metabolites during development and after sexual differentiation

    SciTech Connect

    Ancelin, M.L.; Torpier, G.; Vial, H.J.; Capron, A.

    1987-06-01

    Choline metabolism was investigated in Schistosoma mansoni during the main phases of its development, namely, schistosomula, 11- and 15-day-old worms, and adults. At the physiological choline concentration used in the assay (20 microM), betaine was, along with phosphatidylcholine, one of the most abundant choline metabolites, revealing considerable choline oxidation activity. Very little radioactivity was associated with CDP-choline, whereas a sustained incorporation into phosphocholine occurred. These results provide good evidence that CTP:phosphocholine cytidylyltransferase plays a regulatory role in the de novo pathway of phosphatidylcholine biosynthesis. During development, the incorporation of choline into its various metabolites was maximal in 11-day-old worms. At this stage, the oxidative pathway predominated over the Kennedy pathway, whereas at all other stages the de novo phosphatidylcholine biosynthesis was predominant. Furthermore, choline incorporation into betaine was much more important in the adult female worm than in the male, indicating a major difference in choline incorporation and distribution between the 2 sexes of the adult worms.

  8. Assessment of phospholipid deacylation-reacylation cycles by H/sub 2/ /sup 18/O incorporation

    SciTech Connect

    Kuwae, T.; Schmid, P.C.; Johnson, S.B.; Schmid, H.H.O.

    1987-05-01

    Quantitative estimation of metabolic turnover involving hydrolysis is possible with the use of H/sub 2/ /sup 18/O which readily equilibrates throughout subcellular compartments and thus obviates many problems associated with the use of exogenous metabolites. Incubation of cells in H/sub 2/ /sup 18/O results in the time-dependent incorporation of /sup 18/O into ester carbonyls of glycerophospholipids. Preparation of methyl esters by alkaline methanolysis preserves the carbonyl oxygen and hydrogenation prior to analysis by gas-chromatography/mass-spectrometry simplifies determination of /sup 18/O//sup 16/O ratios. Mouse peritoneal exudate cells incorporate /sup 18/O from H/sub 2/ /sup 18/O into acyl groups of phosphatidylcholine (PC) and phosphatidylinositol (PI), and smaller amounts into phosphatidylethanolamine (PE) and phosphatidylserine (PS). Acyl groups at the sn-1 position of PC and PE show about the same rate of turnover as those at the sn-2 position. Considering the route of /sup 18/O incorporation via free fatty acid derived through ester hydrolysis in 40% H/sub 2/ /sup 18/O, acyl turnover may exceed 20% per hour. Turnover of 2-O-acyl groups in the plasmalogen analog of PE, as assayed by /sup 18/O incorporation, is drastically lower than in diacylPE. This indicates either a much lower rate of hydrolysis, or reacylation of lysoplasmalogen by direct acyl transfer not involving free fatty acid.

  9. Effects of epidermal growth factor on neural crest cells in tissue culture

    SciTech Connect

    Erickson, C.A.; Turley, E.A.

    1987-04-01

    Epidermal growth factor (EGF) stimulates the release of hyaluronic acid (HA) and chondroitin sulfate proteoglycan (CSPG) from quail trunk neural crest cultures in a dose-dependent fashion. It also promotes the expression of cell-associated heparan sulfate proteoglycan (HSPG) as detected by immunofluorescence and immunoprecipitation of the /sup 3/H-labeled proteoglycan. Furthermore, EGF stimulates (/sup 3/H)thymidine incorporation into total cell DNA. These results raise the possibility that EGF or an analogous growth factor is involved in regulation of neural crest cell morphogenesis.

  10. Lymphocyte function in myasthenia gravis.

    PubMed Central

    Kawanami, S; Kanaide, A; Itoyama, Y; Kuroiwa, Y

    1979-01-01

    Mitogen-induced blastoid transformation of peripheral blood lymphocytes from patients with myasthenia gravis was studied using a microplate culture technique and evaluated with 3H-thymidine incorporation. It was found that both phytohaemagglutinin and pokeweed mitogen responses decreased significantly in patients with myasthenia gravis. In myasthenic crisis, indices of stimulation by phytohaemagglutination became very low. The autologous plasma neither inhibited nor facilitated mitogenic responses of lymphocytes. The decreased mitogen responsiveness of lymphocytes suggests that part of the T lymphocyte function is subnormal in myasthenia. PMID:490180

  11. Ultraviolet light-resistant primary transfectants of xeroderma pigmentosum cells are also DNA repair-proficient

    SciTech Connect

    Stark, M.; Naiman, T.; Canaani, D. )

    1989-08-15

    In a previous work, an immortal xeroderma pigmentosum cell line belonging to complementation group C was complemented to a UV-resistant phenotype by transfection with a human cDNA clone library. We now report that the primary transformants selected for UV-resistance also acquired normal levels of DNA repair. This was assessed both by measurement of UV-induced ({sup 3}H)thymidine incorporation and by equilibrium sedimentation analysis of repair-DNA synthesis. Therefore, the transduced DNA element which confers normal UV-resistance also corrects the excision repair defect of the xeroderma pigmentosum group C cell line.

  12. Modulation of cytokine secretion by mesenteric lymph node cells from vitamin A-deficient mice during Hymenolepis nana infection.

    PubMed

    Ikeda, K; Matsuo, S; Asano, K; Okamoto, K

    1994-01-01

    The influence of vitamin A deficiency on the development of cellular immune responses was examined using vitamin A-deficient mice (A mice)/Hymenolepis nana system. Mesenteric lymph node cells (MLNC) prepared from both normal BALB/c mice and A mice during H. nana infection proliferated extensively when cultured with soluble egg antigen of H. nana as assessed by 3H-thymidine incorporation. MLNC from normal mice secreted significantly more IL-2 and significantly less IFN-gamma than A mice when the cells were cultured in the presence of soluble egg antigen.

  13. Effects of dexamethasone on human synovial fibroblast-like cells, from osteoarthritic joints, in culture

    SciTech Connect

    Vento, R.; Torregrossa, M.V.; Giuliano, M.; Grecomoro, G.; Piccione, F. )

    1990-01-01

    The effect of Dexamethasone (DEX) on cell division and macromolecular synthesis was investigated in a line (Mc Coy cells, A 9) of synovial fibroblast-like cells derived from human osteoarthritic joints. DEX markedly reduced the proliferation of Mc Coy cells in a time and dose-dependent manner. The maximal inhibition was found at 500 nM DEX 24 h after incubation and was accompanied by the appearance of giant macrophage-like cells. After DEX treatment cells showed increased content of DNA, proteins and RNA together with the reduction of ({sup 3}H)-thymidine incorporation into the TCA-precipitable fraction.

  14. Chloroplast Division and DNA Synthesis in Light-grown Wheat Leaves 1

    PubMed Central

    Boffey, Stephen A.; Ellis, J. Raymond; Selldén, Gun; Leech, Rachel M.

    1979-01-01

    Light-grown 7-day-old wheat seedlings (Triticum aestivum, var. Maris Dove) showed an increase of 200% in plastids per cell between 1.7 and 4.5 centimeters from the leaf base. This increase was the result of divisions of young chloroplasts at various stages of development, and was well separated in distance, and therefore in time from the region of cell division in the basal meristem. [3H]Thymidine was incorporated into plastid DNA throughout the zone of plastid division, but not above it. Images PMID:16660998

  15. Preparation of monoclonal antibodies to antigens of specific murine suppressor T cells

    SciTech Connect

    Chervonskii, A.V.; Suslov, A.P.; Shitin, A.G.; Abronina, I.F.; Brondz, B.D.

    1986-12-01

    The aim of this investigation was to obtain monoclonal antibodies (MCAB) interacting selectively with suppressor T cells. Mice were used in the experiments and antibodies in the culture fluids were determined by radioimmunoassay. Pure rabbit antibodies against rat immunoglobulins (Ig) absorbed with mouse Ig were used as /sup 125/I-labeled antibodies. Activity of specific suppressor T cells was estimated as the index of inhibition of /sup 3/H-thymidine incorporation after incubation. The class and type of the MCAB and their concentration were determined by gel filtration and inhibition of radioimmunoadsorption.

  16. A preclinical assay for chemosensitivity in multiple myeloma

    PubMed Central

    Khin, Zayar P.; Ribeiro, Maria L. C.; Jacobson, Timothy; Hazlehurst, Lori; Perez, Lia; Baz, Rachid; Shain, Kenneth; Silva, Ariosto S.

    2013-01-01

    Accurate preclinical predictions of the clinical efficacy of experimental cancer drugs are highly desired but often haphazard. Such predictions might be improved by incorporating elements of the tumor microenvironment in preclinical models by providing a more physiological setting. In generating improved xenograft models, it is generally accepted that the use of primary tumors from patients are preferable to clonal tumor cell lines. Here we describe an interdisciplinary platform to study drug response in multiple myeloma (MM), an incurable cancer of the bone marrow. This platform uses microfluidic technology to minimize the number of cells per experiment, while incorporating 3D extracellular matrix and mesenchymal cells derived from the tumor microenvironment. We used sequential imaging and a novel digital imaging analysis algorithm to quantify changes in cell viability. Computational models were used convert experimental data into dose-exposure-response "surfaces" which offered predictive utility. Using this platform, we predicted chemosensitivity to bortezomib and melphalan, two clinical MM treatments, in 3 MM cell lines and 7 patient-derived primary MM cell populations. We also demonstrated how this system could be used to investigate environment-mediated drug resistance and drug combinations that target it. This interdisciplinary preclinical assay is capable of generating quantitative data that can be used in computational models of clinical response, demonstrating its utility as a tool to contribute to personalized oncology. Major Findings By designing an experimental platform with the specific intent of generating experimental parameters for a computational clinical model of personalized therapy in multiple myeloma, while taking in consideration the limitations of working with patient primary cells, and the need to incorporate elements of the tumor microenvironment, we have generated patient-individualized estimations of initial response and time to relapse

