Science.gov

Sample records for 3h-thymidine incorporation assays

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

    SciTech Connect

    Winding, A. )

    1992-08-01

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

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

    SciTech Connect

    Jeffrey, W.H.

    1989-01-01

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

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

    SciTech Connect

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

    1988-09-01

    The conversion factor for the calculation of bacterial production from rates of ({sup 3}H)thymidine incorporation was examined with diluted batch cultures of freshwater bacteria. Natural bacterial assemblages were grown in aged, normal, and enriched media at 10 to 20{degree}C. The generation time during 101 growth cycles covered a range from 4 to >200 h. The average conversion factor was 2.15 {times} 10{sup 18} cells mol{sup {minus}1} of thymidine incorporated into the trichloroacetic acid (TCA) precipitate, when the generation time exceeded 20 h. At generation times of <20 h, the average conversion factor was 11.8 {times} 10{sup 18} cells mol{sup {minus}1} of thymidine incorporated into TCA precipitate. The amount of radioactivity in purified DNA increased with decreasing generation time and increasing conversion factor (calculated from the TCA precipitate), corresponding to a decrease in the percentage in protein. The conversion factors calculated from purified DNA or from the TCA precipitate gave the same variability. Conversion factors did not change significantly with the medium, but were significantly higher at 20{degree}C that at 15 and 10{degree}C. Results suggests that incorporation of ({sup 3}H)thymidine into DNA is probably limited by uptake during period with generation times of <20 h and that freshwater bacterioplankton cell production sometimes is underestimated when a conversion factor of 2.15 {times} 10{sup 18} cells mol{sup {minus}1} of thymidine incorporated is used.

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

    SciTech Connect

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

    1990-01-01

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

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

    SciTech Connect

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

    1987-08-01

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

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

    SciTech Connect

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

    1989-01-01

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

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

    SciTech Connect

    Bern, L.

    1985-01-01

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

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

    SciTech Connect

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

    1990-11-01

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

  9. 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.

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

    SciTech Connect

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

    1987-05-01

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

  11. 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.

  12. 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.

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

    PubMed Central

    Duque, Alvaro; Rakic, Pasko

    2011-01-01

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

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

    SciTech Connect

    Overbye, L.J.

    1989-01-01

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

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

    SciTech Connect

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

    1985-11-01

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

  16. 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).

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

    SciTech Connect

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

    1984-11-01

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

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

    PubMed

    Hellman, B

    1986-01-01

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

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

    PubMed

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

    2004-10-01

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

  20. 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.

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

    PubMed

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

    2009-01-01

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

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

    PubMed

    Martirosyan, Varsik; Baghdasaryan, Naira; Ayrapetyan, Sinerik

    2013-03-01

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

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

    PubMed

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

    1995-01-01

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

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

    SciTech Connect

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

    1984-01-01

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

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

    SciTech Connect

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

    1989-01-01

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

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

    PubMed

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

    1988-01-01

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

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

    SciTech Connect

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

    1983-03-01

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

  8. 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.

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

    SciTech Connect

    Newman, C.N.; Hagler, M.

    1986-04-01

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

  10. Labeling cells in microtiter plates for determination of [3H]thymidine uptake.

    PubMed

    Shevach, E M

    2001-05-01

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

  11. 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.

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

    SciTech Connect

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

    1990-04-01

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

  13. 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.

  14. Assays for in vitro monitoring of proliferation of human airway smooth muscle (ASM) and human pulmonary arterial vascular smooth muscle (VSM) cells.

    PubMed

    Goncharova, Elena A; Lim, Poay; Goncharov, Dmitry A; Eszterhas, Andrew; Panettieri, Reynold A; Krymskaya, Vera P

    2006-01-01

    Vascular and airway remodeling, which are characterized by airway smooth muscle (ASM) and pulmonary arterial vascular smooth muscle (VSM) proliferation, contribute to the pathology of asthma, pulmonary hypertension, restenosis and atherosclerosis. To evaluate the proliferation of VSM and ASM cells in response to mitogens, we perform a [3H]thymidine incorporation assay. The proliferation protocol takes approximately 48 h and includes stimulating cells synchronized in G0/G1 phase of the cell cycle with agonists, labeling cells with [3H]thymidine and examining levels of [3H]thymidine incorporation by scintillation counting. Although using radiolabeled [3H]thymidine incorporation is a limitation, the greatest benefit of the assay is providing reliable and statistically significant data. PMID:17406550

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

    PubMed

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

    2010-06-01

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

  16. 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.

  17. 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.

  18. 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.

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

    SciTech Connect

    Bell, R.T.

    1986-11-01

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

  20. 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.

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

    SciTech Connect

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

    1990-05-01

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

  2. 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.

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

    SciTech Connect

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

    1990-07-01

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

  4. 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.

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

    PubMed

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

    2014-12-15

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

  6. 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. PMID:24909732

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

    PubMed Central

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

    1992-01-01

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

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

    SciTech Connect

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

    1990-05-01

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

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

    PubMed Central

    Chrzanowski, Thomas H.

    1988-01-01

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

  10. MUTAGENICITY OF NITRODIBENZOPYANONES IN THE SALMONELLA PLATE-INCORPORATION AND MICROSUSPENSION ASSAYS

    EPA Science Inventory

    The mutagenicity of three isomers of nitro-6-H-dibenzo[b,d]pyran-6-one (NDBP), 3-NDBP and 4-NDBP, was characterized in the plate-incorporation (P1) and microsuspension (MS) assays using Salmonella typhimurium tester stains in the presence or absence of S9 mix. n both assays, all ...

  11. 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.

  12. A novel microculture kinetic assay (MiCK assay) for malignant cell growth and chemosensitivity.

    PubMed

    Kravtsov, V D

    1994-01-01

    The THERMOmax microplate reader was adapted for monitoring the growth kinetics of human leukaemic OCI/AML-2 and mouse tumour J-774.1 cell lines in continuous culture. Fluid evaporation from wells, CO2 escape and contamination were prevented by hermetic sealing of the microcultures in wells of a 96-well microplate, thus enabling the cells to grow exponentially for 72 h under the conditions of the incubated microplate reader. For both OCI/AML-2 cells, which grow in suspension, and adherent J-774.1 cells, a linear correlation was demonstrated between the number of unstained cells seeded in a given microplate well and the optical density (OD) of that well. Therefore, the OD/time curve of the culture could be deemed to be its growth curve. By the use of the linear fit equation, the actual number of the cells in the wells was computable at any time point of the assay. In the chemosensitivity test, an inhibitory effect of ARA-C on the growth of the cells could be estimated by viewing of the growth curves plotted on the screen. The maximum kinetic rates (Vmax) of the curves in the control and the ARA-C-treated wells were compared, yielding a growth inhibition index (GII). Comparison of results of the kinetic chemosensitivity assay with those of a [3H]thymidine incorporation assay revealed that the novel assay is suitable for precise quantitation of the cell chemosensitivity, is more informative and has the added technical advantage of performance without recourse to radioactive or chemically hazardous substances. PMID:7833120

  13. 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.

  14. An enzyme-linked immunosorbent assay for bromodeoxyuridine incorporation using fixed microcultures

    SciTech Connect

    Muir, D.; Varon, S.; Manthorpe, M. )

    1990-03-01

    We report a quantitative method by which a single microculture can be examined for cell morphology; cell number; DNA synthesis; and expression of cell antigens. This method first involves measuring by enzyme-linked immunosorbent assay (ELISA) the total bromodeoxyuridine (BrdU) incorporation into DNA by monolayer microcultures. The BrdU-ELISA measurement was followed by simultaneous immunostaining for BrdU-positive nuclei and for a cytoplasmic antigen. The method was applied to the measurement of mitogen-induced proliferation of rat sciatic nerve Schwann cell and cerebral astroglia microcultures. The ELISA measurement of BrdU incorporation compares favorably with measurements of tritiated thymidine incorporation and offers the additional advantages that the same microculture can subsequently be examined for cell number, for cell morphology, and for the percentage of cells having BrdU-labeled nuclei and other antigens.

  15. 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.

  16. B cell increases and ex vivo IL-2 production as secondary endpoints for the detection of sensitizers in non-radioisotopic local lymph node assay using flow cytometry.

    PubMed

    Jung, Kyoung-Mi; Jang, Won-Hee; Lee, Yong-Kyoung; Yum, Young Na; Sohn, Soojung; Kim, Bae-Hwan; Chung, Jin-Ho; Park, Young-Ho; Lim, Kyung-Min

    2012-03-25

    Non-radioisotopic local lymph node assay (LLNA) using 5-bromo-2'-deoxyuridine (BrdU) with flow cytometry (FCM) is gaining attention since it is free from the regulatory issues in traditional LLNA (tLLNA) accompanying in vivo uses of radioisotope, (3)H-thymidine. However, there is also concern over compromised performance of non-radioisotopic LLNA, raising needs for additional endpoints to improve the accuracy. With the full 22 reference substances enlisted in OECD Test Guideline No. 429, we evaluated the performance of LLNA:BrdU-FCM along with the concomitant measurements of B/T cell ratio and ex vivo cytokine production from isolated lymph node cells (LNCs) to examine the utility of these markers as secondary endpoints. Mice (Balb/c, female) were topically treated with substances on both ears for 3 days and then, BrdU was intraperitoneally injected on day 5. After a day, lymph nodes were isolated and undergone FCM to determine BrdU incorporation and B/T cell sub-typing with B220+ and CD3e+. Ex vivo cytokine production by LNCs was measured such as IL-2, IL-4, IL-6, IL-12, IFN-γ, MCP-1, GM-CSF and TNFα. Mice treated with sensitizers showed preferential increases in B cell population and the selective production of IL-2, which matched well with the increases in BrdU incorporation. When compared with guinea pig or human data, BrdU incorporation, B cell increase and IL-2 production ex vivo could successfully identify sensitizers with the accuracy comparable to tLLNA, suggesting that these markers may be useful for improving the accuracy of LLNA:BrdU-FCM or as stand-alone non-radioisotopic endpoints. PMID:22245253

  17. Incorporation of Slow Off-Rate Modified Aptamers Reagents in Single Molecule Array Assays for Cytokine Detection with Ultrahigh Sensitivity.

    PubMed

    Wu, Danlu; Katilius, Evaldas; Olivas, Edgar; Dumont Milutinovic, Milena; Walt, David R

    2016-09-01

    Slow off-rate modified aptamers (SOMAmers) are attractive protein recognition reagents due to their high binding affinities, stable chemical structures, easy production, and established selection process. Here, biotinylated SOMAmer reagents were incorporated into single molecule array (Simoa)-based assays in place of traditional detection antibodies for six cytokine targets. Optimization and validation were conducted for TNF-α as a demonstration using a capture antibody/detection-SOMAmer detection scheme to highlight the performance of this approach. The optimized assay has a broad dynamic range (>4 log10 units) and an ultralow detection limit of 0.67 fM (0.012 pg/mL). These results show comparable sensitivity to our antibody pair-based Simoa assays, and tens to thousands-fold enhancement in sensitivity compared with conventional ELISAs. High recovery percentages were observed in a spike-recovery test using human sera, demonstrating the feasibility of this novel Simoa assay in detecting TNF-α in clinically relevant samples. Detection SOMAmers were also used to detect other cytokines, such as IFN-γ, IL-1β, IL-2, IL-6, and IL-10, in human samples. Although not yet demonstrated, in principle it should be possible to eventually replace both the capture and detector antibodies with corresponding SOMAmer pairs in sandwich immunoassays. The combination of the ultrasensitive Simoa platform with the higher reliability of SOMAmer binding reagents will greatly benefit both biomarker discovery and disease diagnostic fields. PMID:27529794

  18. Stopped-flow DNA polymerase assay by continuous monitoring of dNTP incorporation by fluorescence.

    PubMed

    Montgomery, Jesse L; Rejali, Nick; Wittwer, Carl T

    2013-10-15

    DNA polymerase activity was measured by a stopped-flow assay that monitors polymerase extension using an intercalating dye. Double-stranded DNA formation during extension of a hairpin substrate was monitored at 75°C for 2 min. Rates were determined in nucleotides per second per molecule of polymerase (nt/s) and were linear with time and polymerase concentration from 1 to 50 nM. The concentrations of 15 available polymerases were quantified and their extension rates determined in 50 mM Tris, pH 8.3, 0.5 mg/ml BSA, 2 mM MgCl₂, and 200 μM each dNTP as well as their commercially recommended buffers. Native Taq polymerases had similar extension rates of 10-45 nt/s. Three alternative polymerases showed faster speeds, including KOD (76 nt/s), Klentaq I (101 nt/s), and KAPA2G (155 nt/s). Fusion polymerases including Herculase II and Phusion were relatively slow (3-13 nt/s). The pH optimum for Klentaq extension was between 8.5 and 8.7 with no effect of Tris concentration. Activity was directly correlated to the MgCl2 concentration and inversely correlated to the KCl concentration. This continuous assay is relevant to PCR and provides accurate measurement of polymerase activity using a defined template without the need of radiolabeled substrates. PMID:23872003

  19. Evidence from a carbohydrate incorporation assay for direct activation of bone marrow myelopoietic precursor cells by bacterial cell wall constitutents.

    PubMed Central

    Monner, D A; Gmeiner, J; Mühlradt, P F

    1981-01-01

    The stimulation of incorporation of [3H]galactose into membrane glycoconjugates, measured in a precipitation test, was used as a criterion for activation of bone marrow cells. In this assay, purified bacterial lipopolysaccharide, lipoprotein, and murein monomer and dimer fragments all activated rat bone marrow cells in vitro. The response was dose dependent, followed a defined time course, and was not serum dependent. O-Acetylated murein dimer fragments from Proteus mirabilis were much less active than their unsubstituted counterparts, indicating a structural specificity for murein activation. Removal of adherent and phagocytizing cells from the marrow suspensions did not alter these results. The labeled, activated cells constituted a distinct population of buoyant density 1.064 to 1.069 g/cm3 when centrifuged on a continuous gradient of Percoll. Enrichment of the target cell population was achieved by a combination of adherent cell removal and discontinuous density gradient centrifugation to remove granulocytes and erythropoietic cells. It was concluded that a population of myelopoietic precursors could be activated by direct contact with bacterial cell wall constituents. The stimulation of galactose incorporation was not coupled to active deoxyribonucleic acid synthesis in the marrow cells. Thus, the activation was interpreted as an induction of differentiation rather than a mitotic event. PMID:7014467

  20. Inter-laboratory validation of the modified murine local lymph node assay based on 5-bromo-2'-deoxyuridine incorporation.

    PubMed

    Kojima, Hajime; Takeyoshi, Masahiro; Sozu, Takashi; Awogi, Takumi; Arima, Kazunori; Idehara, Kenji; Ikarashi, Yoshiaki; Kanazawa, Yukiko; Maki, Eiji; Omori, Takashi; Yuasa, Atsuko; Yoshimura, Isao

    2011-01-01

    The murine local lymph node assay (LLNA) is a well-established alternative to the guinea pig maximization test (GPMT) or Buehler test (BT) for the assessment of the skin sensitizing ability of a drug, cosmetic material, pesticide or industrial chemical. Instead of radioisotope using in this method, Takeyoshi M. et al. (2001) has developed a modified LLNA based on the 5-bromo-2'-deoxyuridine (BrdU) incorporation (LLNA:BrdU-ELISA). The LLNA:BrdU-ELISA is practically identical to the LLNA methodology excluding the use of BrdU, for which a single intraperitoneal injection of BrdU is made on day 4, and colorimetric detection of cell turnover. We conducted the validation study to evaluate the reliability and relevance of LLNA:BrdU-ELISA. The experiment involved 7 laboratories, wherein 10 chemicals were examined under blinded conditions. In this study, 3 chemicals were examined in all laboratories and the remaining 7 were examined in 3 laboratories. The data were expressed as the BrdU incorporation using an ELISA method for each group, and the stimulation index (SI) for each chemical-treated group was determined as the increase in the BrdU incorporation relative to the concurrent vehicle control group. An SI of 2 was set as the cut-off value for exhibiting skin sensitization activity. The results obtained in the experiments conducted for all 10 chemicals were sufficiently consistent with small variations in their SI values. The sensitivity, specificity, and accuracy of LLNA:BrdU-ELISA against those of GPMT/BT were 7/7 (100%), 3/3 (100%), and 10/10 (100%), respectively. PMID:20677212

  1. 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

  2. Synthesis of thiol-reactive lipopeptide adjuvants. Incorporation into liposomes and study of their mitogenic effect on mouse splenocytes.

    PubMed

    Roth, Audrey; Espuelas, Socorro; Thumann, Christine; Frisch, Benoît; Schuber, Francis

    2004-01-01

    Synthetic analogues of triacylated and diacylated lipopeptides derived from the N-terminal domain of respectively bacterial and mycoplasmal lipoproteins are highly potent immunoadjuvants when administered either in combination with protein antigens or covalently linked to small peptide epitopes. Because of their amphipathic properties, lipopeptides, such as S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-cysteinyl-alanyl-glycine (Pam(3)CAG), can be conveniently incorporated into liposomes and serve as anchors for antigens that are linked to them. To design vaccination constructs based on synthetic peptides and liposomes as vectors. we have accordingly synthesized a series of lipopeptides that differ by the number (Pam(3)C vs Pam(2)C) and nature of the acyl chains (palmitoyl vs oleoyl) and by the presence at their C-terminus of thiol-reactive functions, such as maleimide or bromoacetyl. When incorporated into liposomes, these latter functionalized lipopeptides allow, in aqueous media, a well controlled chemoselective conjugation of HS-peptides to the surface of the vesicles. Using a BALB/c mice splenocyte proliferation assay ([(3)H]thymidine incorporation), we have measured the lymphocyte activation potency of the different lipopeptides. We found that, compared to their free (emulsified) forms, the liposomal lipopeptides were endowed with enhanced mitogenic activities; i.e., up to 2 orders of magnitude for Pam(3)CAG which was more potent than Pam(2)CAG. The impact of functionalization on the cellular activity of Pam(3)CAG was dependent on the thiol-reactive group introduced: whereas the bromoacetyl derivative retained its full activity, the presence of a maleimide group virtually abolished the lymphocyte activation of the lipopeptide. Finally, the substitution of saturated palmitoyl chains by unsaturated oleoyl chains was inhibitory. Thus, thiol-reactive Ol(3)CAG derivatives were the least active mitogens in our assay. Taken together, our findings are of

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

    SciTech Connect

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

    1986-11-01

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

  4. In vitro Plasmodium falciparum drug sensitivity assay: inhibition of parasite growth by incorporation of stomatocytogenic amphiphiles into the erythrocyte membrane.

    PubMed

    Ziegler, Hanne L; Staerk, Dan; Christensen, Jette; Hviid, Lars; Hägerstrand, Henry; Jaroszewski, Jerzy W

    2002-05-01

    Lupeol, which shows in vitro inhibitory activity against Plasmodium falciparum 3D7 strain with a 50% inhibitory concentration (IC50) of 27.7 +/- 0.5 microM, was shown to cause a transformation of the human erythrocyte shape toward that of stomatocytes. Good correlation between the IC50 value and the membrane curvature changes caused by lupeol was observed. Preincubation of erythrocytes with lupeol, followed by extensive washing, made the cells unsuitable for parasite growth, suggesting that the compound incorporates into erythrocyte membrane irreversibly. On the other hand, lupeol-treated parasite culture continued to grow well in untreated erythrocytes. Thus, the antiplasmodial activity of lupeol appears to be indirect, being due to stomatocytic transformation of the host cell membrane and not to toxic effects via action on a drug target within the parasite. A number of amphiphiles that cause stomatocyte formation, but not those causing echinocyte formation, were shown to inhibit growth of the parasites, apparently via a mechanism similar to that of lupeol. Since antiplasmodial agents that inhibit parasite growth through erythrocyte membrane modifications must be regarded as unsuitable as leads for development of new antimalarial drugs, care must be exercised in the interpretation of results of screening of plant extracts and natural product libraries by an in vitro Plasmodium toxicity assay. PMID:11959580

  5. In Vitro Plasmodium falciparum Drug Sensitivity Assay: Inhibition of Parasite Growth by Incorporation of Stomatocytogenic Amphiphiles into the Erythrocyte Membrane

    PubMed Central

    Ziegler, Hanne L.; Stærk, Dan; Christensen, Jette; Hviid, Lars; Hägerstrand, Henry; Jaroszewski, Jerzy W.