  17. Three Enzymatic Steps Required for the Galactosamine Incorporation into Core Lipopolysaccharide*

    PubMed Central

    Aquilini, Eleonora; Azevedo, Joana; Merino, Susana; Jimenez, Natalia; Tomás, Juan M.; Regué, Miguel

    2010-01-01

    The core lipopolysaccharides (LPS) of Proteus mirabilis as well as those of Klebsiella pneumoniae and Serratia marcescens are characterized by the presence of a hexosamine-galacturonic acid disaccharide (αHexN-(1,4)-αGalA) attached by an α1,3 linkage to l-glycero-d-manno-heptopyranose II (l-glycero-α-d-manno-heptosepyranose II). In K. pneumoniae, S. marcescens, and some P. mirabilis strains, HexN is d-glucosamine, whereas in other P. mirabilis strains, it corresponds to d-galactosamine. Previously, we have shown that two enzymes are required for the incorporation of d-glucosamine into the core LPS of K. pneumoniae; the WabH enzyme catalyzes the incorporation of GlcNAc from UDP-GlcNAc to outer core LPS, and WabN catalyzes the deacetylation of the incorporated GlcNAc. Here we report the presence of two different HexNAc transferases depending on the nature of the HexN in P. mirabilis core LPS. In vivo and in vitro assays using LPS truncated at the level of galacturonic acid as acceptor show that these two enzymes differ in their specificity for the transfer of GlcNAc or GalNAc. By contrast, only one WabN homologue was found in the studied P. mirabilis strains. Similar assays suggest that the P. mirabilis WabN homologue is able to deacetylate both GlcNAc and GalNAc. We conclude that incorporation of d-galactosamine requires three enzymes: Gne epimerase for the generation of UDP-GalNAc from UDP-GlcNAc, N-acetylgalactosaminyltransferase (WabP), and LPS:HexNAc deacetylase. PMID:20959463

  18. DNA Methyltransferase Activity Assays: Advances and Challenges

    PubMed Central

    Poh, Wan Jun; Wee, Cayden Pang Pee; Gao, Zhiqiang

    2016-01-01

    DNA methyltransferases (MTases), a family of enzymes that catalyse the methylation of DNA, have a profound effect on gene regulation. A large body of evidence has indicated that DNA MTase is potentially a predictive biomarker closely associated with genetic disorders and genetic diseases like cancer. Given the attention bestowed onto DNA MTases in molecular biology and medicine, highly sensitive detection of DNA MTase activity is essential in determining gene regulation, epigenetic modification, clinical diagnosis and therapeutics. Conventional techniques such as isotope labelling are effective, but they often require laborious sample preparation, isotope labelling, sophisticated equipment and large amounts of DNA, rendering them unsuitable for uses at point-of-care. Simple, portable, highly sensitive and low-cost assays are urgently needed for DNA MTase activity screening. In most recent technological advances, many alternative DNA MTase activity assays such as fluorescent, electrochemical, colorimetric and chemiluminescent assays have been proposed. In addition, many of them are coupled with nanomaterials and/or enzymes to significantly enhance their sensitivity. Herein we review the progress in the development of DNA MTase activity assays with an emphasis on assay mechanism and performance with some discussion on challenges and perspectives. It is hoped that this article will provide a broad coverage of DNA MTase activity assays and their latest developments and open new perspectives toward the development of DNA MTase activity assays with much improved performance for uses in molecular biology and clinical practice. PMID:26909112

  19. Incorporating LCA tools in integrated simulation environments

    SciTech Connect

    Pal, Vineeta; Papamichael, Konstantinos; Bourassa, Norman; Loffeld, John J.

    2001-02-01

    In this paper we address the issue of building data schema evolution in integrated simulation environments, as seen from the perspective of incorporating LCA tools within these environments. First we describe the key features of an integrated simulation environment designed for expandability, focusing on (a) the mechanism for the expansion of the integrated environment, and (b) its overall system architecture that allows processes and data to be added to the system without modifications or restructuring of existing code. We then focus on how the data schema allows the inclusion and maintenance of specialized construction objects bearing LCA data. Finally, we discuss various integration issues that arise from modeling capabilities and idiosyncrasies of individual simulation and analysis tools.

  20. Active containment systems incorporating modified pillared clays

    SciTech Connect

    Lundie, P. |; McLeod, N.

    1997-12-31

    The application of treatment technologies in active containment systems provides a more advanced and effective method for the remediation of contaminated sites. These treatment technologies can be applied in permeable reactive walls and/or funnel and gate systems. The application of modified pillared clays in active containment systems provides a mechanism for producing permeable reactive walls with versatile properties. These pillared clays are suitably modified to incorporate reactive intercalatants capable of reacting with both a broad range of organic pollutants of varying molecular size, polarity and reactivity. Heavy metals can be removed from contaminated water by conventional ion-exchange and other reactive processes within the clay structure. Complex contamination problems can be addressed by the application of more than one modified clay on a site specific basis. This paper briefly describes the active containment system and the structure/chemistry of the modified pillared clay technology, illustrating potential applications of the in-situ treatment process for contaminated site remediation.

  1. Incorporating Pharmacy Scholarship to Management Responsibilities

    PubMed Central

    Hertig, John B.; Weber, Robert J.

    2015-01-01

    Practice advancement demands innovation. Amidst professional change, pharmacy leaders have the opportunity to collaborate with colleagues to develop transformational ideas, implement these solutions, and share those successes with professionals around the state, country, and world. Scholarship, defined as contributing to the literature through publications, presentations, and other writings, is an ideal way to advance innovation within the profession. It is critical for pharmacy leaders to build scholarship into their professional workflow. Ensuring that successful projects are published or presented may translate into shared best practices. Many pharmacy leaders may find it difficult to participate in scholarship activities because of their busy schedules. This column serves to outline recommendations on how to effectively incorporate writing for publications, making presentations, and other scholarly work into the role of pharmacy leaders and managers. To reduce the barriers to scholarship, pharmacy leaders role can apply project management principles to their work and identify projects that otherwise would not be published and support their development. PMID:26823623

  2. Incorporating immigrant flows into microsimulation models.

    PubMed

    Duleep, Harriet Orcutt; Dowhan, Daniel J

    2008-01-01

    Building on the research on immigrant earnings reviewed in the first article of this series, "Research on Immigrant Earnings," the preceding article, "Adding Immigrants to Microsimulation Models," linked research results to various issues essential for incorporating immigrant earnings into microsimulation models. The discussions of that article were in terms of a closed system. That is, it examined a system in which immigrant earnings and emigration are forecast for a given population represented in the base sample in the microsimulation model. This article, the last in the series, addresses immigrant earnings projections for open systems--microsimulation models that include projections of future immigration. The article suggests a simple method to project future immigrants and their earnings. Including the future flow of immigrants in microsimulation models can dramatically affect the projected Social Security benefits of some groups.

  3. Radiation shielding materials and containers incorporating same

    DOEpatents

    Mirsky, Steven M.; Krill, Stephen J.; Murray, Alexander P.

    2005-11-01

    An improved radiation shielding material and storage systems for radioactive materials incorporating the same. The PYRolytic Uranium Compound ("PYRUC") shielding material is preferably formed by heat and/or pressure treatment of a precursor material comprising microspheres of a uranium compound, such as uranium dioxide or uranium carbide, and a suitable binder. The PYRUC shielding material provides improved radiation shielding, thermal characteristic, cost and ease of use in comparison with other shielding materials. The shielding material can be used to form containment systems, container vessels, shielding structures, and containment storage areas, all of which can be used to house radioactive waste. The preferred shielding system is in the form of a container for storage, transportation, and disposal of radioactive waste. In addition, improved methods for preparing uranium dioxide and uranium carbide microspheres for use in the radiation shielding materials are also provided.

  4. Radiation Shielding Materials and Containers Incorporating Same

    DOEpatents

    Mirsky, Steven M.; Krill, Stephen J.; and Murray, Alexander P.

    2005-11-01

    An improved radiation shielding material and storage systems for radioactive materials incorporating the same. The PYRolytic Uranium Compound (''PYRUC'') shielding material is preferably formed by heat and/or pressure treatment of a precursor material comprising microspheres of a uranium compound, such as uranium dioxide or uranium carbide, and a suitable binder. The PYRUC shielding material provides improved radiation shielding, thermal characteristic, cost and ease of use in comparison with other shielding materials. The shielding material can be used to form containment systems, container vessels, shielding structures, and containment storage areas, all of which can be used to house radioactive waste. The preferred shielding system is in the form of a container for storage, transportation, and disposal of radioactive waste. In addition, improved methods for preparing uranium dioxide and uranium carbide microspheres for use in the radiation shielding materials are also provided.

  5. [Effectiveness of incorporating a quality management system].

    PubMed

    Seki, Akira; Hankins, Raleigh W; Miya, Tetsumasa

    2010-01-01

    In 2003, the ISO 15189 international standardization program on the quality and competence of the clinical reference laboratory was introduced. To date, 46 facilities have committed themselves to providing a higher level of medical service by incorporating a quality management system (QMS) and acquiring accreditation. QMS is defined as "setting up a policy and goals pertaining to quality, and adopting an appropriate system," and is a scheme that includes all managerial and technical factors that can affect test results. Regarding the Health Sciences Research Institute Group, 4 facilities have previously received the accreditation described above, but in the process of implementing the QMS, a number of problems have been identified. Here, we report on the effectiveness of adopting such a QMS based on the results of employee questionnaires, internal audits, customer complaint analyses, and external audits by the Japan Accreditation Board for Conformity Assessment (JAB), the official inspection body for accreditation.

  6. Solar cells incorporating light harvesting arrays

    DOEpatents

    Lindsey, Jonathan S.; Meyer, Gerald J.

    2003-07-22

    A solar cell incorporates a light harvesting array that comprises: (a) a first substrate comprising a first electrode; and (b) a layer of light harvesting rods electrically coupled to the first electrode, each of the light harvesting rods comprising a polymer of Formula I: ##EQU1## wherein m is at least 1, and may be from two, three or four to 20 or more; X.sup.1 is a charge separation group (and preferably a porphyrinic macrocycle, which may be one ligand of a double-decker sandwich compound) having an excited-state of energy equal to or lower than that of X.sup.2 ; and X.sup.2 through X.sup.m+1 are chromophores (and again are preferably porphyrinic macrocycles).

  7. Solar cells incorporating light harvesting arrays

    DOEpatents

    Lindsey, Jonathan S.; Meyer, Gerald J.

    2002-01-01

    A solar cell incorporates a light harvesting array that comprises: (a) a first substrate comprising a first electrode; and (b) a layer of light harvesting rods electrically coupled to the first electrode, each of the light harvesting rods comprising a polymer of Formula I: X.sup.1.paren open-st.X.sup.m+1).sub.m (I) wherein m is at least 1, and may be from two, three or four to 20 or more; X.sup.1 is a charge separation group (and preferably a porphyrinic macrocycle, which may be one ligand of a double-decker sandwich compound) having an excited-state of energy equal to or lower than that of X.sup.2 ; and X.sup.2 through X.sup.m+1 are chromophores (and again are preferably porphyrinic macrocycles).

  8. Jay Carter Enterprises, Incorporated steam engine

    NASA Technical Reports Server (NTRS)

    1981-01-01

    The Small Community Solar Thermal Power Experiment (SCSE) selected an organic rankine cycle (ORC) engine driving a high speed permanent magnet alternator (PMA) as the baseline power conversion subsystem (PCS) design. The back-up conceptual PCS design is a steam engine driving an induction alternator delivering power directly to the grid. The development of the automotive reciprocating simple rankine cycle steam engine and how an engine of similar design might be incorporated into the SCSE is discussed. A description of the third generation automotive engine is included along with some preliminary test data. Tests were conducted with the third generation engine driving an induction alternator delivering power directly to the grid. The purpose of these tests is to further verify the effects of expander inlet temperature, input thermal power level, expansion ratio, and other parameters affecting engine performance to aid in the development of an SCSE PCS.

  9. Upgrading carbon dioxide by incorporation into heterocycles.

    PubMed

    Yu, Bing; He, Liang-Nian

    2015-01-01

    Carbon dioxide is commonly regarded as the primary greenhouse gas, but from a synthetic standpoint can be utilized as an alternative and sustainable C1 synthon in organic synthesis rather than a waste. This results in the production of organic carbonates, carboxylic acids, and derivatives. Recently, CO2 has emerged as an appealing tool for heterocycle synthesis under mild conditions without using stoichiometric amounts of organometallic reducing reagents. This Minireview summarizes recent advances on methodologies for CO2 incorporation into N-, O-, and C-nucleophiles to provide various heterocycles, including cyclic carbamates, benzoxazine-2-one, 4-hydroxyquinolin-2-one, quinazoline-2,4(1 H,3 H)-diones, benzimidazolones, α-alkylidene cyclic carbonate.

  10. Incorporating global components into ethics education.

    PubMed

    Wang, George; Thompson, Russell G

    2013-03-01

    Ethics is central to science and engineering. Young engineers need to be grounded in how corporate social responsibility principles can be applied to engineering organizations to better serve the broader community. This is crucial in times of climate change and ecological challenges where the vulnerable can be impacted by engineering activities. Taking a global perspective in ethics education will help ensure that scientists and engineers can make a more substantial contribution to development throughout the world. This paper presents the importance of incorporating the global and cross culture components in the ethic education. The authors bring up a question to educators on ethics education in science and engineering in the globalized world, and its importance, necessity, and impendency. The paper presents several methods for discussion that can be used to identify the differences in ethics standards and practices in different countries; enhance the student's knowledge of ethics in a global arena.

  11. SkinSensDB: a curated database for skin sensitization assays.

    PubMed

    Wang, Chia-Chi; Lin, Ying-Chi; Wang, Shan-Shan; Shih, Chieh; Lin, Yi-Hui; Tung, Chun-Wei

    2017-01-01

    Skin sensitization is an important toxicological endpoint for chemical hazard determination and safety assessment. Prediction of chemical skin sensitizer had traditionally relied on data from rodent models. The development of the adverse outcome pathway (AOP) and associated alternative in vitro assays have reshaped the assessment of skin sensitizers. The integration of multiple assays as key events in the AOP has been shown to have improved prediction performance. Current computational models to predict skin sensitization mainly based on in vivo assays without incorporating alternative in vitro assays. However, there are few freely available databases integrating both the in vivo and the in vitro skin sensitization assays for development of AOP-based skin sensitization prediction models. To facilitate the development of AOP-based prediction models, a skin sensitization database named SkinSensDB has been constructed by curating data from published AOP-related assays. In addition to providing datasets for developing computational models, SkinSensDB is equipped with browsing and search tools which enable the assessment of new compounds for their skin sensitization potentials based on data from structurally similar compounds. SkinSensDB is publicly available at http://cwtung.kmu.edu.tw/skinsensdb.

  12. The evolution of chemotaxis assays from static models to physiologically relevant platforms.

    PubMed

    Toetsch, Stephanie; Olwell, Peter; Prina-Mello, Adriele; Volkov, Yuri

    2009-02-01

    The role of chemotactic gradients in the immunological response is an area which elicits a lot of attention due to its impact on the outcome of the inflammatory process. Consequently there are numerous standard in vitro designs which attempt to mimic chemotactic gradients, albeit in static conditions, and with no control over the concentration of the chemokine gradient. In recent times the design of the standard chemotaxis assay has incorporated modern microfluidic platforms, computer controlled flow devices and cell tracking software. Assays under fluid flow which use biochips have provided data which highlight the importance of shear stress on cell attachment and migration towards a chemokine gradient. However, the in vivo environment is far more complex in comparison to conventional cell assay chambers. The designs of biochips are therefore in constant flux as advances in technology permit ever greater imitations of in vivo conditions. Researchers are focused on designing a generation of new biochips and enhancing the physiological relevance of the current assays. The challenge is to combine a shear flow with a 3D scaffold containing the endothelial layer and permitting a natural diffusion of chemokines through a tissue-like basal matrix. Here we review the latest range of chemotaxis assays and assess the innovative features of their designs which enable them to better imitate the in vivo environment. We also present some alternative designs that were initially employed in tissue engineering which could potentially be used in the establishment of novel chemotaxis assays.

  13. Incorporation of RAM techniques into simulation modeling

    SciTech Connect

    Nelson, S.C. Jr.; Haire, M.J.; Schryver, J.C.

    1995-07-01

    This work concludes that reliability, availability, and maintainability (RAM) analytical techniques can be incorporated into computer network simulation modeling to yield an important new analytical tool. This paper describes the incorporation of failure and repair information into network simulation to build a stochastic computer model represents the RAM Performance of two vehicles being developed for the US Army: The Advanced Field Artillery System (AFAS) and the Future Armored Resupply Vehicle (FARV). The AFAS is the US Army`s next generation self-propelled cannon artillery system. The FARV is a resupply vehicle for the AFAS. Both vehicles utilize automation technologies to improve the operational performance of the vehicles and reduce manpower. The network simulation model used in this work is task based. The model programmed in this application requirements a typical battle mission and the failures and repairs that occur during that battle. Each task that the FARV performs--upload, travel to the AFAS, refuel, perform tactical/survivability moves, return to logistic resupply, etc.--is modeled. Such a model reproduces a model reproduces operational phenomena (e.g., failures and repairs) that are likely to occur in actual performance. Simulation tasks are modeled as discrete chronological steps; after the completion of each task decisions are programmed that determine the next path to be followed. The result is a complex logic diagram or network. The network simulation model is developed within a hierarchy of vehicle systems, subsystems, and equipment and includes failure management subnetworks. RAM information and other performance measures are collected which have impact on design requirements. Design changes are evaluated through ``what if`` questions, sensitivity studies, and battle scenario changes.

  14. Methods for threshold determination in multiplexed assays

    DOEpatents

    Tammero, Lance F. Bentley; Dzenitis, John M; Hindson, Benjamin J

    2014-06-24

    Methods for determination of threshold values of signatures comprised in an assay are described. Each signature enables detection of a target. The methods determine a probability density function of negative samples and a corresponding false positive rate curve. A false positive criterion is established and a threshold for that signature is determined as a point at which the false positive rate curve intersects the false positive criterion. A method for quantitative analysis and interpretation of assay results together with a method for determination of a desired limit of detection of a signature in an assay are also described.

  15. Nondestructive assay confirmatory assessment experiments: mixed oxide

    SciTech Connect

    Lemming, J.F.

    1980-04-30

    The confirmatory assessment experiments demonstrate traceable nondestructive assay (NDA) measurements of plutonium in mixed oxide powder using commercially available spontaneous-fission assay systems. The experiments illustrate two major concepts: the production of calibration materials using calorimetric assay, and the use of paired measurements for measurement assurance. Two batches of well-characterized mixed oxide powder were used to establish the random and systematic error components. The major components of an NDA measurement assurance technique to establish and maintain traceability are identified and their functions are demonstrated. 20 refs., 10 figs., 10 tabs.

  16. Assuring reliable performance of antibiotic assay media.

    PubMed

    Freeman, K A; Johnson, D P; Garth, M A

    1977-11-01

    The Microbiological Assay Branch of the National Center for Antibiotics Analysis assays over 100,000 samples of antibiotic products annually, using more than 1000 Ib dehydrated media. The media must be consistently dependable to produce accurate, reliable test results. To assure that the supply of media will meet the established requirements, each lot before purchase is subjected to a series of trials designed to examine growth support, sensitivity, and behavioral and physical factors. Actual antibiotic assays are conducted with the test medium, and performance is rated against a control medium. Controls on the system reduce the variables to allow appraisal of the medium itself.

  17. Phytosphingosine stimulates the differentiation of human keratinocytes and inhibits TPA-induced inflammatory epidermal hyperplasia in hairless mouse skin.