    2002-01-01

    Lupeol, which shows in vitro inhibitory activity against Plasmodium falciparum 3D7 strain with a 50% inhibitory concentration (IC50) of 27.7 ± 0.5 μM, was shown to cause a transformation of the human erythrocyte shape toward that of stomatocytes. Good correlation between the IC50 value and the membrane curvature changes caused by lupeol was observed. Preincubation of erythrocytes with lupeol, followed by extensive washing, made the cells unsuitable for parasite growth, suggesting that the compound incorporates into erythrocyte membrane irreversibly. On the other hand, lupeol-treated parasite culture continued to grow well in untreated erythrocytes. Thus, the antiplasmodial activity of lupeol appears to be indirect, being due to stomatocytic transformation of the host cell membrane and not to toxic effects via action on a drug target within the parasite. A number of amphiphiles that cause stomatocyte formation, but not those causing echinocyte formation, were shown to inhibit growth of the parasites, apparently via a mechanism similar to that of lupeol. Since antiplasmodial agents that inhibit parasite growth through erythrocyte membrane modifications must be regarded as unsuitable as leads for development of new antimalarial drugs, care must be exercised in the interpretation of results of screening of plant extracts and natural product libraries by an in vitro Plasmodium toxicity assay. PMID:11959580

  6. Evaluation of intestinal absorption of ginsenoside Rg1 incorporated in microemulison using parallel artificial membrane permeability assay.

    PubMed

    Han, Min; Fu, Shao; Gao, Jian-Qing; Fang, Xiao-Ling

    2009-06-01

    In the present study, ginsenoside Rg(1) (Rg(1)), a naturally occurring drug which is hardly absorbed in gastrointestinal (GI) tract due to its high hydrophilicity and low membrane permeability, was incorporated in different compositions of water-in-oil microemulsions (MEs). And parallel artificial membrane permeability assay (PAMPA) that have been mainly utilized for the evaluation of in vitro permeability of early drug candidates was introduced in present study, as well as rat in vivo pharmacokinetics and in vitro permeability measurements, to investigate the effect of w/o ME on Rg(1) absorption. Correlation between various models as mentioned above was further performed to estimate the feasibility of PAMPA in the application of pharmaceutical preparation studies. After being administrated intraduodenally to rats, most of MEs can enhance the intestinal absorption of Rg(1) to various extents with relative bioavailability (F(re)) ranging from 268 to 1270% using drug solution as control. This enhanced absorption of Rg(1) may be related to its increased membrane permeability induced by ME as exhibited in the PAMPA and rat in vitro permeability measurements. Meanwhile, rat in vivo pharmacokinetics-PAMPA correlation (r(2)=0.6082) is significant (p<0.05) for ME, representing a potential prospect for the application of PAMPA in the study of pharmaceutical preparation in some conditions. PMID:19483317

  7. 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. PMID:27285358

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

    PubMed Central

    Brittain, Andrew M.; Karl, David M.

    1990-01-01

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

  9. Re-exploration of the Codon Context Effect on Amber Codon-Guided Incorporation of Noncanonical Amino Acids in Escherichia coli by the Blue-White Screening Assay.

    PubMed

    Xu, Huan; Wang, Yan; Lu, Jiaqi; Zhang, Bo; Zhang, Ziwei; Si, Longlong; Wu, Ling; Yao, Tianzhuo; Zhang, Chuanling; Xiao, Sulong; Zhang, Lihe; Xia, Qing; Zhou, Demin

    2016-07-01

    The effect of codon context on amber codon-guided incorporation of noncanonical amino acids (NAAs) has been previously examined by antibiotic selection. Here, we re-explored this effect by screening a library in which three nucleotides upstream and downstream of the amber codon were randomised, and inserted within the lacZ-α gene. Thousands of clones were obtained and distinguished by the depth of blue colour upon exposure to X-gal. Large-scale sequencing revealed remarkable preferences in nucleotides downstream of the amber codon, and moderate preferences for upstream nucleotides. Nucleotide preference was quantified by a dual-luciferase assay, which verified that the optimum context for NAA incorporation, AATTAGACT, was applicable to different proteins. Our work provides a general guide for engineering amber codons into genes of interest in bacteria. PMID:27028123

  10. Development and evaluation of a human T-cell leukemia virus type I serologic confirmatory assay incorporating a recombinant envelope polypeptide.

    PubMed

    Lillehoj, E P; Alexander, S S; Dubrule, C J; Wiktor, S; Adams, R; Tai, C C; Manns, A; Blattner, W A

    1990-12-01

    A recombinant protein derived from the gp21 region of the human T-cell leukemia virus type I (HTLV-I) env gene was synthesized in Escherichia coli and purified by reversed-phase high-performance liquid chromatography. The purified protein was free of contaminating bacterial proteins and retained reactivity with human HTLV-I- and HTLV-II-positive sera and a gp21 monoclonal antibody. An immunoblot procedure using the recombinant polypeptide in conjunction with native viral proteins was more sensitive than the conventional immunoblot and radioimmunoprecipitation confirmatory assays for detection of antibodies to HTLV-I and HTLV-II env-encoded gene products. The recombinant protein was equally reactive with sera from polymerase chain reaction-confirmed HTLV-I or HTLV-II infections. Furthermore, on the basis of the differential reactivities of gp21-positive sera with the HTLV-I p19 and p24 gag-encoded proteins, an algorithm was proposed to distinguish exposure to HTLV-I from exposure to HTLV-II. These results establish the utility of a modified immunoblot assay incorporating a recombinant envelope polypeptide as an alternative to existing HTLV-I-confirmatory assays. PMID:2279997

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

    PubMed Central

    Wardell, William L.

    1975-01-01

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

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

    SciTech Connect

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

    1995-12-31

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

  13. Quantitative In Vitro Assay for “Corncob” Formation

    PubMed Central

    Lancy, P.; Appelbaum, B.; Holt, S. C.; Rosan, B.

    1980-01-01

    The interaction of Bacterionema matruchotii with strains of Streptococcus sanguis produces a structure which morphologically resembles a corncob. To determine the specific bacterial surface receptors involved in the interaction, we developed a quantitative assay. The assay consisted of mixing saline suspensions of [CH3-3H]thymidine-labeled streptococci and B. matruchotii, incubating at 37°C for 2 h, and filtering the mixture through a 5-μm polycarbonate membrane filter. The free cocci and filaments passed through the filter, but the corncobs were retained. Estimates of the number of corncobs formed were obtained by quantitating the radioactivity retained on the membranes relative to that of controls of streptococci alone. Although saturation of the Bacterionema occurred at a ratio of streptococci to Bacterionema of 10:1 (Klett units), a 2:1 ratio was chosen because of the increased sensitivity of the assay at this ratio. The percentage of streptococci binding at this ratio was 18.6 ± 8.1 (standard deviation). All five Bacterionema strains tested formed corncobs; in contrast, only three strains of S. sanguis were positive. These were serotype 1 strains which had localized surface “fuzz.” Although scanning electron microscopic observations revealed an almost random distribution of cocci along the filament surface, transmission electron microscopy revealed that the streptococci were attached to the Bacterionema by the surface fuzz. No differences in corncob formation were observed in sodium phosphate buffer, pH 6 to 8, at phosphate concentrations ranging from 0.005 to 0.05 M. Concentrations of NaCl or KCl up to 0.25 M did not affect corncob formation, and low concentrations of CaCl2 increased corncob formation slightly, whereas MgCl2, ethylenediaminetetraacetic acid, and citrate buffers reduced the number of streptococci binding to the filaments. These results suggest that divalent cations may play a role in this process. ImagesFig. 3Fig. 4Fig. 5 PMID:7011981

  14. Relative impact of incorporating pharmacokinetics on predicting in vivo hazard and mode of action from high-throughput in vitro toxicity assays.

    PubMed

    Wetmore, Barbara A; Wambaugh, John F; Ferguson, Stephen S; Li, Lili; Clewell, Harvey J; Judson, Richard S; Freeman, Kimberly; Bao, Wenjun; Sochaski, Mark A; Chu, Tzu-Ming; Black, Michael B; Healy, Eric; Allen, Brittany; Andersen, Melvin E; Wolfinger, Russell D; Thomas, Russell S

    2013-04-01

    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 extrapolation was used to estimate the daily dose in a rat, called the oral equivalent dose, which would result in steady-state in vivo blood concentrations equivalent to the AC 50 or lowest effective concentration (LEC) across more than 600 ToxCast phase I in vitro assays. Statistical classification analysis was performed using either oral equivalent doses or unadjusted AC 50 /LEC values for the in vitro assays to predict the in vivo effects of the 59 chemicals. Adjusting the in vitro assays for pharmacokinetics did not improve the ability to predict in vivo effects as either a discrete (yes or no) response or a low effect level (LEL) on a continuous dose scale. Interestingly, a comparison of the in vitro assay with the lowest oral equivalent dose with the in vivo endpoint with the lowest LEL suggested that the lowest oral equivalent dose may provide a conservative estimate of the point of departure for a chemical in a dose-response assessment. Furthermore, comparing the oral equivalent doses for the in vitro assays with the in vivo dose range that resulted in adverse effects identified more coincident in vitro assays across chemicals than expected by chance, suggesting that the approach may also be used to identify potential molecular initiating events leading to adversity. PMID:23358191

  15. 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...

  16. New chemotherapeutic drug sensitivity assay for colon carcinomas in monolayer culture.

    PubMed

    Schroy, P C; Cohen, A; Winawer, S J; Friedman, E A

    1988-06-01

    Ten previously untreated colon carcinomas were tested for chemotherapeutic drug sensitivity in primary monolayer culture. Colon carcinomas were partly digested to groups of epithelial cells which plated with a mean efficiency of 42 +/- 9% (SE) on a collagen I-bovine serum albumin substrate in serum-free medium, producing patches of tightly adherent epithelial cells. The cultured cells were judged epithelial by the presence of cytokeratins, an epithelial cell surface epitope, junctional complexes, and brush borders. Each carcinoma was plated in 40 to 60 Petri dishes (35 mm), yielding a mean of 28 +/- 8 (SE) colonies per dish (6832 +/- 1952 cells). Drugs tested in duplicate plates were mitomycin C, cisplatin, streptozotocin, and 5-fluorouracil at 0.1, 1, 10, and 100 micrograms/ml, and at 0.1, 1, and 2x the peak tolerated drug concentration in serum. Twenty-four h after plating, any nonadherent cells were removed, and the adherent tumor cells were continuously exposed to the drugs for 3 days. Each drug induced colony lysis in a dose-dependent manner in responsive tumors. Drug-resistant, cycling cells were identified by [3H]thymidine incorporation in colonies which were not lysed by drug treatment. Each of the ten carcinomas exhibited inherent resistance to one or more chemotherapy drugs within the concentration ranges clinically achievable. PMID:2966672

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

    EPA Science Inventory

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

  18. 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.

  19. 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.

  20. 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. PMID:22824340

  1. 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.

  2. Chemosensitivity testing of primary cultures of Merkel cell cancer.

    PubMed

    Kearsley, J H; Hurst, T; Khoo, S K

    1993-10-01

    Twenty-seven tumor specimens from patients with Merkel cell carcinoma (MCC) were tested for chemosensitivity against a battery of nine cytotoxic drugs in a short-term antimetabolic assay measuring inhibition of thymidine incorporation. Dose-response curves were constructed by plotting drug concentration in micrograms/ml versus % control [3H]thymidine incorporation. Specimens were considered 'sensitive' to a drug if, at the approximate peak plasma concentration (PPC), the inhibition of [3H]thymidine was greater than 50% when compared with untreated control primary cultures. The assay revealed a 'sensitive' tumor in 19 of 20 specimens and 16 of 17 patients had a tumor that was 'sensitive' to at least one drug tested in the assay system. The highest sensitivity in order of frequency was found with doxorubicin, epirubicin, cyclophosphamide, etoposide and cisplatin. At least 40% of the tumors were 'sensitive' to these five drugs. Cyclophosphamide was chosen as the most active drug (at PPC) in 10 of 19 assays (53%), etoposide in seven of 17 (41%), doxorubicin in four of 19 (21%), chlorambucil in one of 12 (8%) and cisplatin in one of 18 (5%) of assays. Though our results are preliminary, we have identified for the first time a range of cytotoxic drugs which appear effective against MCC in vitro. Our main task now is to determine whether our in vitro predictive assay will correlate with clinical benefit to the patient. PMID:8292815

  3. Effect of urokinase on the proliferation of primary cultures of human prostatic cells

    SciTech Connect

    Kirchheimer, J.C.; Wojta, J.; Hienert, G.; Christ, G.; Heger, M.E.; Pflueger, H.B.; Binder, B.R.

    1987-11-01

    The effects of exogenously added urokinase type plasminogen activator, tissue type plasminogen activator, plasmin and thrombin on the proliferation of primary cultures of cells derived from prostatic hyperplasia or prostatic carcinomas were investigated by measuring the incorporation of /sup 3/H-thymidine into the cultures. Addition of urokinase type plasminogen activator (1.35 x 10(-9) M) or thrombin (10(-7) M) to the culture medium caused a two-fold increase of /sup 3/H-thymidine incorporation, regardless of the origin of the prostatic cells. Tissue type plasminogen activator did not alter the rate of /sup 3/H-thymidine incorporation, whereas plasmin caused a 25% decrease of /sup 3/H-thymidine incorporation in all cultures.

  4. 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

  5. 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.

  6. 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. PMID:12677096

  7. Animal model for evaluation of topical photoprotection against ultraviolet A (320-380 nm) radiation

    SciTech Connect

    Chew, S.; DeLeo, V.A.; Harber, L.C.

    1987-10-01

    Recent studies reporting UVA (ultraviolet A radiation 320-380 nm) as an integral part of the cumulative sun-induced damage in human skin have prompted an interest in developing effective UVA photoprotective agents. The development of such compounds has been impeded by the absence of a clinically relevant animal model for evaluating their efficacy. This report describes the development and use of such a laboratory animal system. Selected concentrations of oxybenzone (2-hydroxy-4-methoxybenzophenone) in vehicle (0.1% to 6%) or vehicle alone were applied to the depilated dorsal skin of 30 Hartley strain female albino guinea pigs. The skin was irradiated with solar simulated UVA from a xenon light source. Acute radiation-induced damage was assayed by erythema grading and inhibition of (/sup 3/H)thymidine incorporation into epidermal DNA. Data from erythema grading studies indicated that a significant degree of photoprotection was achieved with 6%, 3%, and 1% solutions of benzophenone compared with the control vehicle; the 6% solution was significantly more photoprotective than the 3% and 1% solutions. A 6% solution afforded significant photoprotection when assayed by (/sup 3/H)thymidine incorporation.

  8. 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.

  9. In vitro incorporation of LNA nucleotides.

    PubMed

    Veedu, Rakesh N; Vester, Birte; Wengel, Jesper

    2007-01-01

    An LNA modified nucleoside triphosphate 1 was synthesized in order to investigate its potential to act as substrate for DNA strand synthesis by polymerases. Primer extension assays for the incorporation experiments revealed that Phusion High Fidelity DNA polymerase is an efficient enzyme for incorporation of the LNA nucleotide and for extending strand to full length. It was also observed that pfu DNA polymerase could incorporate the LNA nucleotide but it failed to extend the strand to a full length product. PMID:18058567

  10. Appraisal of within- and between-laboratory reproducibility of non-radioisotopic local lymph node assay using flow cytometry, LLNA:BrdU-FCM: comparison of OECD TG429 performance standard and statistical evaluation.

    PubMed

    Yang, Hyeri; Na, Jihye; Jang, Won-Hee; Jung, Mi-Sook; Jeon, Jun-Young; Heo, Yong; Yeo, Kyung-Wook; Jo, Ji-Hoon; Lim, Kyung-Min; Bae, SeungJin

    2015-05-01

    Mouse local lymph node assay (LLNA, OECD TG429) is an alternative test replacing conventional guinea pig tests (OECD TG406) for the skin sensitization test but the use of a radioisotopic agent, (3)H-thymidine, deters its active dissemination. New non-radioisotopic LLNA, LLNA:BrdU-FCM employs a non-radioisotopic analog, 5-bromo-2'-deoxyuridine (BrdU) and flow cytometry. For an analogous method, OECD TG429 performance standard (PS) advises that two reference compounds be tested repeatedly and ECt(threshold) values obtained must fall within acceptable ranges to prove within- and between-laboratory reproducibility. However, this criteria is somewhat arbitrary and sample size of ECt is less than 5, raising concerns about insufficient reliability. Here, we explored various statistical methods to evaluate the reproducibility of LLNA:BrdU-FCM with stimulation index (SI), the raw data for ECt calculation, produced from 3 laboratories. Descriptive statistics along with graphical representation of SI was presented. For inferential statistics, parametric and non-parametric methods were applied to test the reproducibility of SI of a concurrent positive control and the robustness of results were investigated. Descriptive statistics and graphical representation of SI alone could illustrate the within- and between-laboratory reproducibility. Inferential statistics employing parametric and nonparametric methods drew similar conclusion. While all labs passed within- and between-laboratory reproducibility criteria given by OECD TG429 PS based on ECt values, statistical evaluation based on SI values showed that only two labs succeeded in achieving within-laboratory reproducibility. For those two labs that satisfied the within-lab reproducibility, between-laboratory reproducibility could be also attained based on inferential as well as descriptive statistics. PMID:25732604

  11. Helicase Assays

    PubMed Central

    Wang, Xin; Li, Jing; Diaz, Jason; You, Jianxin

    2016-01-01

    Helicases are a class of enzymes which are motor proteins using energy derived from ATP hydrolysis to move directionally along a nucliec acid phosphodiester backbone (such as DNA, RNA and DNA-RNA hybrids) and separate two annealed nucleic acid strands. Many cellular processes, such as transcription, DNA replication, recombination and DNA repair involve helicase activity. Here, we provide a protocol to analyze helicase activities in vitro. In this protocol, the DNA helicase protein Merkel cell polyomavirus large T-antigen was expressed in the mammalian cell line HEK293 and immoblized on an IgG resin. The helicase assay is performing while the protein is immoblized on IgG resin.

  12. A novel herbal formulation "LiverCare" differentially regulates primary rat hepatocyte and hepatocarcinoma cell proliferation in vitro.