    PubMed

    Kim, Sujong; Hong, Il; Hwang, Jung Sun; Choi, Jin Kyu; Rho, Ho Sik; Kim, Duck Hee; Chang, Ihseop; Lee, Seung Hun; Lee, Mi-Ock; Hwang, Jae Sung

    2006-01-01

    The binding of sphingoid bases to peroxisome proliferator-activated receptor (PPAR) has been detected in a solid-phase binding assay. However, sphingoid base-induced changes in PPAR transactivation activity have not been examined. In this report, we show by reporter gene analyses that phytosphingosine (PS), a natural sphingoid base, activates the transcriptional activity of PPARs in the immortalized human keratinocyte, HaCaT. Real-time PCR analyses showed that the mRNA level of PPARgamma was increased after PS treatment in HaCaT cells in a dose- and time-dependent manner. Because PPARs play important roles in skin barrier homeostasis by regulating epidermal cell growth, terminal differentiation, and inflammatory response, we examined the effect of PS on normal human epidermal keratinocytes (NHEKs) and mouse skin. PS increased the production of cornified envelope in NHEKs by approximately 1.8-fold compared with controls. Epidermal differentiation marker proteins such as involucrin, loricrin, and keratin1 were also increased in PS-treated NHEKs, by ELISA or Western blotting analysis. A [(3)H]thymidine incorporation assay showed that PS inhibited DNA synthesis in NHEKs to 20% compared with controls. The antiproliferative and anti-inflammatory effects of PS were examined in a mouse model of irritant contact dermatitis produced by topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA). PS blocked epidermal thickening and edema and the infiltration of inflammatory cells into the dermis in the skin of TPA-treated hairless mice. The anti-inflammatory effects of PS were confirmed by the observation that PS blocked the TPA-induced generation of prostaglandin E(2) in peripheral mononuclear leukocytes. Taken together, our results provide an insight into the multiple regulatory roles of PS in epidermal homeostasis, and furthermore point to the potential use of PS as a therapeutic agent in the treatment of inflammatory and proliferative cutaneous diseases.

  18. Phytosphingosine Stimulates the Differentiation of Human Keratinocytes and Inhibits TPA-Induced Inflammatory Epidermal Hyperplasia in Hairless Mouse Skin

    PubMed Central

    Kim, Sujong; Hong, Il; Hwang, Jung Sun; Choi, Jin Kyu; Rho, Ho Sik; Kim, Duck Hee; Chang, Ihseop; Lee, Seung Hun; Lee, Mi-Ock; Hwang, Jae Sung

    2006-01-01

    The binding of sphingoid bases to peroxisome proliferator-activated receptor (PPAR) has been detected in a solid-phase binding assay. However, sphingoid base–induced changes in PPAR transactivation activity have not been examined. In this report, we show by reporter gene analyses that phytosphingosine (PS), a natural sphingoid base, activates the transcriptional activity of PPARs in the immortalized human keratinocyte, HaCaT. Real-time PCR analyses showed that the mRNA level of PPARγ was increased after PS treatment in HaCaT cells in a dose- and time-dependent manner. Because PPARs play important roles in skin barrier homeostasis by regulating epidermal cell growth, terminal differentiation, and inflammatory response, we examined the effect of PS on normal human epidermal keratinocytes (NHEKs) and mouse skin. PS increased the production of cornified envelope in NHEKs by approximately 1.8-fold compared with controls. Epidermal differentiation marker proteins such as involucrin, loricrin, and keratin1 were also increased in PS-treated NHEKs, by ELISA or Western blotting analysis. A [3H]thymidine incorporation assay showed that PS inhibited DNA synthesis in NHEKs to 20% compared with controls. The antiproliferative and anti-inflammatory effects of PS were examined in a mouse model of irritant contact dermatitis produced by topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA). PS blocked epidermal thickening and edema and the infiltration of inflammatory cells into the dermis in the skin of TPA-treated hairless mice. The anti-inflammatory effects of PS were confirmed by the observation that PS blocked the TPA-induced generation of prostaglandin E2 in peripheral mononuclear leukocytes. Taken together, our results provide an insight into the multiple regulatory roles of PS in epidermal homeostasis, and furthermore point to the potential use of PS as a therapeutic agent in the treatment of inflammatory and proliferative cutaneous diseases. PMID:16838068

  19. Use of growth-hormone-releasing peptide-6 (GHRP-6) for the prevention of multiple organ failure.

    PubMed

    Cibrián, Danay; Ajamieh, Hussam; Berlanga, Jorge; León, Olga S; Alba, Jose S; Kim, Micheal J-T; Marchbank, Tania; Boyle, Joseph J; Freyre, Freya; Garcia Del Barco, Diana; Lopez-Saura, Pedro; Guillen, Gerardo; Ghosh, Subrata; Goodlad, Robert A; Playford, Raymond J

    2006-05-01

    Novel therapies for the treatment of MOF (multiple organ failure) are required. In the present study, we examined the effect of synthetic GHRP-6 (growth hormone-releasing peptide-6) on cell migration and proliferation using rat intestinal epithelial (IEC-6) and human colonic cancer (HT29) cells as in vitro models of injury. In addition, we examined its efficacy when given alone and in combination with the potent protective factor EGF (epidermal growth factor) in an in vivo model of MOF (using two hepatic vessel ischaemia/reperfusion protocols; 45 min of ischaemia and 45 min of reperfusion or 90 min of ischaemia and 120 min of reperfusion). In vitro studies showed that GHRP-6 directly influenced gut epithelial function as its addition caused a 3-fold increase in the rate of cell migration of IEC-6 and HT29 cells (P<0.01), but did not increase proliferation ([3H]thymidine incorporation). In vivo studies showed that, compared with baseline values, ischaemia/reperfusion caused marked hepatic and intestinal damage (histological scoring), neutrophilic infiltration (myeloperoxidase assay; 5-fold increase) and lipid peroxidation (malondialdehyde assay; 4-fold increase). Pre-treatment with GHRP-6 (120 microg/kg of body weight, intraperitoneally) alone truncated these effects by 50-85% (all P<0.05) and an additional benefit was seen when GHRP-6 was used in combination with EGF (1 mg/kg of body weight, intraperitoneally). Lung and renal injuries were also reduced by these pre-treatments. In conclusion, administration of GHRP-6, given alone or in combination with EGF to enhance its effects, may provide a novel simple approach for the prevention and treatment of MOF and other injuries of the gastrointestinal tract. In view of these findings, further studies appear justified.

  20. Rabbit renotropic system

    SciTech Connect

    Areas, J.; Yun, G.C.; Rahmat, J.; Gersten, D.; Goel, R.; Preuss, H.G.

    1988-04-01

    Elevated levels of a specific renal growth factor, renotropin, have been associated with spontaneous hypertension. To examine this association more closely, we have undertaken the development of a better assay system to characterize and purify renotropin. Sera from rabbits prior to operation (control) and at a specified time after unilateral nephrectomy (uni) were examined for renotropic activity. Comparing the effects of uni to control sera in the same rabbit, significant stimulation of 3H-thymidine incorporation into the DNA of primary rabbit kidney cultures incubated in D-valine medium to eliminate fibroblast growth was noted: at 3 days postoperatively 73% (n = 13), at 7 days 103% (n = 39), at 10 days 130% (n = 31), at 21 days 101% (n = 24), at 42 days 89% (n = 13). All values were at least P less than 0.01. The stimulatory properties were dose-dependent but reached a plateau at high serum concentrations. Comparing CPM/mg protein in uni/control in different concentrations of sera 7 days postoperatively, uni versus control were 67/44 at 5% v/v, 139/72 at 10% v/v, 261/161 at 20% v/v, and 243/136 at 40% v/v. The renotropic effect of uni sera remained after dialysis in incubation medium and after sera were heated in boiling water for 5 minutes. Renal extracts obtained from growing kidneys 7 days postnephrectomy augmented renotropic activity. Atrial natriuretic factor, ouabain, PGF2 alpha, PGE1, and cAMP did not possess renotropic activity. We conclude that the primary rabbit kidney culture assay for renotropin is highly sensitive and will be an important tool to comprehend the role of renotropin in the pathogenesis of hypertension.

  1. Novel substituted methylenedioxy lignan suppresses proliferation of cancer cells by inhibiting telomerase and activation of c-myc and caspases leading to apoptosis

    PubMed Central

    Giridharan, P; Somasundaram, S T; Perumal, K; Vishwakarma, R A; Karthikeyan, N P; Velmurugan, R; Balakrishnan, A

    2002-01-01

    Conventional solvent fractionation and bioactivity based target assays were used to identify a new anti-cancer molecule from Phyllanthus urinaria, a herbal medicinal plant used in South India. At each step of the purification process the different fractions that were isolated were tested for specific anti-proliferative activity by assays measuring the inhibition of [3H]thymidine incorporation, and trypan blue drug exclusion. The ethyl acetate fraction that contained the bioactivity was further purified and resolved by HPLC on a preparative column. The purity of each of the fractions and their bioactivity were checked. Fraction 3 demonstrated a single spot on TLC and showed maximum anti-proliferative activity. This fraction was further purified and the structure was defined as 7′-hydroxy-3′,4′,5,9,9′-pentamethoxy-3,4-methylene dioxy lignan using NMR and mass spectrometry analysis. The pure compound and the crude ethyl acetate fraction which showed anti-proliferative activities were examined for ability to target specific markers of apoptosis like bcl2, c-myc and caspases and for effects on telomerase. Four specific cancer cell lines HEp2, EL-1 monocytes, HeLa and MCP7 were used in this study. The results indicate that 7′-hydroxy-3′,4′,5,9,9′-pentamethoxy-3,4-methylene dioxy lignan was capable of inhibiting telomerase activity and also could inhibit bcl2 and activate caspase 3 and caspase 8 whose significance in the induction of apoptosis is well known. We believe that this compound could serve as a valuable chemotherapeutic drug after further evaluations. British Journal of Cancer (2002) 87, 98–105. doi:10.1038/sj.bjc.6600422 www.bjcancer.com © 2002 Cancer Research UK PMID:12085264

  2. Novel substituted methylenedioxy lignan suppresses proliferation of cancer cells by inhibiting telomerase and activation of c-myc and caspases leading to apoptosis.