    PubMed

    Vidyashankar, Satyakumar; Varma, Sandeep R; Azeemudin, Mohammed; Godavarthi, Ashok; Krishna, Nandakumar S; Patki, Pralhad Sadashiv

    2011-09-01

    Hepatocyte growth factor (HGF) plays an important role in hepatocyte proliferation. HGF expression is regulated by various signaling molecules and nuclear receptors. In the present study, LiverCare(®) (LC), a novel polyherbal formulation (The Himalaya Drug Company, Bangalore, India), was evaluated for its efficacy, using co-cultures of primary rat hepatocytes-non-parenchymal cells (NPCs) and human hepatocellular carcinoma cells (HepG2). The rate of primary hepatocyte co-culture proliferation was significantly and dose-dependently increased by LC as determined by [(3)H]thymidine incorporation into newly synthesized DNA and cell proliferation assay. LC also increased HGF expression in primary hepatocyte co-culture. Albumin and urea content remained constant during proliferation of hepatocyte co-cultures in the presence of LC with decreased activity of alanine aminotransferase. It is interesting that LC inhibited incorporation of [(3)H]thymidine into DNA in HepG2 cells. LC enhanced peroxisome proliferator-activated receptor-α expression during hepatocyte proliferation, whereas tumor necrosis factor-α expression remained unaffected. In conclusion, our study clearly showed that LC differentially regulates primary rat hepatocytes and human hepatocarcinoma cell proliferation. LC may be a promising candidate for treating degenerative liver diseases by enhancing liver regeneration. PMID:21812649

  13. Metronidazole Decreases Viability of DLD-1 Colorectal Cancer Cell Line

    PubMed Central

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

    2013-01-01

    Abstract 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. PMID:23777253

  14. Liver hyperplasia and regression after lead nitrate administration

    SciTech Connect

    Columbano, A.; Ledda-Columbano, G.M.; Coni, P.P.; Vargiu, M.; Faa, G.; Pani, P.

    1984-01-01

    The effect of a single intravenous injection of lead nitrate on liver, was investigated in male Wistar rats. Lead nitrate at 5 and 10 mumoles/100 g of body weight stimulated a 19-fold increase in the incorporation of /sup 3/H-thymidine into liver DNA and resulted in temporal changes in DNA synthesis, as determined by assays of specific activity. Thirty-six hours after lead nitrate administration, the incorporation of /sup 3/H-thymidine reached its maximum and returned to normal levels within 3 days. A significant increase in the number of cells entering mitosis at 36 hours indicated the capacity of lead to stimulate liver cell proliferation. Enlargement of the liver after lead treatment was also observed in both female Wistar rats as well in male Fischer rats. This stimulatory effect of lead on liver growth was reversible; during the involution of the liver, cell death morphologically similar to the one described as apoptosis was observed in histological sections of liver from animals sacrificed 4-7 days after lead treatment.

  15. Transforming growth factor-beta and transforming growth factor beta-receptor expression in human meningioma cells.

    PubMed Central

    Johnson, M. D.; Federspiel, C. F.; Gold, L. I.; Moses, H. L.

    1992-01-01

    The transforming growth factor-beta (TGF beta) family in mammals includes three closely related peptides that influence proliferation and numerous physiologic processes in most mesenchymal cells. In this study, Northern blots, immunohistochemistry, TGF beta radioreceptor assays, TGF beta receptor affinity labeling and [3H] thymidine incorporation were used to evaluate whether primary cell cultures of human meningiomas synthesize the three TGF beta isoforms, bear TGF beta receptors, and respond to TGF beta. Transcripts for TGF beta 1 and 2 were detected in the three cases analyzed. Transforming growth factor-beta 1 immunoreactivity was detected in three of six cases, and TGF beta 2 and 3 immunoreactivity were detected in each case analyzed. Media conditioned by cells cultured from six meningiomas also contained latent TGF beta-like activity. Transforming growth factor-beta receptor cross-linking studies identified TGF beta binding sites corresponding to the type 1, type 2, and type 3 receptors on meningioma cells. Treatment with active TGF beta 1 produced a statistically significant reduction in [3H] thymidine incorporation after stimulation with 10% fetal calf serum and epidermal growth factor in all six cases studied. Images Figure 1 Figure 2 Figure 4 PMID:1325741

  16. Angiogenesis Assays.

    PubMed

    Nambiar, Dhanya K; Kujur, Praveen K; Singh, Rana P

    2016-01-01

    Neoangiogenesis constitutes one of the first steps of tumor progression beyond a critical size of tumor growth, which supplies a dormant mass of cancerous cells with the required nutrient supply and gaseous exchange through blood vessels essentially needed for their sustained and aggressive growth. In order to understand any biological process, it becomes imperative that we use models, which could mimic the actual biological system as closely as possible. Hence, finding the most appropriate model is always a vital part of any experimental design. Angiogenesis research has also been much affected due to lack of simple, reliable, and relevant models which could be easily quantitated. The angiogenesis models have been used extensively for studying the action of various molecules for agonist or antagonistic behaviour and associated mechanisms. Here, we have described two protocols or models which have been popularly utilized for studying angiogenic parameters. Rat aortic ring assay tends to bridge the gap between in vitro and in vivo models. The chorioallantoic membrane (CAM) assay is one of the most utilized in vivo model system for angiogenesis-related studies. The CAM is highly vascularized tissue of the avian embryo and serves as a good model to study the effects of various test compounds on neoangiogenesis. PMID:26608294

  17. The mechanism of inhibition of endothelin-1-induced stimulation of DNA synthesis in rat articular chondrocytes.

    PubMed

    Khatib, A M; Ribault, D; Quintero, M; Barbara, A; Fiet, J; Mitrovic, D R

    1997-09-19

    Endothelin-1 (ET-1) is a potent mitogen for rat articular chondrocytes (AC) in short term culture (24 h). Prolonged incubation (72 h) of AC with ET-1 resulted in inhibition of [3H]thymidine incorporation. This inhibition seemed to be mediated by prostaglandins (PGs) released in response to ET-1, since indomethacin (INDO) enhanced ET-1-induced [3H]thymidine incorporation. In agreement with this hypothesis, exogenous prostaglandins (PGE2, PGF2alpha and TxB2) blocked all basal, ET-1-induced and ET-1 induced-INDO-enhanced [3H]thymidine incorporation and ET-1 stimulated PGE2 release in a time and concentration-dependent manner. INDO also blocked cGMP production and 6-anilino-5,8-quinolinedione, a relatively specific inhibitor of cGMP formation, enhanced the stimulation and suppressed the inhibition of ET-1-induced DNA synthesis. In addition, 8-bromo-cGMP, an analogue of cGMP, blocked at all time periods studied, both basal and ET-1-induced incorporations of [3H]thymidine. Thus, PGs produced in response to ET-1 counteract the ET-1-induced stimulation of [3H]thymidine incorporation into rat AC by increasing cGMP production. PMID:9324043

  18. HIV-1 Fusion Assay

    PubMed Central

    Cavrois, Marielle; Neidleman, Jason; Greene, Warner C.

    2016-01-01

    The HIV-1 fusion assay measures all steps in the HIV-1 life cycle up to and including viral fusion. It relies on the incorporation of a β-lactamase Vpr (BlaM-Vpr) protein chimera into the virion and the subsequent transfer of this chimera into the target cell by fusion (Figure 1). The transfer is monitored by the enzymatic cleavage of CCF2, a fluorescent dye substrate of β-lactamase, loaded into the target cells. Cleavage of the β-lactam ring in CCF2 by β-lactamase changes the fluorescence emission spectrum of the dye from green (520 nm) to blue (447 nm). This change reflects virion fusion and can be detected by flow cytometry (Figure 2).

  19. Mitogenic effect of Parkia speciosa seed lectin on human lymphocytes.

    PubMed

    Suvachittanont, W; Jaranchavanapet, P

    2000-12-01

    Mitogenic activity of a lectin, purified from Parkia speciosa seeds, on the isolated peripheral blood lymphocytes taken from normal blood donors and patients with esophageal carcinoma was examined using [3H]thymidine incorporation. The lectin increases the incorporation of [3H]thymidine into DNA of human lymphocytes. The activity of the lectin increased as its concentration was increased and then declined once the concentration passed an optimum point. The stimulant effect was also expressed using a proliferation index (PI): the ratio of [3H]thymidine incorporated into lymphocytes in the presence and absence of the lectin. The mitogenic activity of the lectin is comparable to those of the known T-cell mitogens, such as concanavalin A, phytohaemagglutinin, and pokeweed mitogen. Only slightly less responsiveness was observed in the case of lymphocytes from esophageal cancer compared to lymphocytes from normal donors. PMID:11199124

  20. HaCaT cell proliferation influenced by melatonin.

    PubMed

    Hipler, U-C; Fischer, T W; Elsner, P

    2003-01-01

    The hormone melatonin is characterized by numerous pharmacological effects. The influence of melatonin on the growth of the human hair follicle was shown in previous investigations. In the present study, the effects of melatonin were investigated by means of proliferation tests of HaCaT keratinocytes using the [3H]thymidine incorporation, a fluorescence assay with Hoechst dye 33342 and the ATP bioluminescence assay. The aim of the study was to find melatonin concentrations suitable for treatments of the skin and whether there is a cytotoxic effect on HaCaT cells. The different proliferative activity of melatonin depending on its concentration and the time of incubation could be shown in all investigations. PMID:14528062

  1. 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.

  2. 40 CFR 63.841 - Incorporation by reference.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... October 7, 1997, in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. These materials are incorporated as... 40 Protection of Environment 11 2014-07-01 2014-07-01 false Incorporation by reference. 63.841...) ASTM D 2986-95A, Standard Practice for Evaluation of Air Assay Media by the Monodisperse DOP...

  3. 40 CFR 63.841 - Incorporation by reference.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... October 7, 1997, in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. These materials are incorporated as... 40 Protection of Environment 11 2012-07-01 2012-07-01 false Incorporation by reference. 63.841...) ASTM D 2986-95A, Standard Practice for Evaluation of Air Assay Media by the Monodisperse DOP...

  4. 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. PMID:23088321

  5. Radiometric assays for glycerol, glucose, and glycogen.

    PubMed

    Bradley, D C; Kaslow, H R

    1989-07-01

    We have developed radiometric assays for small quantities of glycerol, glucose and glycogen, based on a technique described by Thorner and Paulus (1971, J. Biol. Chem. 246, 3885-3894) for the measurement of glycerokinase activity. In the glycerol assay, glycerol is phosphorylated with [32P]ATP and glycerokinase, residual [32P]ATP is hydrolyzed by heating in acid, and free [32P]phosphate is removed by precipitation with ammonium molybdate and triethylamine. Standard dose-response curves were linear from 50 to 3000 pmol glycerol with less than 3% SD in triplicate measurements. Of the substances tested for interference, only dihydroxyacetone gave a slight false positive signal at high concentration. When used to measure glycerol concentrations in serum and in media from incubated adipose tissue, the radiometric glycerol assay correlated well with a commonly used spectrophotometric assay. The radiometric glucose assay is similar to the glycerol assay, except that glucokinase is used instead of glycerokinase. Dose response was linear from 5 to 3000 pmol glucose with less than 3% SD in triplicate measurements. Glucosamine and N-acetylglucosamine gave false positive signals when equimolar to glucose. When glucose concentrations in serum were measured, the radiometric glucose assay agreed well with hexokinase/glucose-6-phosphate dehydrogenase (H/GDH)-based and glucose oxidase/H2O2-based glucose assays. The radiometric method for glycogen measurement incorporates previously described isolation and digestion techniques, followed by the radiometric assay of free glucose. When used to measure glycogen in mouse epididymal fat pads, the radiometric glycogen assay correlated well with the H/GDH-based glycogen assay. All three radiometric assays offer several practical advantages over spectral assays. PMID:2817333

  6. Radiometric assays for glycerol, glucose, and glycogen

    SciTech Connect

    Bradley, D.C.; Kaslow, H.R. )

    1989-07-01

    We have developed radiometric assays for small quantities of glycerol, glucose and glycogen, based on a technique described by Thorner and Paulus for the measurement of glycerokinase activity. In the glycerol assay, glycerol is phosphorylated with (32P)ATP and glycerokinase, residual (32P)ATP is hydrolyzed by heating in acid, and free (32P)phosphate is removed by precipitation with ammonium molybdate and triethylamine. Standard dose-response curves were linear from 50 to 3000 pmol glycerol with less than 3% SD in triplicate measurements. Of the substances tested for interference, only dihydroxyacetone gave a slight false positive signal at high concentration. When used to measure glycerol concentrations in serum and in media from incubated adipose tissue, the radiometric glycerol assay correlated well with a commonly used spectrophotometric assay. The radiometric glucose assay is similar to the glycerol assay, except that glucokinase is used instead of glycerokinase. Dose response was linear from 5 to 3000 pmol glucose with less than 3% SD in triplicate measurements. Glucosamine and N-acetylglucosamine gave false positive signals when equimolar to glucose. When glucose concentrations in serum were measured, the radiometric glucose assay agreed well with hexokinase/glucose-6-phosphate dehydrogenase (H/GDH)-based and glucose oxidase/H2O2-based glucose assays. The radiometric method for glycogen measurement incorporates previously described isolation and digestion techniques, followed by the radiometric assay of free glucose. When used to measure glycogen in mouse epididymal fat pads, the radiometric glycogen assay correlated well with the H/GDH-based glycogen assay. All three radiometric assays offer several practical advantages over spectral assays.

  7. Bacterial mutagenicity assays: test methods.

    PubMed

    Gatehouse, David

    2012-01-01

    The most widely used assays for detecting chemically induced gene mutations are those employing bacteria. The plate incorporation assay using various Salmonella typhimurium LT2 and E. coli WP2 strains is a short-term bacterial reverse mutation assay specifically designed to detect a wide range of chemical substances capable of causing DNA damage leading to gene mutations. The test is used worldwide as an initial screen to determine the mutagenic potential of new chemicals and drugs.The test uses several strains of S. typhimurium which carry different mutations in various genes of the histidine operon, and E. coli which carry the same AT base pair at the critical mutation site within the trpE gene. These mutations act as hot spots for mutagens that cause DNA damage via different mechanisms. When these auxotrophic bacterial strains are grown on a minimal media agar plates containing a trace of the required amino-acid (histidine or tryptophan), only those bacteria that revert to amino-acid independence (His(+) or Tryp(+)) will grow to form visible colonies. The number of spontaneously induced revertant colonies per plate is relatively constant. However, when a mutagen is added to the plate, the number of revertant colonies per plate is increased, usually in a dose-related manner.This chapter provides detailed procedures for performing the test in the presence and absence of a metabolic activation system (S9-mix), including advice on specific assay variations and any technical problems. PMID:22147566

  8. PHARMACOLOGIC AND IMMUNOLOGIC APPROACHES TO THE PROBLEMS OF POSTTRAUMATIC GLIAL PROLIFERATION FOLLOWING CNS (CENTRAL NERVOUS SYSTEM) DAMAGE

    EPA Science Inventory

    The authors have devised a pharmacologic approach to block the proliferation of glial cells (gliosis) which follows various forms of trauma to nervous tissue. A method was devised using the incorporation of 3H-thymidine incorporation into DNA of glial cells as a proliferative ind...

  9. 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.

  10. 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. PMID:436117

  11. Synchronization in G0/G1 enhances the mitogenic response of cells overexpressing the human insulin receptor A isoform to insulin

    PubMed Central

    Nelander, Gitte-Mai; Hansen, Bo Falck; Jensen, Pia; Krabbe, Jonas S.; Jensen, Marianne B.; Hegelund, Anne Charlotte; Svendsen, Jette E.; Oleksiewicz, Martin B.

    2009-01-01

    Evaluating mitogenic signaling specifically through the human insulin receptor (IR) is relevant for the preclinical safety assessment of developmental insulin analogs. It is known that overexpression of IR sensitizes cells to the mitogenic effects of insulin, but it is essentially unknown how mitogenic responses can be optimized to allow practical use of such recombinant cell lines for preclinical safety testing. We constitutively overexpressed the short isoform of the human insulin receptor (hIR-A, exon 11-negative) in L6 rat skeletal myoblasts. Because the mitogenic effect of growth factors such as insulin is expected to act in G0/G1, promoting S-phase entry, we developed a combined topoinhibition + serum deprivation strategy to explore the effect of G0/G1 synchronization as an independent parameter in the context of serum deprivation, the latter being routinely used to reduce background in mitogenicity assays. G0/G1 synchronization significantly improved the mitogenic responses of L6-hIR cells to insulin, measured by 3H-thymidine incorporation. Comparison with the parental L6 cells using phospho-mitogen-activated protein kinase, phospho-AKT, as well as 3H-thymidine incorporation end points supported that the majority of the mitogenic effect of insulin in L6-hIR cells was mediated by the overexpressed hIR-A. Using the optimized L6-hIR assay, we found that the X-10 insulin analog was more mitogenic than native human insulin, supporting that X-10 exhibits increased mitogenic signaling through the hIR-A. In summary, this study provides the first demonstration that serum deprivation may not be sufficient, and G0/G1 synchronization may be required to obtain optimal responsiveness of hIR-overexpressing cell lines for preclinical safety testing. PMID:19898946

  12. Absolute nuclear material assay

    DOEpatents

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

    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.

  13. 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.

  14. A lateral electrophoretic flow diagnostic assay

    PubMed Central

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

    2015-01-01

    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. PMID:25608872

  15. 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.

  16. Boron incorporation into mullite

    NASA Astrophysics Data System (ADS)

    Griesser, K. J.; Beran, A.; Voll, D.; Schneider, H.

    2008-03-01

    Boron-doped mullites were synthesized using aluminium nitrate-nonahydrate, tetraethoxysilane and boric acid in a sol gel process with subsequent annealing at 950 and 1300 °C for five hours. Two different bulk compositions with constant Al2O3 contents (60 and 70 mol%, respectively) and varying SiO2 plus B2O3 contents were investigated. X-ray powder diffraction analyses yielded a linear decrease of the lattice parameters with increasing bulk B2O3 content, which was interpreted as to be due to boron incorporation. Related to the increasing boron content, corresponding infrared spectra revealed a slight and continuous shift for most of the absorption bands. These data show that mullite is able to incorporate large amounts of boron into its structure (up to about 20 mol% B2O3 depending on the bulk composition of the starting materials). Infrared analyses suggest that boron is incorporated into the mullite structure in form of planar three-fold coordinated BO3 groups.

  17. 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.

  18. 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) ...