    PubMed

    Giridharan, P; Somasundaram, S T; Perumal, K; Vishwakarma, R A; Karthikeyan, N P; Velmurugan, R; Balakrishnan, A

    2002-07-01

    Conventional solvent fractionation and bioactivity based target assays were used to identify a new anti-cancer molecule from Phyllanthus urinaria, a herbal medicinal plant used in South India. At each step of the purification process the different fractions that were isolated were tested for specific anti-proliferative activity by assays measuring the inhibition of [(3)H]thymidine incorporation, and trypan blue drug exclusion. The ethyl acetate fraction that contained the bioactivity was further purified and resolved by HPLC on a preparative column. The purity of each of the fractions and their bioactivity were checked. Fraction 3 demonstrated a single spot on TLC and showed maximum anti-proliferative activity. This fraction was further purified and the structure was defined as 7'-hydroxy-3',4',5,9,9'-pentamethoxy-3,4-methylene dioxy lignan using NMR and mass spectrometry analysis. The pure compound and the crude ethyl acetate fraction which showed anti-proliferative activities were examined for ability to target specific markers of apoptosis like bcl2, c-myc and caspases and for effects on telomerase. Four specific cancer cell lines HEp2, EL-1 monocytes, HeLa and MCP7 were used in this study. The results indicate that 7'-hydroxy-3',4',5,9,9'-pentamethoxy-3,4-methylene dioxy lignan was capable of inhibiting telomerase activity and also could inhibit bcl2 and activate caspase 3 and caspase 8 whose significance in the induction of apoptosis is well known. We believe that this compound could serve as a valuable chemotherapeutic drug after further evaluations.

  3. Endothelin-1 activation of ETB receptors leads to a reduced cellular proliferative rate and an increased cellular footprint

    SciTech Connect

    Wilson, Jamie L.; Taylor, Linda; Polgar, Peter

    2012-06-10

    Endothelin-1 (ET-1) is a vasoactive peptide which signals through two G-protein coupled receptors, endothelin receptor A (ETA) and B (ETB). We determined that ET-1 activation of its ETB receptor in stably cDNA transfected CHO cells leads to a 55% reduction in cell number by end-point cell counting and a 35% decrease in cell growth by a real-time cell-substrate impedance-based assay after 24 h of cell growth. When CHO ETB cells were synchronized in the late G1 cell cycle phase, ET-1 delayed their S phase progression compared to control by 30% as determined by [{sup 3}H]-thymidine incorporation. On the other hand, no such delay was observed during late G2/M to G1 transit when cells were treated with ET-1 after release from mitotic arrest. Using the cell-substrate impedance-based assay, we observed that ET-1 induces opposing morphological changes in CHO ETA and CHO ETB cells with ETB causing an increase in the cell footprint and ETA a decrease. Likewise, in pulmonary artery smooth muscle cells, which express both ETA and ETB receptors, ET-1 induces an ETA-dependent contraction and an ETB dependent dilation. These results are shedding light on a possible beneficial role for ETB in diseases involving ET-1 dysfunction such as pulmonary hypertension. -- Highlights: Black-Right-Pointing-Pointer ET- hinders cell proliferation in CHO cells transfected with ETB. Black-Right-Pointing-Pointer ET-1 also decreases the rate of DNA synthesis in CHO ETB cells. Black-Right-Pointing-Pointer JNK and PI3K appear to be involved in this reduction of DNA synthesis. Black-Right-Pointing-Pointer ETB activation in CHO ETB cells and hSMCs leads to dilatory morphological changes. Black-Right-Pointing-Pointer In CHO ETA and hSMCs, ETA activation leads to constrictive morphological changes.

  4. Over-expression of neuron-derived orphan receptor-1 (NOR-1) exacerbates neointimal hyperplasia after vascular injury.

    PubMed

    Rodríguez-Calvo, Ricardo; Guadall, Anna; Calvayrac, Olivier; Navarro, María A; Alonso, Judith; Ferrán, Beatriz; de Diego, Alicia; Muniesa, Pedro; Osada, Jesús; Rodríguez, Cristina; Martínez-González, José

    2013-05-15

    We have previously shown that NOR-1 (NR4A3) modulates the proliferation and survival of vascular cells in culture. However, in genetically modified animal models, somewhat conflicting results have been reported concerning the involvement of NOR-1 in neointimal formation after vascular injury. The aim of this study was to generate a transgenic mouse model over-expressing NOR-1 in smooth muscle cells (SMCs) and assess the consequence of a gain of function of this receptor on intimal hyperplasia after vascular injury. The transgene construct (SM22-NOR1) was prepared by ligating the full-length human NOR-1 cDNA (hNOR-1) and a mouse SM22α minimal promoter able to drive NOR-1 expression to SMC. Two founders were generated and two stable transgenic mouse lines (TgNOR-1) were established by backcrossing the transgene-carrying founders with C57BL/6J mice. Real-time PCR and immunohistochemistry confirmed that hNOR-1 was mainly targeted to vascular beds such as aorta and carotid arteries, and was similar in both transgenic lines. Vascular SMC from transgenic animals exhibit increased NOR-1 transcriptional activity (assessed by electrophoretic mobility shift assay and luciferase assays), increased mitogenic activity (determined by [(3)H]-thymidine incorporation; 1.58-fold induction, P < 0.001) and increased expression of embryonic smooth muscle myosin heavy chain (SMemb) than wild-type cells from control littermates. Using the carotid artery ligation model, we show that neointima formation was increased in transgenic versus wild-type mice (2.36-fold induction, P < 0.01). Our in vivo data support a role for NOR-1 in VSMC proliferation and vascular remodelling. This NOR-1 transgenic mouse could be a useful model to study fibroproliferative vascular diseases.

  5. Autocrine/paracrine proliferative effect of ovarian GH and IGF-I in chicken granulosa cell cultures.

    PubMed

    Ahumada-Solórzano, S Marisela; Martínez-Moreno, Carlos G; Carranza, Martha; Ávila-Mendoza, José; Luna-Acosta, José Luis; Harvey, Steve; Luna, Maricela; Arámburo, Carlos

    2016-08-01

    It is known that growth hormone (GH) and its receptor (GHR) are expressed in granulosa cells (GC) and thecal cells during the follicular development in the hen ovary, which suggests GH is involved in autocrine/paracrine actions in the female reproductive system. In this work, we show that the knockdown of local ovarian GH with a specific cGH siRNA in GC cultures significantly decreased both cGH mRNA expression and GH secretion to the media, and also reduced their proliferative rate. Thus, we analyzed the effect of ovarian GH and recombinant chicken GH (rcGH) on the proliferation of pre-hierarchical GCs in primary cultures. Incubation of GCs with either rcGH or conditioned media, containing predominantly a 15-kDa GH isoform, showed that both significantly increased proliferation as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, proliferating cell nuclear antigen (PCNA) quantification and ((3)H)-thymidine incorporation ((3)H-T) assays in a dose response fashion. Both, locally produced GH and rcGH also induced the phosphorylation of Erk1/2 in GC cultures. Furthermore, GH increased IGF-I synthesis and its release into the GC culture incubation media. These results suggest that GH may act through local IGF-I to induce GC proliferation, since IGF-I immunoneutralization completely abolished the GH-induced proliferative effect. These data suggest that GH and IGF-I may play a role as autocrine/paracrine regulators during the follicular development in the hen ovary at the pre-hierarchical stage.

  6. Cannabinoid receptor-independent cytotoxic effects of cannabinoids in human colorectal carcinoma cells: synergism with 5-fluorouracil.

    PubMed

    Gustafsson, Sofia B; Lindgren, Theres; Jonsson, Maria; Jacobsson, Stig O P

    2009-03-01

    Cannabinoids (CBs) have been found to exert antiproliferative effects upon a variety of cancer cells, including colorectal carcinoma cells. However, little is known about the signalling mechanisms behind the antitumoural effect in these cells, whether the effects are shared by endogenous lipids related to endocannabinoids, or whether such effects are synergistic with treatment paradigms currently used in the clinic. The aim of this preclinical study was to investigate the effect of synthetic and endogenous CBs and their related fatty acids on the viability of human colorectal carcinoma Caco-2 cells, and to determine whether CB effects are synergistic with those seen with the pyrimidine antagonist 5-fluorouracil (5-FU). The synthetic CB HU 210, the endogenous CB anandamide, the endogenous structural analogue of anandamide, N-arachidonoyl glycine (NAGly), as well as the related polyunsaturated fatty acids arachidonic acid and eicosapentaenoic acid showed antiproliferative and cytotoxic effects in the Caco-2 cells, as measured by using [(3)H]-thymidine incorporation assay, the CyQUANT proliferation assay and calcein-AM fluorescence. HU 210 was the most potent compound examined, followed by anandamide, whereas NAGly showed equal potency and efficacy as the polyunsaturated fatty acids. Furthermore, HU 210 and 5-FU produced synergistic effects in the Caco-2 cells, but not in the human colorectal carcinoma cell lines HCT116 or HT29. The compounds examined produced cytotoxic, rather than antiproliferative effects, by a mechanism not involving CB receptors, since the CB receptor antagonists AM251 and AM630 did not attenuate the effects, nor did pertussis toxin. However, alpha-tocopherol and the nitric oxide synthase inhibitor L-NAME attenuated the CB toxicity, suggesting involvement of oxidative stress. It is concluded that the CB system may provide new targets for the development of drugs to treat colorectal cancer.