  19. Induction of Angiogenesis in Zebrafish Embryos and Proliferation of Endothelial Cells by an Active Fraction Isolated from the Root of Astragalus membranaceus using Bioassay-guided Fractionation

    PubMed Central

    Lai, Patrick Kwok-Kin; Chan, Judy Yuet-Wa; Kwok, Hin-Fai; Cheng, Ling; Yu, Hua; Lau, Ching-Po; Leung, Ping-Chung; Fung, Kwok-Pui; Lau, Clara Bik-San

    2014-01-01

    The objective of the study was to identify the active fraction(s) from AR aqueous extract responsible for promoting angiogenesis using bioassay-guided fractionation. The angiogenic activity was screened by monitoring the increase of sprout number in sub-intestinal vessel (SIV) of the transgenic zebrafish embryos after they were treated with 0.06-0.25 mg/ml of AR aqueous extract or its fraction(s) for 96 h. Furthermore, the angiogenic effect was evaluated in treated zebrafish embryos by measuring the gene expression of angiogenic markers (VEGFA, KDR, and Flt-1) using real-time polymerase chain reaction (RT-PCR), and in human microvascular endothelial cell (HMEC-1) by measuring cell proliferation using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 3H-thymidine uptake assay, and cell cycle analysis. A major active fraction (P1-1-1), which was identified as glycoproteins, was found to significantly stimulate sprout formation (2.03 ± 0.27) at 0.125 mg/ml (P < 0.001) and up-regulate the gene expression of VEGFA, KDR, and Flt-1 by 2.6-fold to 8.2-fold. Additionally, 0.031-0.125 mg/ml of P1-1-1 was demonstrated to significantly stimulate cell proliferation by increasing cell viability (from 180% to 205%), 3H-thymidine incorporation (from 126% to 133%) during DNA synthesis, and the shift of cell population to S phase of cell cycle. A major AR active fraction consisting of glycoproteins was identified, and shown to promote angiogenesis in zebrafish embryos and proliferation of endothelial cells in vitro. PMID:25379465

  20. Immunochromatographic assay on thread.

    PubMed

    Zhou, Gina; Mao, Xun; Juncker, David

    2012-09-18

    Lateral-flow immunochromatographic assays are low-cost, simple-to-use, rapid tests for point-of-care screening of infectious diseases, drugs of abuse, and pregnancy. However, lateral flow assays are generally not quantitative, give a yes/no answer, and lack multiplexing. Threads have recently been proposed as a support for transporting and mixing liquids in lateral-flow immunochromatographic assays, but their use for quantitative high-sensitivity immunoassays has yet to be demonstrated. Here, we introduce the immunochromatographic assay on thread (ICAT) in a cartridge format that is suitable for multiplexing. The ICAT is a sandwich assay performed on a cotton thread knotted to a nylon fiber bundle, both of which are precoated with recognition antibodies against one target analyte. Upon sample application, the assay results become visible to the eye within a few minutes and are quantified using a flatbed scanner. Assay conditions were optimized, the binding curves for C-reactive protein (CRP) in buffer and diluted serum were established and a limit of detection of 377 pM was obtained. The possibility of multiplexing was demonstrated using three knotted threads coated with antibodies against CRP, osteopontin, and leptin proteins. The performance of the ICAT was compared with that of the paper-based and conventional assays. The results suggest that thread is a suitable support for making low-cost, sensitive, simple-to-use, and multiplexed diagnostic tests. PMID:22889381

  1. Gabapentin-induced mitogenic activity in rat pancreatic acinar cells.

    PubMed

    Dethloff, L; Barr, B; Bestervelt, L; Bulera, S; Sigler, R; LaGattuta, M; de La Iglesia, F

    2000-05-01

    Gabapentin induces pancreatic acinar cell tumors in rats through unknown, yet apparently nongenotoxic mechanisms. The primary objective of this study was to determine whether gabapentin acts as a tumor promoter by stimulating acinar cell proliferation in rat pancreas. To this end, indices of pancreatic growth, including increased pancreatic weight, stimulation of acinar cell proliferation, and/or enhanced expression of immediate-early oncogenes were monitored in rats given gabapentin in the diet at 2 g/kg/day for up to 12 months. Rats fed raw soy flour (RSF), a known inducer of pancreatic acinar cell tumors through cholecystokinin-mediated mitogenic stimulation, were used throughout as positive controls. In addition, recent data suggests that gabapentin binds to the alpha(2)delta subunit of a voltage-gated, L-type calcium channel. Because signaling pathways for proliferative processes in pancreatic acinar cells involve intracellular calcium mobilization, the effects of gabapentin on intracellular calcium mobilization ([Ca(2+)](i)) and (3)H-thymidine incorporation were investigated in pancreatic acinar cells isolated from normal rat pancreas and in the AR42J rat pancreatic tumor cell line. As indicated by BrdU labeling indices, acinar cell proliferation increased 3-fold by Day 3 of RSF treatment and remained slightly greater than controls throughout the experiment. Pancreatic weights of RSF-fed rats were 32 to 56% greater than controls throughout the experiment. In contrast, gabapentin had no effect on pancreatic weight or acinar cell labeling index, and therefore had no apparent effect on pancreatic growth. In isolated pancreatic acinar cells, however, gabapentin induced mobilization of intracellular calcium and caused a slight increase in (3)H-thymidine incorporation. The data suggest that gabapentin may possess low level mitogenic activity, which is not easily detectable in in vivo assays. PMID:10788559

  2. BMP-2 gene expression and effects on human vascular smooth muscle cells.

    PubMed

    Willette, R N; Gu, J L; Lysko, P G; Anderson, K M; Minehart, H; Yue, T

    1999-01-01

    Bone morphogenetic proteins (BMPs) and their serine/threonine kinase receptors have been identified in atherosclerotic arteries and vascular smooth muscle cells, respectively. Thus, BMPs (the largest subfamily of the TGF-beta superfamily) have been implicated in the pathogenesis of atherosclerosis. However, the origins of BMP biosynthesis and the functional roles of BMP in blood vessels are unclear. The present study explored BMP-2 gene expression in various human blood vessels and vascular cell types. Functional in vitro studies were also performed to determine the effects of recombinant human BMP-2 on migration (transwell assay) and proliferation ([3H]-thymidine incorporation) of human aortic vascular smooth muscle cells (HASMC). RT-PCR experiments revealed BMP-2 gene expression in normal and atherosclerotic human arteries as well as cultured human aortic and coronary vascular smooth muscle cells, human umbilical vein endothelial cells (HUVECs) and human macrophages. In cellular migration studies, incubation with BMP-2 produced efficacious (3H]-thymidine incorporation in HASMC at chemotaxic concentrations (

  3. Epidermal growth factor does not act as a primary cue for inducing developmental changes in suckling mouse jejunum.

    PubMed

    Ménard, D; Arsenault, P; Gallo-Payet, N

    1986-01-01

    The direct influence of epidermal growth factor (EGF) on the differentiation and proliferation of small intestine was studied in organ culture. Eight-day-old mouse small intestine was cultured during 2 days in serum-free Leibovitz L-15 medium alone or supplemented with EGF (50, 100, and 500 ng/ml) either at room temperature or at 37 degrees C. Brush border membrane hydrolytic activities, namely, sucrase, lactase, glucoamylase, trehalase, maltase, and alkaline phosphatase, were assayed in the intestinal tissue as well as in the culture medium. None of the brush border enzymic activities was affected by the addition of EGF to the culture medium. This lack of effect is not temperature dependent since it occurred both at room temperature and at 37 degrees C. The addition of hydrocortisone (10(-6) M) to the culture medium induced the appearance of sucrase activity and increased the activity of the other brush border enzymes. The simultaneous addition of EGF with hydrocortisone did not influence the response of the intestinal explants to hydrocortisone. The deoxyribonucleic acid (DNA) content was determined while DNA synthesis was evaluated by the incorporation of (3H)-thymidine. The addition of EGF did not affect DNA content or (3H)-thymidine incorporation into DNA either at room temperature or at 37 degrees C. The EGF binding to epithelial cells did not significantly vary throughout the culture period and a down-regulation process occurred in presence of EGF. These observations strongly suggest that EGF does not act as a primary cue for inducing developmental changes in suckling mouse small intestine. It is proposed that EGF induces a systemic reaction in vivo that then influences the neonatal small intestine. PMID:3491891

  4. Inhibitory effect of benzene metabolites on nuclear DNA synthesis in bone marrow cells

    SciTech Connect

    Lee, E.W.; Johnson, J.T.; Garner, C.D. )

    1989-01-01

    Effects of endogenously produced and exogenously added benzene metabolites on the nuclear DNA synthetic activity were investigated using a culture system of mouse bone marrow cells. Effects of the metabolites were evaluated by a 30-min incorporation of ({sup 3}H)thymidine into DNA following a 30-min interaction with the cells in McCoy's 5a medium with 10% fetal calf serum. Phenol and muconic acid did not inhibit nuclear DNA synthesis. However, catechol, 1,2,4-benzenetriol, hydroquinone, and p-benzoquinone were able to inhibit 52, 64, 79, and 98% of the nuclear DNA synthetic activity, respectively, at 24 {mu}M. In a cell-free DNA synthetic system, catechol and hydroquinone did not inhibit the incorporation of ({sup 3}H)thymidine triphosphate into DNA up to 24 {mu}M but 1,2,4-benzenetriol and p-benzoquinone did. The effect of the latter two benzene metabolites was completely blocked in the presence of 1,4-dithiothreitol (1 mM) in the cell-free assay system. Furthermore, when DNA polymerase {alpha}, which requires a sulfhydryl (SH) group as an active site, was replaced by DNA polymerase 1, which does not require an SH group for its catalytic activity, p-benzoquinone and 1,2,4-benzenetriol were unable to inhibit DNA synthesis. Thus, the data imply the p-benzoquinone and 1,2,4-benzenetriol inhibited DNA polymerase {alpha}, consequently resulting in inhibition of DNA synthesis in both cellular and cell-free DNA synthetic systems. The present study identifies catechol, hydroquinone, p-benzoquinone, and 1,2,4-benzenetriol as toxic benzene metabolites in bone marrow cells and also suggests that their inhibitory action on DNA synthesis is mediated by mechanism(s) other than that involving DNA damage as a primary cause.

  5. Vasoactive peptides upregulate mRNA expression and secretion of vascular endothelial growth factor in human airway smooth muscle cells.

    PubMed

    Alagappan, Vijay K T; Willems-Widyastuti, Anna; Seynhaeve, Ann L B; Garrelds, Ingrid M; ten Hagen, Timo L M; Saxena, Pramod R; Sharma, Hari S

    2007-01-01

    Airway remodeling and associated angiogenesis are documented features of asthma, of which the molecular mechanisms are not fully understood. Angiotensin (ANG)II and endothelin (ET)-1 are potent vasoconstricting circulatory hormones implicated in asthma. We investigated the effects of ANG II and ET-1 on human airway smooth muscle (ASM) cells proliferation and growth and examined the mRNA expression and release of the angiogenic peptide, vascular endothelial growth factor (VEGF). Serum deprived (48 h) human ASM cells were incubated with ANG II (100 nM) or ET-1 (10 nM) for 30 min, 1, 2, 4, 8, 16, and 24 h and the endogenous synthesis of VEGF was examined in relation to control cells receiving serum free culture medium. ET-1 induced time dependent DNA biosynthesis as determined by [3H]-thymidine incorporation assay. Using northern blot hybridization, we detected two mRNA species of 3.9 and 1.7 kb encoding VEGF in the cultured smooth muscle cells. Both ANG II and ET-1 induced the mRNA expression (two- to threefold) and secretion (1.8- to 2.8-fold) of VEGF reaching maximal levels between 4-8 h of incubation. Induced expression and release of VEGF declined after 8 h of ANG II incubation while levels remained elevated in the case of ET-1. The conditioned medium derived from ET-1-treated ASM cells induced [3H]-thymidine incorporation and cell number in porcine pulmonary artery endothelial as well as human umbilical vein endothelial cells. Moreover, the VEGF tyrosine kinase receptor inhibitor blocked the conditioned medium induced mitogenesis in endothelial cells. Our results suggest a potential role for ANG II and ET-1 in ASM cell growth and upregulation of VEGF that may participate in endothelial cell proliferation via paracrine mechanisms and thus causing pathological angiogenesis and vascular remodelling seen during asthma. PMID:17406064

  6. Rapid mercury assays

    SciTech Connect

    Szurdoki, S.; Kido, H.; Hammock, B.D.

    1996-10-01

    We have developed rapid assays with the potential of detecting mercury in environmental samples. our methods combine the simple ELISA-format with the selective, high affinity complexation of mercuric ions by sulfur-containing ligands. The first assay is based on a sandwich chelate formed by a protein-bound ligand immobilized on the wells of a microliter plate, mercuric ion of the analyzed sample, and another ligand conjugated to a reporter enzyme. The second assay involves competition between mercuric ions and an organomercury-conjugate to bind to a chelating conjugate. Several sulfur containing chelators (e.g., dithiocarbamates) and organomercurials linked to macromolecular carriers have been investigated in these assay formats. The assays detect mercuric ions in ppb/high ppt concentrations with high selectivity.

  7. Carbazole incorporated ratiometric chemosensor for Zn2+

    NASA Astrophysics Data System (ADS)

    Helal, Aasif; Kim, Hong-Seok

    2013-03-01

    An electron donating carbazole incorporated thiazole (3) based Zn2+ selective intrinsic chemosensor has been synthesized and investigated. It was found that electron donating substituents such as methyl and carbazole on chemosensor (1) produce remarkable red shift in emission upon complexation with Zn2+. The sensor shows a selective fluorescence response with Zn2+ over biologically relevant cations (Ca2+, Mg2+, Na+, and K+) and biologically non-relevant cations (Cd2+, In3+ and Ga3+) in an aqueous ethanol system. It also produce an enhancement in the quantum yield and a longer emission wavelength shift on Zn2+ binding with the potential of a ratiometric assay.

  8. 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.

  9. Magnesium incorporation into hydroxyapatite.

    PubMed

    Laurencin, Danielle; Almora-Barrios, Neyvis; de Leeuw, Nora H; Gervais, Christel; Bonhomme, Christian; Mauri, Francesco; Chrzanowski, Wojciech; Knowles, Jonathan C; Newport, Robert J; Wong, Alan; Gan, Zhehong; Smith, Mark E

    2011-03-01

    The incorporation of Mg in hydroxyapatite (HA) was investigated using multinuclear solid state NMR, X-ray absorption spectroscopy (XAS) and computational modeling. High magnetic field (43)Ca solid state NMR and Ca K-edge XAS studies of a ∼10% Mg-substituted HA were performed, bringing direct evidence of the preferential substitution of Mg in the Ca(II) position. (1)H and (31)P solid state NMR show that the environment of the anions is disordered in this substituted apatite phase. Both Density Functional Theory (DFT) and interatomic potential computations of Mg-substituted HA structures are in agreement with these observations. Indeed, the incorporation of low levels of Mg in the Ca(II) site is found to be more favourable energetically, and the NMR parameters calculated from these optimized structures are consistent with the experimental data. Calculations provide direct insight in the structural modifications of the HA lattice, due to the strong contraction of the M⋯O distances around Mg. Finally, extensive interatomic potential calculations also suggest that a local clustering of Mg within the HA lattice is likely to occur. Such structural characterizations of Mg environments in apatites will favour a better understanding of the biological role of this cation. PMID:21144581

  10. Microbiological assay of ketoconazole in shampoo.

    PubMed

    Staub, Inara; Schapoval, Elfrides E S; Bergold, Ana M

    2005-03-23

    Ketoconazole, an anti-fungal agent, is often incorporated in several pharmaceutical forms and in shampoo formulation it is known to be effective against fungal infection on the scalp. This paper describes a method to quantify ketoconazole in shampoo by comparing the cylinder plate assay and the HPLC method. The test organism used for the agar diffusion assay was Candida albicans ATCC 10231. Three different concentrations of ketoconazole were used for the diffusion assay. A mean zone diameter was obtained for each concentration. A standard curve was obtained by plotting the three values derived from the zone diameters. A prospective validation of the method showed that the method was linear (r = 0.9982), precise (R.S.D. = 2.57%) and accurate. The results obtained by the two methods were statistically evaluated by analysis of variance (ANOVA) and the results obtained indicate that there is no significant difference between these two methods. PMID:15725566

  11. 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

  12. 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.

  13. 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. PMID:27154596

  14. 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.

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

    SciTech Connect

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

    1988-12-01

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

  16. Implant of polymer containing pentacyclic triterpenes from Eugenia punicifolia inhibits inflammation and activates skeletal muscle remodeling.

    PubMed

    Leite, Paulo Emílio C; Lima-Araújo, Katia G; França, Guilherme R; Lagrota-Candido, Jussara; Santos, Wilson C; Quirico-Santos, Thereza

    2014-12-01

    Sustained chronic inflammation induces activation of genes involved in cellular proliferation and apoptosis, thereby causing skeletal muscle degeneration. To investigate in vitro effects of isolated pentacyclic triterpenes from Eugenia punicifolia (Ep-CM) upon signaling pathways involved in the regulation of skeletal muscle cell line proliferation, and in vivo muscular tissue remodeling. C2C12 cells were seeded on eight-well plates and [(3)H]-thymidine incorporation, TUNEL assays, mitochondria viability, zymography for matrix metalloproteases (MMPs), Western blot analysis for MAPKinase signaling pathway, NFκB activation and HMGB1 production subsequently determined under basal conditions and after Ep-CM treatment. A polymer containing Ep-CM was implanted on the volar surface of gastrocnemius muscles subjected to acute injury induced by bupivacaine for local slow and gradual release of bioactive compounds, and mice killed 4 days after surgery. Ep-CM inhibited proliferation of C2C12 myoblast cell line in a dose-dependent manner, confirmed by reduction of [(3)H]-thymidine uptake without affecting cell viability or inducing apoptosis. The cytostatic effect of Ep-CM occurred mainly via inhibition of phosphorylated extracellular signal-regulated kinase (pERK) activation and DNA synthesis, possibly inhibiting the G1 phase of the cell cycle, since Ep-CM increased pAkt and p27(kip1) but reduced Cyclin D1. Ep-CM in vitro treatment increased MMP-9 and MMP-2 activities of C2C12 myoblast cells, but reduced in vivo MMP-9 activity and acute muscular inflammation. Besides cytostatic and anti-inflammatory effects, Ep-CM pentacyclic triterpenes also contributed to degradation of basement membrane components by activating mechanisms of skeletal muscle remodeling in response to local injury. PMID:24830560

  17. An improved molecular assay for Tritrichomonas foetus.

    PubMed

    Grahn, R A; BonDurant, R H; van Hoosear, K A; Walker, R L; Lyons, L A

    2005-01-01

    Tritrichomonas foetus (T. foetus) is the causative agent of bovine trichomonosis, a sexually transmitted disease leading to abortion (from 1 to 8 months gestation), infertility, and occasional pyometra. The annual losses to the U.S. beef industry are estimated to be in the hundreds of millions of dollars. Currently, the "gold standard" diagnostic test for trichomonosis in most countries is the cultivation of live organisms from reproductive secretions. The cultured organisms can then be followed by PCR assays with primers that amplify T. foetus to the exclusion of all other trichomonad species. Thus, negative results present as null data, indistinguishable from failed PCR amplification during T. foetus specific amplification. Our newly developed assay improves previously developed PCR based techniques by using diagnostic size variants from within the internal transcribed spacer 1 (ITS1) region that is between the 18S rRNA and 5.8S rRNA subunits. This new PCR assay amplifies trichomonad DNA from a variety of genera and positively identifies the causative agent in the bovine trichomonad infection. This approach eliminates false negatives found in some current assays as well as identifying the causative agent of trichomonad infection. Additionally, our assay incorporates a fluorescently labeled primer enabling high sensitivity and rapid assessment of the specific trichomonad species. Moreover, electrophoretic separation of amplified samples can be outsourced, thus eliminating the need for diagnostic laboratories to purchase expensive analysis equipment. PMID:15619373

  18. 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.

  19. Against vaccine assay secrecy

    PubMed Central

    Herder, Matthew; Hatchette, Todd F; Halperin, Scott A; Langley, Joanne M

    2015-01-01

    Increasing the transparency of the evidence base behind health interventions such as pharmaceuticals, biologics, and medical devices, has become a major point of critique, conflict, and policy focus in recent years. Yet the lack of publicly available information regarding the immunogenicity assays upon which many important, widely used vaccines are based has received no attention to date. In this paper we draw attention to this critical public health problem by reporting on our efforts to secure vaccine assay information in respect of 10 vaccines through Canada's access to information law. We argue, under Canadian law, that the public health interest in having access to the methods for these laboratory procedures should override claims by vaccine manufacturers and regulators that this information is proprietary; and, we call upon several actors to take steps to ensure greater transparency with respect to vaccine assays, including regulators, private firms, researchers, research institutions, research funders, and journal editors. PMID:25826194

  20. 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.

  1. 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.

  2. 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.