  7. Effect of a bispidinone analog on mitochondria‑mediated apoptosis in HeLa cells.

    PubMed

    Yi, Myeongjin; Parthiban, Paramasivam; Hwang, Jiyoung; Zhang, Xin; Jeong, Hyunjin; Park, Dong Ho; Kim, Dong-Kyoo

    2014-01-01

    The present study was carried out to investigate the effect of 2,4,6,8-tetraaryl-3,7-diazabicyclo[3.3.1]nonan-9-one (bispidinone) analogs on the in vitro growth of human cervical carcinoma (HeLa) cells. A series of 11 bispidinone analogs was synthesized with substituents, e.g., fluoro/methyl/ethyl/isopropyl/thiomethyl/methoxy groups, at various positions. These compounds were synthesized to identify which substituent and position induced the strongest cytotoxic effect in cancer cells. Among these synthetics, analog 9, which contains methoxy groups, had the most significant cytotoxic effect on HeLa cells, and its IC50 value was less than 13 µM. A WST-8 assay also showed that analog 9 inhibited the proliferation of HeLa cells. By using DNA content analysis, we found that analog 9 induced sub-G1 and G1 phase arrest in a time-dependent manner. A [3H]-thymidine incorporation assay suggested that analog 9 inhibited DNA replication in HeLa cells. On performing light microscopy, morphological changes such as cellular shrinkage and disruption, which are apoptotic features, were observed in HeLa cells. Annexin V/propidium iodide double staining and rhodamine-123 staining showed that analog 9 induced apoptosis and disrupted the intracellular mitochondrial membrane potential in HeLa cells. The western blot analysis results suggested that analog 9 induced mitochondria-mediated apoptosis. In addition, we have shown that analog 9 may play a role in the Fas signaling apoptotic pathway.

  8. Effects of mebendazole on protein biosynthesis and secretion in human-derived fibroblast cultures.

    PubMed

    Soto, H; Massó, F; Cano, S; Díaz de León, L

    1996-07-26

    Previous results of our group revealed that mebendazole, a broad spectrum anthelmintic drug with antimicrotubular properties, used for the treatment of liver cirrhosis, decreased total collagen content and biosynthesis in liver upon treatment. In the present study, we have evaluated the effects of mebendazole (5-50 micrograms/mL) on protein synthesis, secretion, and deposition in human-derived fibroblast cultures. The results showed a decrease in cell viability (18.5 +/- 0.9%) at 50 micrograms/mL. [3H]Thymidine incorporation diminished gradually with increasing mebendazole concentrations, reaching a plateau (53.67%) between 30 and 50 micrograms/mL. In late logarithmic phase cultures, the drug caused a decrease of [3H]proline incorporation (43.10%) and collagen biosynthesis (58.61%) in the extracellular matrix. This correlated with an increase in radioactivity in total proteins (51.28%) of the intracellular fraction. Similar results were obtained when mebendazole was assayed in post-confluent fibroblast cultures. The electrophoretic patterns of the extracellular matrix showed a decrease of radioactive collagenous components (alpha chains and beta dimers). By contrast, in the intracellular fraction an increase of radioactive collagen precursors (pro alpha chains) was observed. Immunofluorescence studies and immunotransfer analysis, using polyclonal anti-type I collagen antibodies, revealed an accumulation of intracellular collagen which included: collagen pro alpha chains, alpha chains, and low molecular weight peptides. The results obtained suggest that mebendazole interferes with the transcellular mobilization of proteins, resulting in a decrease of secretion and deposition of extracellular matrix proteins, and an accumulation of intracellular collagenous components. The intracellular accumulation of newly synthesized proteins could cause a feedback regulation in fibroblast cultures.

  9. Evidence for ovarian tumor necrosis factor

    SciTech Connect

    Roby, K.F.

    1989-01-01

    Ovarian folliculogenesis and luteal formation occur concomitantly with the development of new blood vessels that function in nutritional support of the developing follicles. As follicles undergo atresia and the corpus luteum regresses, blood vessels supplying these tissues degenerate. The first study determined if the ovary contained factors that might regulate ovarian angiogenesis. The bovine ovary was subjected to ammonium sulfate (AS) precipitation and the precipitates (ppt.) were assayed in vitro for effects on endothelial cell (CPAE) and fibroblast (3T3 and L929) incorporation of {sup 3}H-thymidine. Heparin sepharose (HS) chromatography of the 80% AS ppt. revealed the inhibitory activity on CPAE and L929 cells did not bind to HS but was found in the HS column breakthrough (80% BT). Sizing chromatography of the 80% BT indicated thymidine incorporation inhibitory activity exhibited a molecular weight of 30,000-50,000 Daltons. TNF was immunohistochemically localized in the human, bovine and rat ovary. Frozen sections were incubated with polyclonal antibody to human recombinant TNF. Antigen-antibody binding was visualized using a Biotin-StreptAvidin peroxidase technique. Immunoreactive TNF (I-TNF) was localized in corpora lutea and the more antral layers of granulosa cells in antral follicles. Incubation of sections with anti-TNF in the presence of excess TNF resulted in lose of immunostaining. Cell blotting and ELISA further indicated I-TNF was present in granulosa cells. In order to determine whether TNF had an effect on follicular steroidogenesis, preovulatory follicles from cyclic proestrus rats were incubated in vitro for up to 24 hours with various doses of human recombinant TNF. Stepwise increases in progesterone (P) accumulation in the incubation media were observed with 30-300 pM TNF.

  10. Agar plate freezing assay for the in situ selection of transformed ice nucleating bacteria.

    PubMed

    Anastassopoulos, Elias

    2006-10-01

    An agar plate freezing assay is described based on the incorporation of fluorescein dye in agar medium. Upon addition of fluorescein the medium becomes transparent. This facilitates the monitoring of the ice nucleation event in vivo and the subsequent in situ selection of transformed ice nucleating bacteria. In comparison with known assays for the screening of transformants, the proposed assay is very accurate and reproducible. It may be applied in environmental samples screening for ice nucleating organisms, or in cDNA or genomic libraries for identifying novel ice nucleation genes. It may also prove useful in comparative studies of the ice nucleation activity, e.g. in directed evolution experiments involving ice nucleation genes.

  11. Aptamer capturing of enzymes on magnetic beads to enhance assay specificity and sensitivity.

    PubMed

    Zhao, Qiang; Li, Xing-Fang; Le, X Chris

    2011-12-15

    Activity and specificity of enzyme molecules are important to enzymatic reactions and enzyme assays. We describe an aptamer capturing approach that improves the specificity and the sensitivity of enzyme detection. An aptamer recognizing the target enzyme molecule is conjugated on a magnetic bead, increasing the local concentration, and serves as an affinity probe to capture and separate minute amounts of the enzyme. The captured enzymes catalyze the subsequent conversion of fluorogenic substrate to fluorescent products, enabling a sensitive measure of the active enzyme. The feasibility of this technique is demonstrated through assays for human alpha thrombin and human neutrophil elastase (HNE), two important enzymes. Thrombin (2 fM) and 100 fM HNE can be detected. The incorporation of two binding events, substrate recognition and aptamer binding, greatly improves assay specificity. With its simplicity, this approach is applicable to biosensing and detection of disease biomarkers.

  12. 27 CFR 21.6 - Incorporations by reference.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Analytical Chemists (13th Edition 1980)” (AOAC) is incorporated by reference in this part. This incorporation... the Association of Official Analytical Chemists, 11 North 19th Street, Suite 210, Arlington,...

  13. 27 CFR 21.6 - Incorporations by reference.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... Analytical Chemists (13th Edition 1980)” (AOAC) is incorporated by reference in this part. This incorporation... the Association of Official Analytical Chemists, 11 North 19th Street, Suite 210, Arlington,...

  14. 27 CFR 21.6 - Incorporations by reference.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Analytical Chemists (13th Edition 1980)” (AOAC) is incorporated by reference in this part. This incorporation... the Association of Official Analytical Chemists, 11 North 19th Street, Suite 210, Arlington,...

  15. 27 CFR 21.6 - Incorporations by reference.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... Analytical Chemists (13th Edition 1980)” (AOAC) is incorporated by reference in this part. This incorporation... the Association of Official Analytical Chemists, 11 North 19th Street, Suite 210, Arlington,...

  16. 27 CFR 21.6 - Incorporations by reference.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... Analytical Chemists (13th Edition 1980)” (AOAC) is incorporated by reference in this part. This incorporation... the Association of Official Analytical Chemists, 11 North 19th Street, Suite 210, Arlington,...

  17. Incorporation of pollution prevention principles in environmental methods

    SciTech Connect

    Erickson, M.D.; Alvarado, J.S.; Lu, C.-S.; Peterson, D.P.; Silzer, J.

    1996-07-01

    The principles of pollution prevention (P2) have not been sufficiently incorporated into analytical methods. In this paper, we focus on the needs for and the benefits of incorporating the principles of P2 into environmental analytical methods.

  18. Evidence for preadipocyte proliferation during culture of subcutaneous and intramuscular adipose tissues from Angus and Wagyu crossbred steers.

    PubMed

    May, S G; Savell, J W; Lunt, D K; Wilson, J J; Laurenz, J C; Smith, S B

    1994-12-01

    The primary objective of this study was to provide evidence for preadipocyte proliferation during culture of adipose tissue explants; a secondary objective was to compare the lipogenic activity and cellularity of adipose tissues from American Wagyu crossbred steers. Subcutaneous (s.c.) and intramuscular (i.m.) adipose tissues were obtained at slaughter from the 2nd to 6th lumbar region of the loin from Angus (n = 10) and Wagyu crossbred steers (n = 10) that had been fed for 552 d by typical Japanese production standards. Adipose tissue explants were incubated 36 h with [3H]thymidine in the absence and presence of aphidicolin (a specific inhibitor of genomic DNA replication). Adipocytes were liberated by collagenase treatment and [3H]thymidine incorporation into DNA was measured. Whereas there were no significant differences between adipose tissue depots, Wagyu s.c. and i.m. preadipocytes and stromal-vascular cells exhibited greater (P < .05) [3H]thymidine incorporation into DNA than adipocytes from Angus steers. Intramuscular adipose tissue from both breeds exhibited lower (P < .05) rates of lipogenesis from acetate both before and after long-term (36-h) incubation than s.c. adipose tissue. Furthermore, i.m. adipocytes were smaller (P < .05) than s.c. adipocytes. The activities of fatty acid synthetase and glucose-6-phosphate dehydrogenase were greater (P < .05) in Wagyu s.c. adipose tissue and less in Wagyu i.m. adipose tissue than in corresponding Angus tissues. There were no differences between breed types (P = .17) in rates of lipogenesis from acetate, either before or after explant culture.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Estradiol receptor binding to the epithelium of uterine lumen and glands: region- and time-related changes during preimplantation and periimplantation periods studied by autoradiography.

    PubMed

    Zorn, Telma M T; Soto-Suazo, Mauricio; Pellegrini, Cleusa R; Oliveira, José G; Stumpf, Walter E

    2003-07-01

    The presence and changes of estradiol nuclear binding and related functions in uterine luminal and glandular epithelium were studied before and after blastocyst implantation using receptor autoradiography with (3)H-estradiol-17beta in association with (3)H-thymidine incorporation and immunocytochemical binding of antibody to estrogen receptor ER-alpha. (3)H-estradiol nuclear binding is present but variable during days 1.5-7.5 of pregnancy. Sites of strong nuclear binding of (3)H-estradiol exhibit strong immunocytochemical staining with ER-alpha antibody. Qualitative and quantitative evaluation of autoradiograms reveal that there is a general increase of nuclear (3)H-estradiol binding during the first 3 days after fertilization in both luminal and glandular epithelium. The binding of estradiol is stronger in glandular epithelium from day 2.5 to day 7.5, paralleled by a rise in (3)H-thymidine incorporation on day 2.5. By comparison, in the epithelium of the uterine lumen (3)H-estradiol nuclear binding is low, but relatively high in epithelial cells at lateral branching of the lumen where the increase in (3)H-estradiol binding corresponds to an increased labeling index with (3)H-thymidine. A highly differentiated binding of (3)H-estradiol to luminal and glandular epithelium was demonstrated with region- and time-specific changes of related effects on cell proliferation, differentiation, and secretion, probably involving involution and remodeling. The strong (3)H-estradiol binding to glandular epithelium suggests that estradiol exerts pronounced effects on glandular activities in the periimplantation period.

  20. Cooperative cell invasion: matrix metalloproteinase–mediated incorporation between cells

    PubMed Central

    Mitchell, Camilla B.; O’Neill, Geraldine M.

    2016-01-01

    Progression to metastatic disease is a leading cause of cancer death. Tumors are a complex mixture of cell types, both genetically heterogeneous malignant cells and associated nonmalignant cells. Models mimicking this heterogeneous cell environment have revealed that invasive cell populations can induce dissemination by otherwise poorly/noninvasive tumor cells, known as cooperative invasion. Neuroblastoma tumors arise in children and are characterized by mixed cellular populations in vivo, consisting chiefly of neuronal (N)-type and substrate (S)-type cells. The S-type cells have all the hallmarks of invasive leader cell populations and have been coisolated with N-type cells from metastatic bone lesions, but to date their ability to induce cooperative invasion has not been investigated. Therefore, in the present study, we analyzed the invasive behavior of mixed N-type and S-type multicellular spheroids embedded in three-dimensional collagen gels. Our analyses show that S-type cells induce invasion of either single cells or small cell clusters of N-type cells. In contrast to other reports of cooperative invasion in which mixed cultures exhibit a follow-the-leader mechanism, we show coincident emergence of S- and N-type cells from mixed spheroids. Our data suggest mutual effects between the two cell types. Thus, whereas coculture with S-type cells induces N-type invasion, coculture with N-type cells slows S-type invasion. Using matrix metalloproteinase (MMP) inhibitors and cell incorporation assays, we demonstrate that MMP activity is required for S-type cells to insert into layers of N-type cells. Our study therefore highlights an important role for S-type neuroblastoma cells in the invasion process and reveals a new mechanism of cooperative invasion. PMID:27605703

  1. Human somatic mutation assays as biomarkers of carcinogenesis

    SciTech Connect

    Compton, P.J.E.; Smith, M.T. ); Hooper, K. )

    1991-08-01

    This paper describes four assays that detect somatic gene mutations in humans: the hypoxanthine-guanine phosphoribosyl transferase assay, the glycophorin A assay, the HLA-A assay, and the sickle cell hemoglobin assay. Somatic gene mutations can be considered a biomarker of carcinogenesis, and assays for somatic mutation may assist epidemiologists in studies that attempt to identify factors associated with increased risks of cancer. Practical aspects of the use of these assays are discussed.

  2. FACS binding assay for analysing GDNF interactions.

    PubMed

    Quintino, Luís; Baudet, Aurélie; Larsson, Jonas; Lundberg, Cecilia

    2013-08-15

    Glial cell-line derived neurotrophic factor (GDNF) is a secreted protein with great therapeutic potential. However, in order to analyse the interactions between GDNF and its receptors, researchers have been mostly dependent of radioactive binding assays. We developed a FACS-based binding assay for GDNF as an alternative to current methods. We demonstrated that the FACS-based assay using TGW cells allowed readily detection of GDNF binding and displacement to endogenous receptors. The dissociation constant and half maximal inhibitory concentration obtained were comparable to other studies using standard binding assays. Overall, this FACS-based, simple to perform and adaptable to high throughput setup, provides a safer and reliable alternative to radioactive methods.

  3. Linearization of the bradford protein assay.

    PubMed

    Ernst, Orna; Zor, Tsaffrir

    2010-04-12

    Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. This most common assay enables rapid and simple protein quantification in cell lysates, cellular fractions, or recombinant protein samples, for the purpose of normalization of biochemical measurements. However, an intrinsic nonlinearity compromises the sensitivity and accuracy of this method. It is shown that under standard assay conditions, the ratio of the absorbance measurements at 590 nm and 450 nm is strictly linear with protein concentration. This simple procedure increases the accuracy and improves the sensitivity of the assay about 10-fold, permitting quantification down to 50 ng of bovine serum albumin. Furthermore, the interference commonly introduced by detergents that are used to create the cell lysates is greatly reduced by the new protocol. A linear equation developed on the basis of mass action and Beer's law perfectly fits the experimental data.

  4. LINE-1 Cultured Cell Retrotransposition Assay.

    PubMed

    Kopera, Huira C; Larson, Peter A; Moldovan, John B; Richardson, Sandra R; Liu, Ying; Moran, John V

    2016-01-01

    The Long INterspersed Element-1 (LINE-1 or L1) retrotransposition assay has facilitated the discovery and characterization of active (i.e., retrotransposition-competent) LINE-1 sequences from mammalian genomes. In this assay, an engineered LINE-1 containing a retrotransposition reporter cassette is transiently transfected into a cultured cell line. Expression of the reporter cassette, which occurs only after a successful round of retrotransposition, allows the detection and quantification of the LINE-1 retrotransposition efficiency. This assay has yielded insight into the mechanism of LINE-1 retrotransposition. It also has provided a greater understanding of how the cell regulates LINE-1 retrotransposition and how LINE-1 retrotransposition impacts the structure of mammalian genomes. Below, we provide a brief introduction to LINE-1 biology and then detail how the LINE-1 retrotransposition assay is performed in cultured mammalian cells.

  5. BIOMARKER ASSAYS IN NIPPLE APIRATE FLUID

    EPA Science Inventory

    ABSTRACT

    The noninvasive technique of nipple aspiration as a potential source of biomarkers of breast cancer risk was evaluated. The feasibility of performing mutagenesis assays, amplifying DNA and performing protein electrophoresis on nipple aspirate fluid was explored. ...

  6. Developmental Toxicity Assays Using the Drosophila Model

    PubMed Central

    Rand, Matthew D.; Montgomery, Sara L.; Prince, Lisa; Vorojeikina, Daria

    2014-01-01

    The fruit fly (Drosophila melanogaster) has long been a premier model for developmental biologists and geneticists. The utility of Drosophila for toxicology studies has only recently gained broader recognition as a tool to elaborate molecular genetic mechanisms of toxic substances. In this article two practical applications of Drosophila for developmental toxicity assays are described. The first assay takes advantage of newly developed methods to render the fly embryo accessible to small molecules, toxicants and drugs. The second assay engages straightforward exposures to developing larvae and easy to score outcomes of adult development. With the extensive collections of flies that are publicly available and the ease with which to create transgenic flies, these two assays have a unique power for identifying and characterizing molecular mechanisms and cellular pathways specific to the mode of action of a number of toxicants and drugs. PMID:24789363

  7. 49 CFR 384.107 - Matter incorporated by reference.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 5 2014-10-01 2014-10-01 false Matter incorporated by reference. 384.107 Section... COMPLIANCE WITH COMMERCIAL DRIVER'S LICENSE PROGRAM General § 384.107 Matter incorporated by reference. (a) Incorporation by reference. This part includes references to certain matter or materials. The text of...

  8. 49 CFR 385.4 - Matter incorporated by reference.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 5 2012-10-01 2012-10-01 false Matter incorporated by reference. 385.4 Section 385.4 Transportation Other Regulations Relating to Transportation (Continued) FEDERAL MOTOR CARRIER... FITNESS PROCEDURES General § 385.4 Matter incorporated by reference. (a) Incorporation by...

  9. 49 CFR 384.107 - Matter incorporated by reference.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 5 2010-10-01 2010-10-01 false Matter incorporated by reference. 384.107 Section... COMPLIANCE WITH COMMERCIAL DRIVER'S LICENSE PROGRAM General § 384.107 Matter incorporated by reference. (a) Incorporation by reference. This part includes references to certain matter or materials. The text of...