  3. Macroautophagic cargo sequestration assays.

    PubMed

    Seglen, Per O; Luhr, Morten; Mills, Ian G; Sætre, Frank; Szalai, Paula; Engedal, Nikolai

    2015-03-01

    Macroautophagy, the process responsible for bulk sequestration and lysosomal degradation of cytoplasm, is often monitored by means of the autophagy-related marker protein LC3. This protein is linked to the phagophoric membrane by lipidation during the final steps of phagophore assembly, and it remains associated with autophagic organelles until it is degraded in the lysosomes. The transfer of LC3 from cytosol to membranes and organelles can be measured by immunoblotting or immunofluorescence microscopy, but these assays provide no information about functional macroautophagic activity, i.e., whether the phagophores are actually engaged in the sequestration of cytoplasmic cargo and enclosing this cargo into sealed autophagosomes. Moreover, accumulating evidence suggest that macroautophagy can proceed independently of LC3. There is therefore a need for alternative methods, preferably effective cargo sequestration assays, which can monitor actual macroautophagic activity. Here, we provide an overview of various approaches that have been used over the last four decades to measure macroautophagic sequestration activity in mammalian cells. Particular emphasis is given to the so-called "LDH sequestration assay", which measures the transfer of the autophagic cargo marker enzyme LDH (lactate dehydrogenase) from the cytosol to autophagic vacuoles. The LDH sequestration assay was originally developed to measure macroautophagic activity in primary rat hepatocytes. Subsequently, it has found use in several other cell types, and in this article we demonstrate a further validation and simplification of the method, and show that it is applicable to several cell lines that are commonly used to study autophagy. PMID:25576638

  4. 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

  5. Incorporation of copper into lysyl oxidase.

    PubMed

    Kosonen, T; Uriu-Hare, J Y; Clegg, M S; Keen, C L; Rucker, R B

    1997-10-01

    Lysyl oxidase is a copper-dependent enzyme involved in extracellular processing of collagens and elastin. Although it is known that copper is essential for the functional activity of the enzyme, there is little information on the incorporation of copper. In the present study we examined the insertion of copper into lysyl oxidase using 67Cu in cell-free transcription/translation assays and in normal skin fibroblast culture systems. When a full-length lysyl oxidase cDNA was used as a template for transcription/translation reactions in vitro, unprocessed prolysyl oxidase appeared to bind copper. To examine further the post-translational incorporation of copper into lysyl oxidase, confluent skin fibroblasts were incubated with inhibitors of protein synthesis (cycloheximide, 10 microg/ml), glycosylation (tunicamycin, 10 microg/ml), protein secretion (brefeldin A, 10 microg/ml) and prolysyl oxidase processing (procollagen C-peptidase inhibitor, 2.5 microg/ml) together with 300 microCi of carrier-free 67Cu. It was observed that protein synthesis was a prerequisite for copper incorporation, but inhibition of glycosylation by tunicamycin did not affect the secretion of 67Cu as lysyl oxidase. Brefeldin A inhibited the secretion of 67Ci-labelled lysyl oxidase by 46%, but the intracellular incorporation of copper into lysyl oxidase was not affected. In addition, the inhibition of the extracellular proteolytic processing of prolysyl oxidase to lysyl oxidase had minimal effects on the secretion of protein-bound 67Cu. Our results indicate that, similar to caeruloplasmin processing [Sato and Gitlin (1991) J. Biol. Chem. 266, 5128-5134], copper is inserted into prolysyl oxidase independently of glycosylation. PMID:9355764

  6. Optimized Diagnostic Assays Based on Redox Tagged Bioreceptive Interfaces.

    PubMed

    Bedatty Fernandes, Flavio C; Patil, Amol V; Bueno, Paulo R; Davis, Jason J

    2015-12-15

    Among the numerous label free electronic biomarker assay methodologies now available, impedance based electrochemical capacitance spectroscopy (ECS), based upon mapping the perturbations in interfacial charging of redox elements incorporated into a biologically receptive interface, has recently been shown to be a convenient and highly sensitive mode of transduction and one which, additionally, requires no predoping of analytical solution. We present, herein, a data acquisition and analysis methodology based on frequency resolved immittance function analysis. Ultimately, this enables both a maximization of assay sensitivity and a reduction in assay acquisition time by an order of magnitude. PMID:26583592

  7. Neutral red assay in minimum fungicidal concentrations of antifungal agents.

    PubMed

    Fukuda, T; Naka, W; Tajima, S; Nishikawa, T

    1996-01-01

    We assayed the fungicidal effects of antifungal agents using neutral red staining. Fungal elements of Trichophyton mentagrophytes and T. rubrum were treated with various concentrations of antifungal agents in 96-well filtration plates and then stained with neutral red. The amount of neutral red incorporated by the surviving viable cells was determined from the automated spectrophotometric readings at 550 nm. The minimum fungicidal concentrations (MFCs) of antifungal agents determined by this assay correlated well with those determined by conventional assay. This newly developed procedure should provide a rapid, reproducible, quantitative, qualitative and semi-automated susceptibility test for determination of the MFCs of the fungicidal agents. PMID:8912170

  8. 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.

  9. Comparison of Luminescence ADP Production Assay and Radiometric Scintillation Proximity Assay for Cdc7 Kinase

    PubMed Central

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

    2013-01-01

    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 [33P]-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 [33P]-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. PMID:21564015

  10. Radioreceptor assay for oxyphenonium.

    PubMed

    Ensing, K; de Zeeuw, R A

    1984-01-01

    The development of a radioreceptor assay for the quaternary anticholinergic drug, oxyphenonium, in plasma is reported. It is based on competition between this drug and 3H-dexetimide for binding to muscarinic receptors. After ion pair extraction and reextraction, the drug can be determined in plasma at concentrations down to a value of 100 pg/ml. This permits pharmacokinetic studies to be made after inhalation of oxyphenonium. PMID:6428927

  11. Bioanalytical advances in assays for C-reactive protein.

    PubMed

    Vashist, Sandeep Kumar; Venkatesh, A G; Marion Schneider, E; Beaudoin, Christopher; Luppa, Peter B; Luong, John H T

    2016-01-01

    This review presents advances in assays for human C-reactive protein (CRP), the most important biomarker of infection and inflammation for a plethora of diseases and pathophysiological conditions. Routine assays in clinical settings are based on analyzers, enzyme-linked immunosorbent assays and lateral flow assays. However, assays encompassing novel sensing schemes, improved chemistry, signal enhancement, lab-on-a-chip, microfluidics and smartphone detection, have emerged in recent years. The incorporation of immune-transducing chips or sensing interfaces with nanomaterials enables multiplexing analysis of CRP with co-existing biomarkers. However, there are still considerable challenges in the development of rapid diagnostics for both pentameric and monomeric CRP forms. PMID:26717866

  12. Robust quantitative scratch assay

    PubMed Central

    Vargas, Andrea; Angeli, Marc; Pastrello, Chiara; McQuaid, Rosanne; Li, Han; Jurisicova, Andrea; Jurisica, Igor

    2016-01-01

    The wound healing assay (or scratch assay) is a technique frequently used to quantify the dependence of cell motility—a central process in tissue repair and evolution of disease—subject to various treatments conditions. However processing the resulting data is a laborious task due its high throughput and variability across images. This Robust Quantitative Scratch Assay algorithm introduced statistical outputs where migration rates are estimated, cellular behaviour is distinguished and outliers are identified among groups of unique experimental conditions. Furthermore, the RQSA decreased measurement errors and increased accuracy in the wound boundary at comparable processing times compared to previously developed method (TScratch). Availability and implementation: The RQSA is freely available at: http://ophid.utoronto.ca/RQSA/RQSA_Scripts.zip. The image sets used for training and validation and results are available at: (http://ophid.utoronto.ca/RQSA/trainingSet.zip, http://ophid.utoronto.ca/RQSA/validationSet.zip, http://ophid.utoronto.ca/RQSA/ValidationSetResults.zip, http://ophid.utoronto.ca/RQSA/ValidationSet_H1975.zip, http://ophid.utoronto.ca/RQSA/ValidationSet_H1975Results.zip, http://ophid.utoronto.ca/RQSA/RobustnessSet.zip, http://ophid.utoronto.ca/RQSA/RobustnessSet.zip). Supplementary Material is provided for detailed description of the development of the RQSA. Contact: juris@ai.utoronto.ca Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26722119

  13. Using Models that Incorporate Uncertainty

    ERIC Educational Resources Information Center

    Caulkins, Jonathan P.

    2002-01-01

    In this article, the author discusses the use in policy analysis of models that incorporate uncertainty. He believes that all models should consider incorporating uncertainty, but that at the same time it is important to understand that sampling variability is not usually the dominant driver of uncertainty in policy analyses. He also argues that…

  14. Molecular dissection of membrane-transport proteins: mass spectrometry and sequence determination of the galactose-H+ symport protein, GalP, of Escherichia coli and quantitative assay of the incorporation of [ring-2-13C]histidine and (15)NH(3).

    PubMed Central

    Venter, Henrietta; Ashcroft, Alison E; Keen, Jeffrey N; Henderson, Peter J F; Herbert, Richard B

    2002-01-01

    The molecular mass of the galactose-H(+) symport protein GalP, as its histidine-tagged derivative GalP(His)(6), has been determined by electrospray MS (ESI-MS) with an error of <0.02%. One methionine residue, predicted to be present from the DNA sequence, was deduced to be absent. This is a significant advance on the estimation of the molecular masses of membrane-transport proteins by SDS/PAGE, where there is a consistent under-estimation of the true molecular mass due to anomalous electrophoretic migration. Addition of a size-exclusion chromatography step after Ni(2+)-nitrilotriacetate affinity purification was essential to obtain GalP(His)(6) suitable for ESI-MS. Controlled trypsin, trypsin+chymotrypsin and CNBr digestion of the protein yielded peptide fragments suitable for ESI-MS and tandem MS analysis, and accurate mass determination of the derived fragments resulted in identification of 82% of the GalP(His)(6) protein. Tandem MS analysis of selected peptides then afforded 49% of the actual amino acid sequence of the protein; the absence of the N-terminal methionine was confirmed. Matrix-assisted laser-desorption ionization MS allowed identification of one peptide that was not detected by ESI-MS. All the protein/peptide mass and sequence determinations were in accord with the predictions of amino acid sequence deduced from the DNA sequence of the galP gene. [ring-2-(13)C]Histidine was incorporated into GalP(His)(6) in vivo, and ESI-MS analysis enabled the measurement of a high (80%) and specific incorporation of label into the histidine residues in the protein. MS could also be used to confirm the labelling of the protein by (15)NH(3) (93% enrichment) and [(19)F]tryptophan (83% enrichment). Such MS measurements will serve in the future analysis of the structures of membrane-transport proteins by NMR, and of their topology by indirect techniques. PMID:11931651

  15. Incorporation of reporter-labeled nucleotides by DNA polymerases.

    PubMed

    Anderson, Jon P; Angerer, Bernhard; Loeb, Lawrence A

    2005-02-01

    The incorporation of fluorescently labeled nucleotides into DNA by DNA polymerases has been used extensively for tagging genes and for labeling DNA. However, we lack studies comparing polymerase efficiencies for incorporating different fluorescently labeled nucleotides. We analyzed the incorporation of fluorescent deoxynucleoside triphosphates by 10 different DNA polymerases, representing a cross-section of DNA polymerases from families A, B, and reverse transcriptase. The substitution of one or more different reporter-labeled nucleotides for the cognate nucleotides was initially investigated by using an in vitro polymerase extension filter-binding assay with natural DNA as a template. Further analysis on longer DNA fragments containing one or more nucleotide analogs was performed using a newly developed extension cut assay. The results indicate that incorporation of fluorescent nucleotides is dependent on the DNA polymerase, fluorophore, linker between the nucleotide and the fluorophore, and position for attachment of the linker and the cognate nucleotide. Of the polymerases tested, Taq and Vent exo DNA polymerases were most efficient at incorporating a variety of fluorescently labeled nucleotides. This study suggests that it should be feasible to copy DNA with reactions mixtures that contain all four fluorescently labeled nucleotides allowing for high-density labeling of DNA. PMID:15727132

  16. Macrophage Inflammatory Assay

    PubMed Central

    Ylostalo, Joni H.

    2016-01-01

    Macrophages represent a widely distributed and functionally diverse population of innate myeloid cells involved in inflammatory response to pathogens, tissue homeostasis and tissue repair (Murray and Wynn, 2011). Macrophages can be broadly grouped into two subpopulations with opposing activites: M1 or pro-inflammatory macrophages that promote T-helper type 1 (Th1) cell immunity and tissue damage, and M2 or anti-inflammatory/alternatively activated macrophages implicated in Th2 response and resolution of inflammation. Here we describe a rapid assay we used previously to monitor changes in pro-inflammatory and anti-inflammatory cytokine production by lipopolysaccharide (LPS)-activated macrophages in response to therapeutic paracrine factors produced by adult stem cells (Bartosh et al., 2010; Ylostalo et al., 2012; Bartosh et al., 2013). The assay can be adapted appropriately to test macrophage response to other agents as well that will be referred to herein as ‘test reagents’ or ‘test compounds’. In this protocol, the mouse macrophage cell line J774A.1 is expanded as an adherent monolayer on petri dishes allowing for the cells to be harvested easily without enzymes or cell scrapers that can damage the cells. The macropahges are then stimulated in suspension with LPS and seeded into 12-well cell culture plates containing the test reagents. After 16–18 h, the medium conditioned by the macrophages is harvested and the cytokine profile in the medium determined with enzyme-linked immunosorbent assays (ELISA). We routinely measure levels of the pro-inflammtory cytokine TNF-alpha and the anti-inflammatory cytokine interleukin-10 (IL-10).

  17. C. elegans chemotaxis assay.

    PubMed

    Margie, Olivia; Palmer, Chris; Chin-Sang, Ian

    2013-01-01

    Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior. The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress (1-10). The method described here aims to address these issues by modifying the assay developed by Bargmann et al.(1). A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired. PMID:23644543

  18. 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+.

  19. 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+.

  20. Physicochemical characterization of surfactant incorporating vesicles that incorporate colloidal magnetite.

    PubMed

    de Melo Barbosa, Raquel; Luna Finkler, Christine L; Bentley, Maria Vitória L B; Santana, Maria Helena A

    2013-03-01

    Drug administration through the transdermal route has optimized for the comfort of patients and easy application. However, the main limitation of transdermal drug delivery is the impermeability of the human skin. Recent advances on improvement of drug transport through the skin include elastic liposomes as a penetration enhancer. Entrapment of ferrofluids in the core of liposomes produces magnetoliposomes, which can be driven by a high-gradient magnetic field. The association of both strategies could enhance the penetration of elastic liposomes. This work relies on the preparation and characterization of elastic-magnetic liposomes designed to permeate through the skin. The incorporation of colloidal magnetite and the elastic component, octaethylene glycol laurate (PEG-8-L), in the structure of liposomes were evaluated. The capability of the elastic magnetoliposomes for permeation through nanopores of two stacked polycarbonate membranes was compared to conventional and elastic liposomes. Magnetite incorporation was dependent on vesicle diameter and size distribution as well as PEG-8-L incorporation into liposomes, demonstrating the capability of the fluid bilayer to accommodate the surfactant without disruption. On the contrary, PEG-8-L incorporation into magnetoliposomes promoted a decrease of average diameter and a lower PEG-8-L incorporation percentage as a result of reduction on the fluidity of the bilayer imparted by iron incorporation into the lipid structure. Elastic liposomes demonstrated an enhancement of the deformation capability, as compared with conventional liposomes. Conventional and elastic magnetoliposomes presented a reduced capability for deformation and permeation. PMID:23363304

  1. 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.

  2. TOTAL CULTURABLE VIRUS QUANTAL ASSAY

    EPA Science Inventory

    This chapter describes a quantal method for assaying culturable human enteric viruses from water matrices. The assay differs from the plaque assay described in Chapter 10 (December 1987 Revision) in that it is based upon the direct microscopic viewing of cells for virus-induced ...

  3. INTERIM PROCEDURES FOR CONDUCTING THE 'SALMONELLA'/MICROSOMAL MUTAGENICITY ASSAY (AMES TEST)

    EPA Science Inventory

    The Ames assay is a rapid-screening procedure for determining the presence of mutagenic and carcinogenic pure chemicals, complex environmental mixtures, and commercial products. The procedure described incorporates modifications designed to make Ames testing data more precise and...

  4. DEVELOPMENT OF AN ENZYME-LINKED IMMUNOSORBENT ASSAY FOR THE DETECTION OF ANTIBODY TO EPIZOOTIC HEMORRHAGIC DISEASE OF DEER VIRUS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An enzyme linked immunosorbent assay has been developed to detect antibodies to epizootic hemorrhagic disease of deer virus (EHDV). The assay incorporates a monoclonal antibody to EHDV serotype 2 (EHDV-2) that demonstrates specificity for the viral structural protein, VP7. The assay was evaluated ...

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

    PubMed

    Nakajima, Dai; Tabata, Yasuhiko; Sato, Soh

    2015-09-01

    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. PMID:26481592

  6. 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. PMID:26498795

  7. Molecular inversion probe assay.

    PubMed

    Absalan, Farnaz; Ronaghi, Mostafa

    2007-01-01

    We have described molecular inversion probe technologies for large-scale genetic analyses. This technique provides a comprehensive and powerful tool for the analysis of genetic variation and enables affordable, large-scale studies that will help uncover the genetic basis of complex disease and explain the individual variation in response to therapeutics. Major applications of the molecular inversion probes (MIP) technologies include targeted genotyping from focused regions to whole-genome studies, and allele quantification of genomic rearrangements. The MIP technology (used in the HapMap project) provides an efficient, scalable, and affordable way to score polymorphisms in case/control populations for genetic studies. The MIP technology provides the highest commercially available multiplexing levels and assay conversion rates for targeted genotyping. This enables more informative, genome-wide studies with either the functional (direct detection) approach or the indirect detection approach. PMID:18025701

  8. 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.

  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. PMID:25256511

  10. Depth distribution of bacterial production in a stratified lake with an anoxic hypolimnion

    SciTech Connect

    McDonough, R.J.; Sanders, R.W.; Porter, K.G.; Kirchman, D.L.

    1986-11-01

    The purpose of this study was to determine the depth distribution of bacterial biomass and production in a stratified lake and to test techniques to measure bacterial production in anaerobic waters. Bacterial abundance and incorporation of both (/sup 3/H)thymidine and (/sup 3/H)leucine into protein were highest in the metalimnion, at the depth at which oxygen first became unmeasurable. In contrast, (/sup 3/H)thymidine incorporation into DNA was highest in the epilimnion. Recovery of added (/sup 3/H)DNA was about 90% in waters in which the portion of (/sup 3/H)thymidine incorporation into DNA was about 40%. At least some obligate anaerobic bacteria were capable of assimilating thymidine since aeration of anaerobic hypolimnion waters substantially inhibited thymidine incorporation. The depth profile of bacterial production estimated from total thymidine and leucine incorporation and the frequency of dividing cells were all similar, with maximal rates in the metalimnion. However, estimates of bacterial production based on frequency of dividing cells and leucine incorporation were usually significantly higher than estimates based on thymidine incorporation (using conversion factors from the literature), especially in anaerobic hypolimnion waters. These data indicate that the thymidine approach must be examined carefully if it is to be applied to aquatic systems with low oxygen concentrations. The results also indicate that the interface between the aerobic epilimnion and anaerobic hypolimnion is the site of intense bacterial mineralization and biomass production which deserves further study.

  11. 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

  12. Growth promoting effect of recombinant interleukin I and tumor necrosis factor for human astrocytoma cells

    SciTech Connect

    Giulian, D.; Dinarello, C.A.; Brown, D.C.; Lachman, L.B.

    1986-03-01

    Human IL I has been demonstrated to stimulate the growth of rat astrocytes in vitro. To determine if IL I has a similar growth promoting effect upon human brain cells, two astrocytoma cell lines were tested for their ability to incorporate /sup 3/H-thymidine in response to various types of IL I and tumor necrosis factor (TNF). The U373 astrocytoma was found to respond mitogenically to human native IL I, human recombinant IL I, rat IL I and murine recombinant IL I. The cell line failed to respond to recombinant IL 2 and recombinant ..cap alpha.. and ..gamma.. interferon. The sensitivity of the U373 cells paralleled the murine thymocyte assay for IL I. Interestingly, the U373 responded mitogenically to recombinant TNF prepared by two different companies, thus indicating that TNF stimulates proliferation of this cell line and does not lead to cell death. In the murine thymocyte assay for IL I, TNF was not active. The results indicate that 1) both IL I and TNF are mitogenic for a human astrocytoma cell line and 2) the U373 cells may be used to assay both IL I and TNF in a highly sensitive mitogenic assay.