  10. 49 CFR 385.4 - Matter incorporated by reference.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 5 2014-10-01 2014-10-01 false Matter incorporated by reference. 385.4 Section 385.4 Transportation Other Regulations Relating to Transportation (Continued) FEDERAL MOTOR CARRIER... FITNESS PROCEDURES General § 385.4 Matter incorporated by reference. (a) Incorporation by...

  11. 49 CFR 385.4 - Matter incorporated by reference.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 5 2011-10-01 2011-10-01 false Matter incorporated by reference. 385.4 Section... FITNESS PROCEDURES General § 385.4 Matter incorporated by reference. (a) Incorporation by reference. Part 385 includes references to certain matter or materials, as listed in paragraph (b) of this...

  12. 49 CFR 384.107 - Matter incorporated by reference.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 5 2011-10-01 2011-10-01 false Matter incorporated by reference. 384.107 Section... COMPLIANCE WITH COMMERCIAL DRIVER'S LICENSE PROGRAM General § 384.107 Matter incorporated by reference. (a) Incorporation by reference. This part includes references to certain matter or materials. The text of...

  13. 49 CFR 384.107 - Matter incorporated by reference.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 5 2012-10-01 2012-10-01 false Matter incorporated by reference. 384.107 Section... COMPLIANCE WITH COMMERCIAL DRIVER'S LICENSE PROGRAM General § 384.107 Matter incorporated by reference. (a) Incorporation by reference. This part includes references to certain matter or materials. The text of...

  14. 49 CFR 385.4 - Matter incorporated by reference.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 5 2010-10-01 2010-10-01 false Matter incorporated by reference. 385.4 Section... FITNESS PROCEDURES General § 385.4 Matter incorporated by reference. (a) Incorporation by reference. Part 385 includes references to certain matter or materials, as listed in paragraph (b) of this...

  15. 14 CFR 25.5 - Incorporations by reference.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... AIRWORTHINESS STANDARDS: TRANSPORT CATEGORY AIRPLANES General § 25.5 Incorporations by reference. (a) The materials listed in this section are incorporated by reference in the corresponding sections noted. These...(a) and 1 CFR part 51. These materials are incorporated as they exist on the date of the...

  16. 14 CFR 25.5 - Incorporations by reference.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AIRWORTHINESS STANDARDS: TRANSPORT CATEGORY AIRPLANES General § 25.5 Incorporations by reference. (a) The materials listed in this section are incorporated by reference in the corresponding sections noted. These...(a) and 1 CFR part 51. These materials are incorporated as they exist on the date of the...

  17. 14 CFR 25.5 - Incorporations by reference.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AIRWORTHINESS STANDARDS: TRANSPORT CATEGORY AIRPLANES General § 25.5 Incorporations by reference. (a) The materials listed in this section are incorporated by reference in the corresponding sections noted. These...(a) and 1 CFR part 51. These materials are incorporated as they exist on the date of the...

  18. 14 CFR 25.5 - Incorporations by reference.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... AIRWORTHINESS STANDARDS: TRANSPORT CATEGORY AIRPLANES General § 25.5 Incorporations by reference. (a) The materials listed in this section are incorporated by reference in the corresponding sections noted. These...(a) and 1 CFR part 51. These materials are incorporated as they exist on the date of the...

  19. 14 CFR 25.5 - Incorporations by reference.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AIRWORTHINESS STANDARDS: TRANSPORT CATEGORY AIRPLANES General § 25.5 Incorporations by reference. (a) The materials listed in this section are incorporated by reference in the corresponding sections noted. These...(a) and 1 CFR part 51. These materials are incorporated as they exist on the date of the...

  20. 46 CFR 107.115 - Incorporation by reference.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Incorporation by reference. 107.115 Section 107.115 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) A-MOBILE OFFSHORE DRILLING UNITS INSPECTION AND CERTIFICATION General § 107.115 Incorporation by reference. (a) The standards referred to in this subchapter are incorporated by...

  1. 40 CFR 282.2 - Incorporation by reference.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    .... (a) Material listed as incorporated by reference in part 282 was approved for incorporation by.... Material is incorporated as it exists on the date of the approval, and notice of any change in the material... Courtland St., NE, Atlanta, GA 30365. (5) Region 5 (Illinois, Indiana, Michigan, Minnesota, Ohio,...

  2. 43 CFR 34.7 - Incorporation by operation of law.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 43 Public Lands: Interior 1 2012-10-01 2011-10-01 true Incorporation by operation of law. 34.7... OPPORTUNITY DURING CONSTRUCTION AND OPERATION OF THE ALASKA NATURAL GAS TRANSPORTATION SYSTEM § 34.7 Incorporation by operation of law. (a) The Equal Opportunity Clause shall be deemed incorporated into...

  3. 43 CFR 34.7 - Incorporation by operation of law.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 43 Public Lands: Interior 1 2013-10-01 2013-10-01 false Incorporation by operation of law. 34.7... OPPORTUNITY DURING CONSTRUCTION AND OPERATION OF THE ALASKA NATURAL GAS TRANSPORTATION SYSTEM § 34.7 Incorporation by operation of law. (a) The Equal Opportunity Clause shall be deemed incorporated into...

  4. 43 CFR 34.7 - Incorporation by operation of law.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 43 Public Lands: Interior 1 2014-10-01 2014-10-01 false Incorporation by operation of law. 34.7... OPPORTUNITY DURING CONSTRUCTION AND OPERATION OF THE ALASKA NATURAL GAS TRANSPORTATION SYSTEM § 34.7 Incorporation by operation of law. (a) The Equal Opportunity Clause shall be deemed incorporated into...

  5. 48 CFR 1952.102-2 - Incorporation in full text.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 6 2012-10-01 2012-10-01 false Incorporation in full text... Clauses 1952.102-2 Incorporation in full text. All IAAR provisions and clauses shall be incorporated in solicitations and/or contracts in full text....

  6. 48 CFR 2852.102-270 - Incorporation in full text.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 6 2013-10-01 2013-10-01 false Incorporation in full text... 2852.102-270 Incorporation in full text. JAR provisions or clauses shall be incorporated in solicitations and contracts in full text....

  7. 48 CFR 1952.102-2 - Incorporation in full text.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 6 2013-10-01 2013-10-01 false Incorporation in full text... Clauses 1952.102-2 Incorporation in full text. All IAAR provisions and clauses shall be incorporated in solicitations and/or contracts in full text....

  8. 48 CFR 1952.102-2 - Incorporation in full text.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 6 2014-10-01 2014-10-01 false Incorporation in full text... Clauses 1952.102-2 Incorporation in full text. All IAAR provisions and clauses shall be incorporated in solicitations and/or contracts in full text....

  9. 48 CFR 2852.102-270 - Incorporation in full text.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 6 2011-10-01 2011-10-01 false Incorporation in full text... 2852.102-270 Incorporation in full text. JAR provisions or clauses shall be incorporated in solicitations and contracts in full text....

  10. 48 CFR 1952.102-2 - Incorporation in full text.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 6 2011-10-01 2011-10-01 false Incorporation in full text... Clauses 1952.102-2 Incorporation in full text. All IAAR provisions and clauses shall be incorporated in solicitations and/or contracts in full text....

  11. 48 CFR 2852.102-270 - Incorporation in full text.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 6 2014-10-01 2014-10-01 false Incorporation in full text... 2852.102-270 Incorporation in full text. JAR provisions or clauses shall be incorporated in solicitations and contracts in full text....

  12. 48 CFR 2852.102-270 - Incorporation in full text.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 6 2012-10-01 2012-10-01 false Incorporation in full text... 2852.102-270 Incorporation in full text. JAR provisions or clauses shall be incorporated in solicitations and contracts in full text....

  13. 46 CFR 160.077-5 - Incorporation by reference.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) EQUIPMENT, CONSTRUCTION, AND MATERIALS... Incorporation by reference. (a) Certain materials are incorporated by reference into this subpart with the..., “Material Approved for Incorporation by Reference,” which appears in the Finding Aids section of this...

  14. 46 CFR 109.105 - Incorporation by reference.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 4 2013-10-01 2013-10-01 false Incorporation by reference. 109.105 Section 109.105 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) A-MOBILE OFFSHORE DRILLING UNITS OPERATIONS General § 109.105 Incorporation by reference. (a) Certain material is incorporated by...

  15. 46 CFR 109.105 - Incorporation by reference.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 4 2014-10-01 2014-10-01 false Incorporation by reference. 109.105 Section 109.105 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) A-MOBILE OFFSHORE DRILLING UNITS OPERATIONS General § 109.105 Incorporation by reference. (a) Certain material is incorporated by...

  16. 46 CFR 109.105 - Incorporation by reference.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 4 2012-10-01 2012-10-01 false Incorporation by reference. 109.105 Section 109.105 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) A-MOBILE OFFSHORE DRILLING UNITS OPERATIONS General § 109.105 Incorporation by reference. (a) Certain material is incorporated by...

  17. 46 CFR 109.105 - Incorporation by reference.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 4 2011-10-01 2011-10-01 false Incorporation by reference. 109.105 Section 109.105 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) A-MOBILE OFFSHORE DRILLING UNITS OPERATIONS General § 109.105 Incorporation by reference. (a) Certain material is incorporated by...

  18. 46 CFR 109.105 - Incorporation by reference.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Incorporation by reference. 109.105 Section 109.105 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) A-MOBILE OFFSHORE DRILLING UNITS OPERATIONS General § 109.105 Incorporation by reference. (a) Certain material is incorporated by...

  19. 46 CFR 38.01-3 - Incorporation by reference.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 1 2012-10-01 2012-10-01 false Incorporation by reference. 38.01-3 Section 38.01-3....01-3 Incorporation by reference. (a) Certain standards and specifications are incorporated by... ASME Boiler and Pressure Vessel Code Section V, Nondestructive Examination (1986)...

  20. 48 CFR 1952.102-2 - Incorporation in full text.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 6 2010-10-01 2010-10-01 true Incorporation in full text... Clauses 1952.102-2 Incorporation in full text. All IAAR provisions and clauses shall be incorporated in solicitations and/or contracts in full text....