  13. 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.

  14. An assay for adjuvanticity

    PubMed Central

    Dresser, D. W.

    1968-01-01

    Adult mice injected with an adequate amount of a non-immunogenic antigen progress to a specific state of immunological paralysis, unless a substance with `extrinsic' adjuvanticity is injected before the induction of paralysis is completed. Consequently incipiently paralysed mice can be used to assay substances for adjuvanticity. Conventional adjuvants such as Freund's adjuvant and pertussis possess adjuvanticity; other substances with varying degrees of adjuvanticity are listed in the tables. It has been shown that the adjuvanticity effect of an injection of pertussis lasts for only a few days, although the effect of such an injection of pertussis on phagocytosis of carbon particles does not reach a maximum until 2 weeks after the injection. The dose-effectiveness of alum precipitated (highly phagocytosable) bovine γ-globulin was greatly increased by the intraperitoneal injection of pertussis. The evidence is considered to be incompatible with increased phagocytosis being either an essential factor in the role of pertussis as a conventional adjuvant, or in the adjuvanticity effect of pertussis. PMID:4179956

  15. Membrane Flotation Assay

    PubMed Central

    Vogt, Dorothee A; Ott, Melanie

    2016-01-01

    Many postitive-stranded RNA viruses, such as Hepatitis C virus (HCV), highjack cellular membranes, including the Golgi, ER, mitchondria, lipid droplets, and utilize them for replication of their RNA genome or assembly of new virions. By investigating how viral proteins associate with cellular membranes we will better understand the roles of cellular membranes in the viral life cycle. Our lab has focused specifically on the role of lipid droplets and lipid-rich membranes in the life cycle of HCV. To analyze the role of lipid-rich membranes in HCV RNA replication, we utilized a membrane flotation assay based on an 10–20–30% iodixanol density gradient developed by Yeaman et al. (2001). This gradient results in a linear increase in density over almost the entire length of the gradient, and membrane particles are separated in the gradient based on their buoyant characteristics. To preserve membranes in the lysate, cells are broken mechanically in a buffer lacking detergent. The cell lysate is loaded on the bottom of the gradient, overlaid with the gradient, and membranes float up as the iodixanol gradient self-generates. The lipid content of membranes and the concentration of associated proteins will determine the separation of different membranes within the gradient. After centrifugation, fractions can be sampled from the top of the gradient and analyzed using standard SDS-PAGE and western blot analysis for proteins of interest.

  16. 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…

  17. 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.

  18. 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.

  19. Epiderstatin, a new inhibitor of the mitogenic activity induced by epidermal growth factor. I. Taxonomy, fermentation, isolation and characterization.

    PubMed

    Osada, H; Sonoda, T; Kusakabe, H; Isono, K

    1989-11-01

    Inhibitors of mitogenic activity induced by epidermal growth factor (EGF) were screened from culture broths of soil microorganisms. A strain of actinomycetes has been found to produce a new glutarimide antibiotic named epiderstatin which inhibits the incorporation of [3H]thymidine into quiescent animal cells stimulated by EGF. Taxonomic studies have revealed that the producing strain belongs to a subspecies of Streptomyces pulveraceus, thus the name, Streptomyces pulveraceus subsp. epiderstagenes was given to this strain. The molecular formula (C15H20N2O4) and UV profile (lambda max 295 nm) of the antibiotic are distinct from other known antibiotics. It inhibited the incorporation of [3H]thymidine into quiescent cells stronger than into growing cells. PMID:2584144

  20. Estrogenic regulation of Leydig cell development in the rat

    SciTech Connect

    Myers, R.B.

    1989-01-01

    Initial studies demonstrated that treatment of male rats with estradiol for a period of four days resulted in a reduction in {sup 3}H-thymidine incorporation of isolated interstitial cells. Furthermore, {sup 3}H-thymidine incorporation in interstitial cells of 33 day old rats was inhibited by the addition of estradiol in vitro. Subsequent studies were performed in the ethylene dimethanesulphonate (EDS) treated rat. Leydig cells were rapidly destroyed after EDS administration as determined by hCG binding, steroid synthesis and morphological studies. A significant finding was the production of 5{alpha}-androstane-3{alpha},17{beta}-diol by regeneration Leydig cells of the EDS treated rat. In subsequent studies, rats received daily treatment with estradiol and/or hCG/LH after EDS treatment. Estradiol treatment had no effect on Leydig cell degeneration. Leydig cell regeneration, however, did not occur in the estradiol treated rat.

  1. Challenge assay in vitro using lymphocyte blastogenesis for the contact hypersensitivity assay.

    PubMed

    Kashima, R; Okada, J; Ikeda, Y; Yoshizuka, N

    1993-10-01

    To confirm positivity in routine guinea pig studies, contact allergenicity was investigated by a challenge assay in vitro using a co-culture of autologous lymphocytes passed through a nylon-wool column and antigen-presenting cells (APCs) modified with or without antigen. Proliferation of the lymphocytes primed with ovalbumin and/or 2,4-dinitrochlorobenzene was antigen specific and dependent on the presence of APCs (peripheral blood monocytes, splenic macrophages and macrophages induced by liquid paraffin). For another nine haptens, primed lymphocytes proliferated significantly more than control lymphocytes; the stimulation index (SI; ratio between [3H]methylthymidine ([3H]TdR) incorporation of lymphocytes with antigen-modified APCs and [3H]TdR incorporation of lymphocytes with APCs not modified by antigen) was 1.6-4.8 in sensitized animals whereas it was about 1.0 in control animals. Sodium dodecyl sulfate did not cause lymphocyte proliferation. The SI value in vitro was correlated with both the positive rate in vivo (r = 0.736) and the mean response score in vivo (r = 0.645). Thus, it was possible to confirm that positivity in routine experiments was a true sign of allergy. A combination of this assay and short-term animal studies would provide an efficient assessment of the allergic potential of chemicals. PMID:8225135

  2. 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

  3. 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. PMID:25027375

  4. The assay of diphtheria toxin

    PubMed Central

    Gerwing, Julia; Long, D. A.; Mussett, Marjorie V.

    1957-01-01

    A precise assay of diphtheria toxin is described, based on the linear relationship between the diameter of the skin reaction to, and logarithm of the dose of, toxin. It eliminates the need for preliminary titrations, is economical, provides information about the slope of the log-dose response lines and, therefore, of the validity of the assay, and yields limits of error of potency from the internal evidence of the assay. A study has been made of the effects of avidity, combining power, toxicity and buffering on the assay of diphtheria toxins against the International Standards for both Diphtheria Antitoxin and Schick-Test Toxin. All the toxins assayed against the standard toxin, whatever their other properties might be, gave log-dose response lines of similar slope provided that they were diluted in buffered physiological saline. The assays were therefore valid. These experiments were repeated concurrently in non-immune and in actively immunized guinea-pigs, and comparable figures for potency obtained in both groups. The result was not significantly affected by the avidity or combining power of the toxin. However, non-avid toxins gave low values in Schick units when assayed, by the Römer & Sames technique, in terms of the International Standard for Diphtheria Antitoxin. The problem of the ultimate standard and the implications of these findings are discussed. PMID:13511133

  5. Transwell(®) invasion assays.

    PubMed

    Marshall, John

    2011-01-01

    The need to identify inhibitors of cancer invasion has driven the development of quantitative in vitro invasion assays. The most common assays used are based on the original Boyden assay system. Today commercially available plastic inserts for multi-well plates, which possess a cell-permeable membrane, as typified by Transwell(®) Permeable Supports, permit accurate repeatable invasion assays. When placed in the well of a multi-well tissue culture plate these inserts create a two-chamber system separated by the cell-permeable membrane. To create an invasion assay the pores in the membrane are blocked with a gel composed of extracellular matrix that is meant to mimic the typical matrices that tumour cells encounter during the invasion process in vivo. By placing the cells on one side of the gel and a chemoattractant on the other side of the gel, invasion is determined by counting those cells that have traversed the cell-permeable membrane having invaded towards the higher concentration of chemoattractant. In this chapter, in addition to protocols for performing Transwell invasion assays, there is consideration of the limitations of current assay designs with regard to available matrices and the absence of tumour microenvironment cells. PMID:21748672

  6. Semiconductor Electronic Label-Free Assay for Predictive Toxicology

    PubMed Central

    Mao, Yufei; Shin, Kyeong-Sik; Wang, Xiang; Ji, Zhaoxia; Meng, Huan; Chui, Chi On

    2016-01-01

    While animal experimentations have spearheaded numerous breakthroughs in biomedicine, they also have spawned many logistical concerns in providing toxicity screening for copious new materials. Their prioritization is premised on performing cellular-level screening in vitro. Among the screening assays, secretomic assay with high sensitivity, analytical throughput, and simplicity is of prime importance. Here, we build on the over 3-decade-long progress on transistor biosensing and develop the holistic assay platform and procedure called semiconductor electronic label-free assay (SELFA). We demonstrate that SELFA, which incorporates an amplifying nanowire field-effect transistor biosensor, is able to offer superior sensitivity, similar selectivity, and shorter turnaround time compared to standard enzyme-linked immunosorbent assay (ELISA). We deploy SELFA secretomics to predict the inflammatory potential of eleven engineered nanomaterials in vitro, and validate the results with confocal microscopy in vitro and confirmatory animal experiment in vivo. This work provides a foundation for high-sensitivity label-free assay utility in predictive toxicology. PMID:27117746

  7. Semiconductor Electronic Label-Free Assay for Predictive Toxicology.

    PubMed

    Mao, Yufei; Shin, Kyeong-Sik; Wang, Xiang; Ji, Zhaoxia; Meng, Huan; Chui, Chi On

    2016-01-01

    While animal experimentations have spearheaded numerous breakthroughs in biomedicine, they also have spawned many logistical concerns in providing toxicity screening for copious new materials. Their prioritization is premised on performing cellular-level screening in vitro. Among the screening assays, secretomic assay with high sensitivity, analytical throughput, and simplicity is of prime importance. Here, we build on the over 3-decade-long progress on transistor biosensing and develop the holistic assay platform and procedure called semiconductor electronic label-free assay (SELFA). We demonstrate that SELFA, which incorporates an amplifying nanowire field-effect transistor biosensor, is able to offer superior sensitivity, similar selectivity, and shorter turnaround time compared to standard enzyme-linked immunosorbent assay (ELISA). We deploy SELFA secretomics to predict the inflammatory potential of eleven engineered nanomaterials in vitro, and validate the results with confocal microscopy in vitro and confirmatory animal experiment in vivo. This work provides a foundation for high-sensitivity label-free assay utility in predictive toxicology. PMID:27117746

  8. Evaluation of co-stimulatory effects of Tamarindus indica L. on MNU-induced colonic cell proliferation.

    PubMed

    Shivshankar, Pooja; Shyamala Devi, Chennam Srinivasulu

    2004-08-01

    Colonic cell proliferation is the prerequisite for the genesis of cancer. Experimental and epidemiologic evidence indicate dietary factors to be one of the commonest predisposing factors in the development of several types of cancers including large intestine. Here we have investigated the role of the fruit pulp of Tamarindus indica L. (TI), a tropical plant-derived food material, on the proliferating colonic mucosa using Swiss albino mice. Crypt cell proliferation rate (CCPR), on histological basis and [3H]-thymidine incorporation assay were chosen to evaluate the modulating potential of TI per se and in response to a subacute dose of N-nitroso N'-methyl urea (MNU). Descending colonic segment showed greater rate of cell proliferation than the ascending colon and cecum tissues isolated from the group 2 (TI-per se) when compared with group 1 (negative controls). It also revealed a positive correlation with the incorporation studies. Significant increase in the CCPR and radiolabeled precursor incorporation (p <0.001) was observed in MNU-induced+TI fed group of animals (group 4) in all the three segments when compared with control diet fed normal (group 1) as well as MNU-induced (group 3) animals. This study therefore indicates a co-stimulatory effect of TI on MNU-induced colonic cell kinetics. PMID:15207373

  9. 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.

  10. Cutaneous inflammation and proliferation in vitro: differential effects and mode of action of topical glucocorticoids.

    PubMed

    Lange, K; Kleuser, B; Gysler, A; Bader, M; Maia, C; Scheidereit, C; Korting, H C; Schäfer-Korting, M

    2000-01-01

    The nonhalogenated double ester of prednisolone, prednicarbate (PC), is the first topical glucocorticoid with an improved benefit/risk ratio verified clinically and in vitro. To evaluate if this is due to unique characteristics of this steroid, a new compound created according to an identical concept, prednisolone 17-ethylcarbonate, 21-phenylacetate (PEP), and the new halogenated monoester desoximetasone 21-cinnamate (DCE) were tested and compared to PC, desoximetasone (DM) and betamethasone 17-valerate (BMV). Isolated foreskin keratinocytes served for in vitro investigations of anti-inflammatory processes in the epidermis, fibroblasts of the same origin were used to investigate the atrophogenic potential. Inflammation was induced by TNFalpha, resulting in an increased interleukin 1alpha (Il-1alpha) synthesis. As quantified by ELISA, all drugs significantly reduced Il-1alpha production. But PC and BMV appeared particularly potent, followed by DM and the two new congeners, which revealed minor anti-inflammatory activity. Glucocorticoid esters including PEP are rapidly degraded in keratinocytes (85% within 12 h). Hence, a ribonuclease protection assay of Il-1alpha mRNA was performed allowing short incubation times and thus minimizing biodegradation. This assay confirmed the anti-inflammatory potency of native PC and BMV. In contrary DCE and PEP did not reduce Il-1alpha mRNA to a significant extent. Therefore PEP acts as a prodrug only. In fibroblasts, Il-1alpha and Il-6 syntheses indicate proliferation and inflammation, respectively. Whereas PC and PEP inhibited Il-1alpha and Il-6 production in fibroblasts only to a minor extent, cytokine synthesis was strongly affected by the conventional glucocorticoids BMV and DM, but also by DCE. The minor unwanted effect of PC and PEP on fibroblasts was also reflected by their low influence on cell proliferation as derived from (3)H-thymidine incorporation. Again, more pronounced antiproliferative features were seen with the

  11. Uranium incorporation into amorphous silica.

    PubMed

    Massey, Michael S; Lezama-Pacheco, Juan S; Nelson, Joey M; Fendorf, Scott; Maher, Kate

    2014-01-01

    High concentrations of uranium are commonly observed in naturally occurring amorphous silica (including opal) deposits, suggesting that incorporation of U into amorphous silica may represent a natural attenuation mechanism and promising strategy for U remediation. However, the stability of uranium in opaline silicates, determined in part by the binding mechanism for U, is an important factor in its long-term fate. U may bind directly to the opaline silicate matrix, or to materials such as iron (hydr)oxides that are subsequently occluded within the opal. Here, we examine the coordination environment of U within opaline silica to elucidate incorporation mechanisms. Precipitates (with and without ferrihydrite inclusions) were synthesized from U-bearing sodium metasilicate solutions, buffered at pH ∼ 5.6. Natural and synthetic solids were analyzed with X-ray absorption spectroscopy and a suite of other techniques. In synthetic amorphous silica, U was coordinated by silicate in a double corner-sharing coordination geometry (Si at ∼ 3.8-3.9 Å) and a small amount of uranyl and silicate in a bidentate, mononuclear (edge-sharing) coordination (Si at ∼ 3.1-3.2 Å, U at ∼ 3.8-3.9 Å). In iron-bearing synthetic solids, U was adsorbed to iron (hydr)oxide, but the coordination environment also contained silicate in both edge-sharing and corner-sharing coordination. Uranium local coordination in synthetic solids is similar to that of natural U-bearing opals that retain U for millions of years. The stability and extent of U incorporation into opaline and amorphous silica represents a long-term repository for U that may provide an alternative strategy for remediation of U contamination. PMID:24984107

  12. Incorporation of antimicrobial compounds in mesoporous silica film monolith.

    PubMed

    Izquierdo-Barba, Isabel; Vallet-Regí, María; Kupferschmidt, Natalia; Terasaki, Osamu; Schmidtchen, Artur; Malmsten, Martin

    2009-10-01

    Incorporation of the antimicrobial peptide LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES), as well as low molecular weight antimicrobial chlorhexidine, into mesoporous silica was obtained using an EISA one-pot synthesis method. FTIR confirmed efficient encapsulation of both LL-37 and chlorhexidine into mesoporous silica, while XRD and TEM showed that antimicrobial agent incorporation can be achieved without greatly affecting the structure of the mesoporous silica. The modified mesoporous silica released LL-37 and chlorhexidine slowly, reaching maximum release after about 200 h. The release rate could also be controlled through incorporation of SH groups in the pore walls, adding to pore hydrophobicity and reducing the release rate by about 50% compared to the unmodified mesoporous silica. Mesoporous silica containing either LL-37 or chlorhexidine displayed potent bactericidal properties against both Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. While chlorhexidine-loaded mesoporous silica displayed an accompanying high toxicity, as judged from hemolysis, LDH release, and MTT assay, the corresponding material containing LL-37 showed very low toxicity by all these assays, comparable to that observed for mesoporous silica in the absence of antibacterial drug, as well as to the negative controls in the respective assays. Mesoporous silica containing LL-37 therefore holds potential as an implantable material or a surface coating for such materials, as it combines potent bactericidal action with low toxicity, important features for controlling implant-related infections, e.g., for multi-resistant pathogens or for cases where access to the infection site of systemically administered antibiotics is limited due to collagen capsule formation or other factors. PMID:19628277

  13. Purification of DNA for bacterial productivity estimates. [Escherichia coli

    SciTech Connect

    Burnison, B.K.; Nuttley, D.J. )

    1990-02-01

    (methyl-{sup 3}H)thymidine-labeled DNA from natural populations of aquatic bacteria was completely separated from RNA and protein by hydroxylapatite chromatography. The procedure was validated by monitoring increases in Escherichia coli cell count, A{sup 550}, DNA concentration, and thymidine incorporation into DNA isolated by the proposed technique. The procedure can be used in the field and does not rely on the use of acid-base hydrolysis or volatile organic solvents.

  14. Cycloheximide resistance of Physarum polycephalum

    SciTech Connect

    Evans, T.E.; Evans, H.H.

    1980-08-01

    In the presence of cycloheximide, wild-type plasmodia of Physarum polycephalum exhibit an immediate decrease in deoxyribonucleic acid synthesis, a reduction in the incorporation of (/sup 3/H)thymidine into thymidine triphosphate, and an increase in the level of thymidine triphosphate, as well as a decrease in protein synthesis. In this study, we have utilized a cycloheximide-resistant (Cyc/sup r/) amoebic strain selected from a population of cells mutagenized with nitrosoguanidine.

  15. 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).

  16. 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.

  17. 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. PMID:26832335

  18. 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.

  19. Colorimetric assay of blood coagulation factor XIII in plasma.

    PubMed

    Lee, K N; Birckbichler, P J; Patterson, M K

    1988-05-01

    In this new colorimetric assay for Factor XIII in plasma, 5-(biotinamido)pentylamine is used as the amine substrate. Factor XIII, a zymogen, is transformed by thrombin and Ca2+ to active Factor XIIIa, and the incorporation of 5-(biotinamido)pentylamine into N,N-dimethylcasein is used to measure catalytically active Factor XIIIa. The biotinylated enzymatic product is immobilized onto 96-well microtiter plates, complexed with streptavidin-beta-galactosidase, and the absorbance at 405 nm is monitored for production of p-nitrophenol from p-nitrophenyl-beta-D-galactopyranoside. Concentrations of N,N-dimethylcasein, 5-(biotinamido)pentylamine, Ca2+, and thrombin were chosen to allow near-maximum velocity of amine incorporation. A linear relationship was obtained between assay product and plasma volume, from 0.5 to 50 microL of plasma. Results correlated well (r greater than 0.924) with those from the most frequently utilized radiometric filter-paper assay for Factor XIII. The method appears to be ideal for routine diagnostic estimation of Factor XIII in plasma because of its simplicity, its lack of use of radioisotopes, and its potential for assay of large numbers of samples by use of microtiter plates and automated plate readers. PMID:2897256

  20. Methods to assay Drosophila behavior.

    PubMed

    Nichols, Charles D; Becnel, Jaime; Pandey, Udai B

    2012-01-01

    Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases(1). We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials(2-4). The rapid iterative negative geotaxis (RING) assay(5) has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously

  1. Incorporation of exogenous uracil by Cryptosporidium parvum in vitro.

    PubMed Central

    Upton, S J; Tilley, M; Mitschler, R R; Oppert, B S

    1991-01-01

    Oocysts of Cryptosporidium parvum were used to infect Madin-Darby bovine kidney cells. Cultures were incubated in a reduced-oxygen atmosphere in candle jars or in a 5% CO2-95% air atmosphere. At 72 h, parasites were quantitated microscopically and found to be enhanced 5.5-fold in the reduced-oxygen atmosphere. Using candle jars, we then determined that C. parvum was amenable to [3H]uracil incorporation assays and easily quantitated with this method. Images PMID:2056042

  2. 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).

  3. ASSESSMENT OF STANDARD REFERENCE COMPOUNDS FOR COMPARATIVE STUDIES USING THE SALMONELLA TYPHIMURIUM MUTAGENICITY ASSAY: II. WITH EXOGENOUS ACTIVATION

    EPA Science Inventory

    The purpose of this project was to evaluate the variability in the mutagenic response of potential standard reference chemicals that require exogenous metabolic activation in the standard plate; incorporation Salmonella mutagenicity assay, and to develop ranking criteria for muta...

  4. 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.

  5. Effects of glycyl-histidyl-lysine on Morris hepatoma 7777 cells.

    PubMed

    Barra, R

    1987-01-01

    Glycyl-histidyl-lysine (GHL) has been shown to have growth stimulatory effects on a number of different cell types including hepatocytes and hepatoma cells. In this study, the effects of GHL on Morris hepatoma 7777 cells were investigated. The greatest stimulatory effects on 3H-thymidine and 3H-leucine incorporation were observed at a GHL concentration of 2 ng/ml. In randomly proliferating cells, the incorporation of 3H-thymidine into DNA increased by 50% and that of 3H-leucine into protein by 29%. In addition, synergistic effects were observed when insulin and glucagon were included with GHL in the incubation mixture. Experiments with cells rendered quiescent by serum starvation indicated that cells in the G1 phase of the cell cycle are more sensitive to GHL stimulation. In these experiments, 3H-thymidine incorporation increased earlier and peaked at a higher value than in the control cells. This finding suggests that GHL may play a role in stimulating quiescent cells to re-enter the cell cycle. PMID:3319436

  6. Uranyl incorporation in natural calcite.

    SciTech Connect

    Kelly, S. D.; Newville, M. G.; Cheng, L.; Kemner, K. M.; Sutton, S. R.; Fenter, P.; Sturchio, N. C.; Spotl, C.; Environmental Research; Univ. of Chicago; Univ. of Illiois at Chicago; Univ. of Innsbruck

    2003-01-01

    The occurrence of trace amounts of uranyl in natural calcite has posed a long-standing problem in crystal chemistry because of speculation that the size and shape of the uranyl ion may preclude its incorporation in a stable lattice position in calcite. This also defines an important environmental problem because of its bearing on the transport and sequestration of uranyl released from nuclear facilities and uranium mining operations. Calcite is a nearly ubiquitous mineral in soils and groundwater aquifers. X-ray absorption spectroscopy and X-ray fluorescence microprobe studies of uranium in relatively U-rich {approx}13700-year-old calcite from a speleothem in northernmost Italy indicate substitution of uranyl for a calcium and two adjacent carbonate ions in calcite. These new data imply that uranyl has a stable lattice position in natural calcite, indicating that it may be reliably sequestered in calcite over long time scales.

  7. Measurement of free light chains with assays based on monoclonal antibodies.

    PubMed

    Te Velthuis, Henk; Drayson, Mark; Campbell, John P

    2016-06-01

    Recently, serum free light chain (FLC) assays incorporating anti-kappa (κ) and anti-lambda (λ) FLC monoclonal antibodies have become available: N Latex FLC assay (Siemens) and Seralite® (Abingdon Health). The purpose of this review is to provide an overview of these two new monoclonal antibody-based methods. In doing so, the review will outline the performance characteristics of each method, including a summary of: assay principles, antibody specificity, analytical performance and assay performance in disease. Additionally, the review will describe the potential user benefits of adopting these new generation FLC assays, which are designed to overcome the established limitations of existing polyclonal antibody based FLC assays. PMID:27010775

  8. HIV-1 Capsid Stabilization Assay.

    PubMed

    Fricke, Thomas; Diaz-Griffero, Felipe

    2016-01-01

    The stability of the HIV-1 core in the cytoplasm is crucial for productive HIV-1 infection. Mutations that stabilize or destabilize the core showed defects in HIV-1 reverse transcription and infection. We developed a novel and simple assay to measure stability of in vitro-assembled HIV-1 CA-NC complexes. This assay allowed us to demonstrate that cytosolic extracts strongly stabilize the HIV-1 core (Fricke et al., J Virol 87:10587-10597, 2013). By using our novel assay, one can measure the ability of different drugs to modulate the stability of in vitro-assembled HIV-1 CA-NC complexes, such as PF74, CAP-1, IXN-053, cyclosporine A, Bi2, and the peptide CAI. We also found that purified CPSF6 (1-321) protein stabilizes in vitro-assembled HIV-1 CA-NC complexes (Fricke et al., J Virol 87:10587-10597, 2013). Here we describe in detail the use of this capsid stability assay. We believe that our assay can be a powerful tool to assess HIV-1 capsid stability in vitro. PMID:26714703

  9. Evidence for involvement of 3'-untranslated region in determining angiotensin II receptor coupling specificity to G-protein.

    PubMed Central

    Thekkumkara, Thomas J; Linas, Stuart L

    2003-01-01

    The mRNA 3'-untranslated region (3'-UTR) of many genes has been identified as an important regulator of the mRNA transcript itself as well as the translated product. Previously, we demonstrated that Chinese-hamster ovary-K1 cells stably expressing angiotensin receptor subtypes (AT(1A)) with and without 3'-UTR differed in AT(1A) mRNA content and its coupling with intracellular signalling pathways. Moreover, RNA mobility-shift assay and UV cross-linking studies using the AT(1A) 3'-UTR probe identified a major mRNA-binding protein complex of 55 kDa in Chinese-hamster ovary-K1 cells. In the present study, we have determined the functional significance of the native AT(1A) receptor 3'-UTR in rat liver epithelial (WB) cell lines by co-expressing the AT(1A) 3'-UTR sequence 'decoy' to compete with the native receptor 3'-UTR for its mRNA-binding proteins. PCR analysis using specific primers for the AT(1A) receptor and [(125)I]angiotensin II (AngII)-binding studies demonstrated the expression of the native AT(1A) receptors in WB (B(max)=2.7 pmol/mg of protein, K(d)=0.56 nM). Northern-blot analysis showed a significant increase in native receptor mRNA expression in 3'-UTR decoy-expressing cells, confirming the role of 3'-UTR in mRNA destabilization. Compared with vehicle control, AngII induced DNA and protein synthesis in wild-type WB as measured by [(3)H]thymidine and [(3)H]leucine incorporation respectively. Activation of [(3)H]thymidine and [(3)H]leucine correlated with a significant increase in cell number (cellular hyperplasia). In these cells, AngII stimulated GTPase activity by AT(1) receptor coupling with G-protein alpha i. We also delineated that functional coupling of AT(1A) receptor with G-protein alpha i is an essential mechanism for AngII-mediated cellular hyperplasia in WB by specifically blocking G-protein alpha i activation. In contrast with wild-type cells, stable expression of the 3'-UTR 'decoy' produced AngII-stimulated protein synthesis and cellular

  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. Interleukin-17 is a potent immuno-modulator and regulator of normal human intestinal epithelial cell growth

    SciTech Connect

    Schwartz, S.; Beaulieu, J.F. . E-mail: frank.ruemmele@nck.ap-hop-paris.fr

    2005-11-18

    Upregulation of the T-cell derived cytokine interleukin (IL-17) was reported in the inflamed intestinal mucosa of patients with inflammatory bowel disorders. In this study, we analyzed the effect of IL-17 on human intestinal epithelial cell (HIEC) turnover and functions. Proliferation and apoptosis in response to IL-17 was monitored in HIEC (cell counts, [{sup 3}H]thymidine incorporation method, and annexinV-PI-apoptosis assay). Signalling pathways were analyzed by Western blots, electromobility shift assay, and immunofluorescence studies. IL-17 proved to be a potent inhibitor of HIEC proliferation without any pro-apoptotic/necrotic effect. The growth inhibitory effect of IL-17 was mediated via the p38 stress kinase. Consequently, the p38-SAPkinase-inhibitor SB203580 abrogated this anti-mitotic effect. In parallel, IL-17 provoked the degradation of I{kappa}B{alpha}, allowing nuclear translocation of the p65 NF-{kappa}B subunit and induction of the NF-{kappa}B-controlled genes IL-6 and -8. IL-17 potently blocks epithelial cell turnover while at the same time amplifying an inflammatory response in a positive feedback manner.

  12. Isolation of hormone responsive uterine stromal cells: an in vitro model for stromal cell proliferation and differentiation.

    PubMed

    Rider, Virginia

    2006-01-01

    The female sex hormones estrogen and progesterone stimulate proliferation and differentiation of human and rodent uterine cells. The purpose of this chapter is to provide a method for isolating hormone-responsive rat uterine stromal cell lines that can be used to study steroid control of the cell cycle. Uteri from ovariectomized rats are differentially digested with trypsin to separate epithelial and stromal cells. The stromal cells are cultured in a standard growth medium containing 10% fetal bovine serum. After several passages, the purity of the stromal cell lines is determined using immunocytochemistry. Cell proliferation is studied by culturing the stromal cells in serum-free medium containing sex steroids and other mitogens. Cell cycle progression is assessed by flow cytometry, 3H-thymidine and BrdU incorporation, whereas proliferation is monitored using the MTT assay. Cell cycle regulators are visualized by Northern and Western blotting whereas cyclin-cyclin-dependent kinase activity is monitored using immune complex kinase assays. Uterine stromal cell lines isolated using the methods reported in this chapter provide a suitable model system to investigate the signal transduction events that stimulate hormone-dependent control of the cell cycle. PMID:16251733

  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. PMID:25721086

  14. 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.

  15. Immunomodulatory effects of the botanical compound LCS101: implications for cancer treatment

    PubMed Central

    Rachmut, Itzchak H; Samuels, Noah; Melnick, Steven J; Ramachandran, Cheppail; Sharabi, Yedida; Pavlovsky, Anya; Maimon, Yair; Shoham, Jacob

    2013-01-01

    Objective To examine the effects of LSC101, a botanical compound, on adaptive and innate immunity. Materials and methods LCS101 preparations were tested for batch-to-batch consistency using high-performance liquid chromatography. T-cell activation was quantified in murine spleen cells using 3H-thymidine incorporation, and cytokine production analyzed with enzyme-linked immunosorbent assay. Natural killer cell activity was tested on human blood cells using flow cytometry, and cytotoxicity measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and apoptosis using a FACSCalibur. Effects on interferon-γ production in fluorouracil/doxorubicin-treated mice were tested with enzyme-linked immunosorbent assay. Results High-performance liquid chromatography analysis demonstrated batch-to-batch consistency. T-cell proliferation was increased, and a dose-dependent activation of natural killer cells and macrophage tumor necrosis factor-α secretion were observed with LCS101 treatment. Interferon-γ levels, reduced following fluorouracil treatment, were corrected in treated animals. No toxicity or compromised treatment outcomes were associated with LCS101 exposure. Conclusions LCS101 demonstrated significant effects on a number of immune processes. Further research is needed in order to understand the molecular immunomodulatory pathways affected by this compound, as well as clinical implications for treatment. PMID:23637542

  16. 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.

  17. 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.

  18. Dehydroleucodine inhibits vascular smooth muscle cell proliferation in G2 phase.

    PubMed

    Cruzado, M; Castro, C; Fernandez, D; Gomez, L; Roque, M; Giordano, O E; Lopez, L A

    2005-11-01

    Vascular smooth muscle cell (VSMC) proliferation plays an important role in the development of atherosclerosis and in the vascular changes seen in hypertension. Dehydroleucodine (DhL) is a sesquiterpene lactone that inhibits cell proliferation in plant cells. In this paper, we study the effect of DhL in the proliferation of VSMCs stimulated with 10% fetal bovine serum (FBS). Very low concentrations of DhL (2-6 microM) inhibited VSMC proliferation and induced cell accumulation in G2. DhL did not affect the dynamics of 3H-thymidine incorporation, and did not modify either the activity of DNA polymerase or the incorporation of deoxyribonucleotides in an in vitro assay. Moreover, DhL did not induce apoptosis in VSMCs. These results indicate that DhL, in very low concentration, induces a transient arrest of VSMCs in G2. Our data show that VSMCs are especially sensitive to DhL effect, suggesting that DhL could be potentially useful to prevent the vascular pathological changes seen in hypertension and other vascular diseases. PMID:16309576

  19. 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...

  20. Incorporation of Unnatural Amino Acids into Proteins Expressed in Mammalian Cells.

    PubMed

    Serfling, R; Coin, I

    2016-01-01

    The site-specific incorporation of unnatural amino acids (Uaas) via genetic code expansion provides a powerful method to introduce synthetic moieties into specific positions of a protein directly in the live cell. The technique, first developed in bacteria, is nowadays widely applicable in mammalian cells. In general, different Uaas are incorporated with different efficiency. By comparing the incorporation efficiency of several Uaas recently designed for bioorthogonal chemistry, we present here a facile dual-fluorescence assay to evaluate relative yields of Uaa incorporation. Several biological questions can be addressed using Uaas tools. In recent years, photo-cross-linking Uaas have been extensively applied to map ligand-binding sites on G protein-coupled receptors (GPCRs). We describe a simple and efficient two-plasmid system to incorporate a photoactivatable Uaa into a class B GPCR, and demonstrate cross-linking to its nonmodified natural ligand. PMID:27586329

  1. 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...

  2. 49 CFR 572.190 - Incorporated materials.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... Register approved the materials incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part... 49 Transportation 7 2011-10-01 2011-10-01 false Incorporated materials. 572.190 Section 572.190... Dummy, Small Adult Female § 572.190 Incorporated materials. (a) The following materials are...

  3. 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...

  4. 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...

  5. 49 CFR 572.180 - Incorporated materials.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... approved the materials incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51... 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...

  6. 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...

  7. 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...

  8. 49 CFR 572.190 - Incorporated materials.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Register approved the materials incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part... 49 Transportation 7 2010-10-01 2010-10-01 false Incorporated materials. 572.190 Section 572.190... Dummy, Small Adult Female § 572.190 Incorporated materials. (a) The following materials are...

  9. 49 CFR 572.190 - Incorporated materials.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... Register approved the materials incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part... 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...

  10. 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...

  11. 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…

  12. 49 CFR 572.180 - Incorporated materials.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... approved the materials incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51... 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...

  13. 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... 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...

  14. 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....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 587.5 - Incorporated materials.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 7 2012-10-01 2012-10-01 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...

  16. 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

  17. Determining optimal surface roughness of TiO(2) blasted titanium implant material for attachment, proliferation and differentiation of cells derived from human mandibular alveolar bone.

    PubMed

    Mustafa, K; Wennerberg, A; Wroblewski, J; Hultenby, K; Lopez, B S; Arvidson, K

    2001-10-01

    In the complex process of bone formation at the implant-tissue interface, implant surface roughness is an important factor modulating osteoblastic function. In this study, primary cultures of osteoblast-like cells, derived from human mandibular bone, were used. The aim was to examine the effect of varying surface roughness of titanium implant material on cellular attachment, proliferation and differentiation. A recognized method of increasing surface roughness and enlarging the surface area of titanium implants is by blasting with titanium dioxide particles: the four specimen types in the study comprised surfaces which were machine-turned only, or blasted after turning, with 63-90 microm, 106-180 microm, or 180-300 microm TiO(2) particles, respectively. The specimens were analyzed by scanning electron microscopy and confocal laser scanning. The turned samples had the smoothest surfaces: average height deviation (S(a)) of 0.20 microm. The roughest were those blasted with 180-300 microm particles, S(a) value 1.38 microm. Blasting with intermediate particle sizes yielded S(a) values of 0.72 microm and 1.30 microm, respectively. Cell profile areas were measured using a semiautomatic interactive image analyzer. Figures were expressed as percentage of attachment. DNA synthesis was estimated by measuring the amount of [(3)H]-thymidine incorporation into trichloroacetic acid (TCA) insoluble cell precipitates. The specific activity of alkaline phosphatase was assayed using p-nitrophenylphosphate as a substrate. The ability of the cells to synthesize osteocalcin was investigated in serum-free culture medium using the ELSA-OST-NAT immunoradiometric kit. After 3 h of culture, the percentage of cellular attachment did not differ significantly between specimens blasted with 180-300 micromparticles and the turned specimens. All blasted surfaces showed significantly higher [(3)H]-thymidine incorporation than the turned surfaces (P<0.05), with the highest on the surfaces blasted

  18. Microfluidic multiculture assay to analyze biomolecular signaling in angiogenesis.

    PubMed

    Theberge, Ashleigh B; Yu, Jiaquan; Young, Edmond W K; Ricke, William A; Bushman, Wade; Beebe, David J

    2015-03-17

    Angiogenesis (the formation of blood vessels from existing blood vessels) plays a critical role in many diseases such as cancer, benign tumors, and macular degeneration. There is a need for cell culture methods capable of dissecting the intricate regulation of angiogenesis within the microenvironment of the vasculature. We have developed a microscale cell-based assay that responds to complex pro- and antiangiogenic soluble factors with an in vitro readout for vessel formation. The power of this system over traditional techniques is that we can incorporate the whole milieu of soluble factors produced by cells in situ into one biological readout (vessel formation), even if the identity of the factors is unknown. We have currently incorporated macrophages, endothelial cells, and fibroblasts into the assay, with the potential to include additional cell types in the future. Importantly, the microfluidic platform is simple to operate and multiplex to test drugs targeting angiogenesis in a more physiologically relevant context. As a proof of concept, we tested the effect of an enzyme inhibitor (targeting matrix metalloproteinase 12) on vessel formation; the triculture microfluidic assay enabled us to capture a dose-dependent effect entirely missed in a simplified coculture assay (p < 0.0001). This result underscores the importance of cell-based assays that capture chemical cross-talk occurring between cell types. The microscale dimensions significantly reduce cell consumption compared to conventional well plate platforms, enabling the use of limited primary cells from patients in future investigations and offering the potential to screen therapeutic approaches for individual patients in vitro. PMID:25719435

  19. Ensemble learning incorporating uncertain registration.

    PubMed

    Simpson, Ivor J A; Woolrich, Mark W; Andersson, Jesper L R; Groves, Adrian R; Schnabel, Julia A

    2013-04-01

    This paper proposes a novel approach for improving the accuracy of statistical prediction methods in spatially normalized analysis. This is achieved by incorporating registration uncertainty into an ensemble learning scheme. A probabilistic registration method is used to estimate a distribution of probable mappings between subject and atlas space. This allows the estimation of the distribution of spatially normalized feature data, e.g., grey matter probability maps. From this distribution, samples are drawn for use as training examples. This allows the creation of multiple predictors, which are subsequently combined using an ensemble learning approach. Furthermore, extra testing samples can be generated to measure the uncertainty of prediction. This is applied to separating subjects with Alzheimer's disease from normal controls using a linear support vector machine on a region of interest in magnetic resonance images of the brain. We show that our proposed method leads to an improvement in discrimination using voxel-based morphometry and deformation tensor-based morphometry over bootstrap aggregating, a common ensemble learning framework. The proposed approach also generates more reasonable soft-classification predictions than bootstrap aggregating. We expect that this approach could be applied to other statistical prediction tasks where registration is important. PMID:23288332

  20. Flex joint incorporating enclosed conductors

    SciTech Connect

    Tomek, M.L.

    1989-06-27

    This patent describes a downhole elongate oil tool adapted to flex as required in placing the elongate tool in a crooked well borehole wherein electrical signals are transmitted along conductors along the elongate tool. The elongate tool incorporating a flex tool consists of: (a) a generally spherical knuckle; (b) a surrounding socket engaging the knuckle with a limited rotational range relative to a defined axis through the knuckle and socket; (c) a passage through the knuckle opening at an outlet into the socket; (d) a serially communicated passage through the socket aligned with the knuckle; (e) means cooperative with the knuckle and the socket; (1) permitting flexure in a conic angle relative to an axis through the knuckle and socket; and (2) preventing relative axial rotation between the knuckle and the socket; and (f) means for filling the passages with a pressure isolated oil bath for contacting structural components in the passages, and further wherein the oil bath is pressure isolated by a piston responsive to external fluid pressure in the well borehole to sustain a pressure on the oil bath equal to and in excess of prevailing external pressure.

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

    SciTech Connect

    Swenne, I. )

    1990-01-01

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

  2. Electrostimulation of rat callus cells and human lymphocytes in vitro

    SciTech Connect

    Aro, H.; Eerola, E.; Aho, A.J.; Penttinen, R.

    1984-01-01

    Asymmetrical pulsing low voltage current was supplied via electrodes to cultured rat fracture callus cells and human peripheral blood lymphocytes. The (/sup 3/H)thymidine incorporation of the callus cells and 5-(/sup 125/I)iodo-2'-deoxyuridine incorporation of the lymphocytes were determined. The growth pattern of callus cells (estimated by cellular density) did not respond to electrical stimulation. However, the uptake of (/sup 3/H)thymidine was increased at the early phase of cell proliferation and inhibited at later phases of proliferation. The (/sup 3/H)thymidine uptake of confluent callus cell cultures did not respond to electrical stimulation. Lymphocytes reacted in a similar way; stimulated cells took up more DNA precursor than control cells at the early phase of stimulation. During cell division, induced by the mitogens phytohemagglutinin and Concanavalin-A, the uptake of DNA precursor by stimulated cells was constantly inhibited. The results suggest that electrical stimuli affect the uptake mechanisms of cell membranes. The duality of the effect seems to be dependent on the cell cycle.

  3. 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.

  4. 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.

  5. 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.

  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. Assays for B lymphocyte function.

    PubMed

    Bondada, Subbarao; Robertson, Darrell A

    2003-11-01

    This unit describes the antigenic stimulation of in vitro antibody production by B cells and the subsequent measurement of secreted antibodies. The first basic protocol is a generalized system for inducing in vitro antibody production and can accommodate various types of antigens under study. Secreted antibodies can then be measured with an enzyme-linked immunosorbent assay (ELISA) or other soluble-antibody detection systems. Alternatively, the number of antibody-producing cells can be quantified by plaque-forming cell (PFC) assays presented in this unit: the Cunningham-Szenberg and the Jerne-Nordin techniques. Both methods employ specially prepared slide chambers, described here, in which the antibody-producing B cells are mixed with complement and indicator sheep red blood cells (SRBC), or with trinitrophenol-modified SRBC (TNP-SRBC), with subsequent lysis and counting of plaques. Because IgM antibodies fix complement efficiently, whereas IgG and IgA antibodies do not, unmodified PFC assays measure only IgM antibodies. The assay can be modified, however, to measure all classes of antibodies or to enumerate total immunoglobulin-secreting B cells, as described in alternate protocols. Yet another method of measuring the number of antibody-producing B cells (in a class-specific fashion) is to use the ELISPOT technique described in UNIT 7.14. The resting B cells used in these procedures are prepared as described in the final support protocols for Percoll gradient centrifugation. PMID:18432909

  8. 21 CFR 225.158 - Laboratory assays.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 4 2010-04-01 2010-04-01 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...

  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. 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

  14. Evaluation of Consistency in Spheroid Invasion Assays

    PubMed Central

    Cisneros Castillo, Liliana R.; Oancea, Andrei-Dumitru; Stüllein, Christian; Régnier-Vigouroux, Anne

    2016-01-01

    Multicellular tumor spheroids embedded in a matrix represent invaluable tools to analyze cell invasion. Spheroid sizes and invasiveness are the main observables easily measurable to evaluate effects of biological or pharmaceutical manipulations on invasion. They largely account for these 3-D platforms variability, leading to flaws in data interpretation. No method has been established yet that characterizes this variability and guarantees a reliable use of 3-D platforms. Spheroid initial/end sizes and invasiveness were systematically analyzed and compared in spheroids of U87MG cells generated by three different methods and embedded at different times in a collagen matrix. A normality test was used to characterize size distribution. We introduced the linearity-over-yield analysis as a novel mathematical tool to assess end sizes and invasion reproducibility. We further provide a proof of concept by applying these tools to the analysis of a treatment known to be effective beforehand. We demonstrate that implementation of these statistical and mathematical tools warranted a confident quantification and interpretation of in 3-D conducted assays. We propose these tools could be incorporated in a guideline for generation and use of 3-D platforms. PMID:27334575

  15. 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.

  16. 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.

  17. 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. PMID:26608293

  18. 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.

  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. Simultaneous assay of pigments, carbohydrates, proteins and lipids in microalgae.

    PubMed

    Chen, Yimin; Vaidyanathan, Seetharaman

    2013-05-01

    Biochemical compositional analysis of microbial biomass is a useful tool that can provide insight into the behaviour of an organism and its adaptational response to changes in its environment. To some extent, it reflects the physiological and metabolic status of the organism. Conventional methods to estimate biochemical composition often employ different sample pretreatment strategies and analytical steps for analysing each major component, such as total proteins, carbohydrates, and lipids, making it labour-, time- and sample-intensive. Such analyses when carried out individually can also result in uncertainties of estimates as different pre-treatment or extraction conditions are employed for each of the component estimations and these are not necessarily standardised for the organism, resulting in observations that are not easy to compare within the experimental set-up or between laboratories. We recently reported a method to estimate total lipids in microalgae (Chen, Vaidyanathan, Anal. Chim. Acta, 724, 67-72). Here, we propose a unified method for the simultaneous estimation of the principal biological components, proteins, carbohydrates, lipids, chlorophyll and carotenoids, in a single microalgae culture sample that incorporates the earlier published lipid assay. The proposed methodology adopts an alternative strategy for pigment assay that has a high sensitivity. The unified assay is shown to conserve sample (by 79%), time (67%), chemicals (34%) and energy (58%) when compared to the corresponding assay for each component, carried out individually on different samples. The method can also be applied to other microorganisms, especially those with recalcitrant cell walls. PMID:23601278

  1. 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.

  2. 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.

  3. Biological activities of a synthetic peptide composed of two unlinked domains from a retroviral transmembrane protein sequence.

    PubMed Central

    Wegemer, D E; Kabat, K G; Kloetzer, W S

    1990-01-01

    We report several biological activities of a synthetic peptide whose sequence contains the highly conserved region of feline leukemia virus transmembrane protein (TM) synthetically linked to another short TM-derived sequence particularly rich in polar positive residues. This 29-amino-acid peptide blocked [3H]thymidine uptake 30 to 50% by concanavalin A-stimulated CD4(+)--but not CD8(+)-enriched murine splenocytes. Maximal suppression was detected at 12.5 micrograms (3 microM) to 75 micrograms (19 microM) per ml of growth medium; stimulation of [3H]thymidine uptake was observed at higher peptide concentrations. The synthetic peptide inhibited but did not stimulate [3H]thymidine uptake by mitogen-activated thymocytes and antibody production by splenocytes as determined in a liquid hemolytic plaque assay. Similarities are reported between a consensus sequence of diverse retroviral TMs and a region of alpha interferons shown by others to be important for antiviral and cytostatic properties. The TM sequence-derived synthetic peptide blocked in a nontoxic and sequence-specific manner the release of murine leukemia virus from two chronically infected cell lines. We suggest that some of the biological effects of retroviral TM are mediated through a common pathway shared with alpha interferons. Images PMID:1969500

  4. Axon-Schwann cell interaction in degenerating and regenerating peripheral nerve

    SciTech Connect

    Pellegrino, R.G.

    1984-01-01

    Severance of a peripheral nerve stimulates a characteristic sequence of events in the distal stump, including the dissolution of axons and myelin and the proliferation of Schwann cells within their basal lamina. The first part of this thesis employs the cat tibial nerve to examine the relationship between the spatio-temporal pattern of Schwann cell mitosis, loss of the structural and functional properties of axolemma, synthesis of P/sub 0/, the major myelin glycoprotein, and the clearance of morphological myelin. Induction of S phase was measured by determining the uptake of /sup 3/H thymidine into trichloroacetic acid (TCA) precipitates following a 3 hour in vitro incubation in Krebs-Ringers buffer containing /sup 3/H thymidine. Nerve transection stimulated a monophasic increase in /sup 3/H thymidine uptake that peaked at 4 days post-transection throughout an 80 mm length of distal stump. Light microscope autoradiography revealed prominent incorporation into Schwann cells of myelinated fibers. Nerve transection also produced dramatic changes in the intrafascicular binding of /sup 3/H STX which binds to voltage-sensitive sodium channels STX binding fell precipitously to 20% of normal at 4 days post-transection, concurrent with the peak of /sup 3/H thymidine uptake. In conclusion, these studies suggest: (a) Schwann cells divide more or less contemporaneously throughout the distal stump; (b) changes in axons rather than myelin are likely to stimulate the Schwann cell to divide; (c) mitosis regulates other events during Wallerian degeneration, including myelin degeneration and the clearance of sodium channels from nodal axolemma.

  5. Aqueous Vernomia amygdalina Extracts Alter MCF-7 Cell Membrane Permeability and Efflux

    PubMed Central

    Opata, Michael M.; Izevbigie, Ernest B.

    2006-01-01

    Breast cancer is the second leading cause of cancer related deaths of women in the United States. Several treatment strategies have been developed over the past decade to reduce cancer morbidity and mortality rates. While mortality rates have declined in some ethnic populations, the overall cancer incidence continues to grow. Hence, chemotherapeutic agents are needed to improve cancer treatment outcome. Previous studies show that low concentrations (microgram/ml) of water-soluble leaf extracts of a Nigerian edible plant, V. amygdalina (VA), potently retard the proliferative activities of estrogen receptor positive (ER+) human breast cancerous cells (MCF-7) cells in vitro in a concentration-dependent fashion. The anti-proliferative activities of VA are extracellular signal-regulated kinases 1/2 (ERKs 1/2)-dependent. Cell culture and animal model studies, conducted by other investigators using other plant extracts, have also revealed that plant extract components called thionins may be responsible for their anticancer activities. These thionins are believed to interact with the cells in ways that compromise membrane potential/permeability resulting in the alteration of efflux, cytosolic activities, and subsequent cell death. Therefore, we hypothesized that VA exposure may compromise cell membrane as another mode of action to elicit its anticancer activities in MCF-7 cells. The exposure of cells to VA decreased [3H]thymidine uptake in a concentration-dependent (0, 30, and 100 μg/ml VA) manner (p < 0.05) but increased [3H]thymidine release, expressed as percent of [3H]thymidine incorporated, into the medium (p < 0.05). The amount of [3H]thymidine released into the medium was 1.7, 7.4, and 11.0 % for 0, 30, and 100 μg/ml VA respectively. Thus suggesting the membranes in VA-treated cells were compromised in a concentration-dependent fashion. PMID:16823089

  6. Rohon-beard cells and other large neurons in Xenopus embryos originate during gastrulation.

    PubMed

    Lamborghini, J E

    1980-01-15

    The time of origin (birthday) of Rohon-Beard cells in Xenopus laevis was studied by 3H-thymidine autoradiography. Rohon-Beard cells were selected because they are a morphologically identifiable population of neurons in which the development of chemical and electrical excitability has been studied. A single injection of a radioactive DNA precursor was given to animals in successive stages of development from blastula to late tail bud (Nieuwkoop and Faber stages 8--33/34). The label was available throughout the stage of injection and longer. The labeling pattern was examined when animals had reached stage 42, when Rohon-Beard cells are easily recognized. All neurons including Rohon-Beard cells were labeled in animals injected with 3H-thymidine before stage 10 1/2 (early gastrula). Unlabeled Rohon-Beard cells were observed in animals injected with 3H-thymidine in and after stage 10 1/2. The percentage of unlabeled Rohon-Beard cells increased as development progressed. About 80% were born by the completion of gastrulation (stage 13). The other approximately 20% were born during neurulation and early tail bud stages. By stage 27, no Rohon-Beard neuron incorporated 3H-thymidine. In addition to Rohon-Beard neurons, five other neuronal populations begin generation during gastrulation: Mauthner neurons (Vargas-Lizardi and Lyser, '74), trigeminal ganglion cells, large basal plate cells of the medulla, extramedullary neurons, and primary motor neurons. The first birthdays in any of the six populations are temporally close to but appear to be independent of the others. PMID:7364967

  7. Effect of the cytostatic agent idarubicin on fibroblasts of the human Tenon's capsule compared with mitomycin C

    PubMed Central

    Heilmann, C.; Schonfeld, P.; Schluter, T.; Bohnensack, R.; Behrens-Baumann, W.

    1999-01-01

    BACKGROUND/AIMS—To investigate the in vitro effect of a short time exposure to the anthracycline idarubicin on proliferation, protein synthesis, and motility of human Tenon's capsule fibroblasts in comparison with the antitumour antibiotic mitomycin C.
METHODS—After determination of effective concentrations of idarubicin, fibroblasts of the human Tenon's capsule were exposed to idarubicin or mitomycin C at concentrations ranging from 0.1 µg/ml to 1 µg/ml or from 2.5 µg/ml to 250 µg/ml, respectively, for 0.5, 2, or 5 minutes and cultured for 60 days. Cell death by apoptosis caused by idarubicin treatment was confirmed by Hoechst 33258 staining. Further proliferation was explored by cell counting and by 3H-thymidine uptake. Protein synthesis was measured by 3H-proline uptake and motility was assessed by agarose droplet motility assay.
RESULTS—Idarubicin is able to exert toxicity and to induce apoptosis during a short time exposure of 0.5 minutes at concentrations of 0.3-1 µg/ml resulting in a significant reduction in cell number compared with the control after 60 days. For mitomycin C, higher concentrations and longer expositions were necessary. Even after treatment with 1 µg/ml idarubicin or 250 µg/ml mitomycin C a few cells were able to incorporate 3H-thymidine. 3H-proline uptake up to 10 days after exposure to 0.3 µg/ml idarubicin was found not to be decreased. Cell motility was reduced after treatment with 1 µg/ml idarubicin for 5 minutes or with 250 µg/ml mitomycin C for 2 or 5 minutes. For low mitomycin C concentrations, an increase in motility was found during the first 10 days.
CONCLUSION—Idarubicin reduces proliferation of human Tenons's capsule fibroblasts after incubation for 0.5 minutes at concentrations as low as 0.3-1 µg/ml. In comparison, mitomycin C requires longer exposure times and higher doses for equal results. Therefore, idarubicin may be useful in the prevention of glaucoma filtering surgery failure

  8. Developmental changes in myelin-induced proliferation of cultured Schwann cells

    SciTech Connect

    Yoshino, J.E.; Mason, P.W.; DeVries, G.H.

    1987-03-01

    Schwann cell proliferation induced by a myelin-enriched fraction was examined in vitro. Although nearly all the Schwann cells contained material that was recognized by antisera to myelin basic protein after 24 h, only 1% of the cells were synthesizing DNA. 72 h after the addition of the mitogen a maximum of 10% of the cells incorporated (/sup 3/H)thymidine. If the cultures were treated with the myelin-enriched fraction for 24 h and then washed, the number of proliferating Schwann cells decreased by 75% when compared with those cells that were incubated with the mitogen continuously. When Schwann cells were labeled with (/sup 14/C)thymidine followed by a pulse of (/sup 3/H)thymidine 24 h later, every Schwann cell labeled with (/sup 3/H)thymidine was also labeled with (/sup 14/C)thymidine. Although almost every Schwann cell can metabolize the myelin membranes within 24 h of exposure, a small population of cell initially utilizes the myelin as a mitogen, and this population continues to divide only if myelin is present in the extracellular media. The percentage of the Schwann cells that initially recognize the myelin-enriched fraction as a mitogen is dependent upon the age of the animal from which the cells were prepared.

  9. Hinokitiol, a metal chelator derived from natural plants, suppresses cell growth and disrupts androgen receptor signaling in prostate carcinoma cell lines

    SciTech Connect

    Liu, Shicheng . E-mail: riu@sdsk.co.jp; Yamauchi, Hitoshi

    2006-12-08

    Hinokitiol ({beta}-thujaplicin), a troplone-related compound found in the heartwood of cupressaceous plants, strongly inhibits the proliferation of a broad range of tumor cell lines. This is the first report to demonstrate that hinokitiol, a metal chelator derived from natural plants, suppresses cell growth and disrupts AR signaling in prostate carcinoma cell lines. Our present studies indicate that hinokitiol suppresses androgen/AR-mediated cell growth and androgen-stimulated DNA synthesis by [{sup 3}H]thymidine incorporation in a dose- and time-dependent manner. Hinokitiol simultaneously suppresses the intracellular and secreted PSA levels, a marker for the progression of prostate cancer. Hinokitiol significantly represses the AR mRNA and protein expression in a dose- and time-dependent manner. Additionally, the ligand-binding assay shows that hinokitiol blocks binding of the synthetic androgen [{sup 3}H]R1881 to AR in LNCaP cells. These findings collectively suggest that hinokitiol is potentially effective against prostate cancer in vitro, and thus it might become a novel chemopreventive or chemotherapeutic agent for prostate cancer.

  10. Reactivity of T cells from women with antibodies to the human platelet antigen (HPA)-1a to peptides encompassing the HPA-1 polymorphism

    PubMed Central

    Jackson, DJ; Murphy, MF; Soothill, PW; Lucas, GF; Elson, CJ; Kumpel, BM

    2005-01-01

    The human platelet antigen-1a (HPA-1a) is the most common alloantigenic target in fetal and neonatal alloimmune thrombocytopenia (NAIT). Treatment currently depends on the outcome in previous pregnancies. HPA-1 specific T cell responses were determined in 14 HPA-1a alloimmunized women during or after pregnancies affected by NAIT. Peripheral blood mononuclear cells were incubated with peptides encompassing the Leu33Pro polymorphism (residues 20–39 and 24–45 in both Leu33 (HPA-1a) and Pro33 (HPA-1b) forms) or control recall antigens in the presence of autologous sera and T cell proliferation was measured by 3H-thymidine incorporation. Control antenatal and postpartum sera suppressed T cell proliferation and use of such sera was avoided. Most patients (86%) responded to the HPA-1a peptides with 64% also having weaker T cell proliferation to the HPA-1b peptides; 14% had no activity towards any peptide despite responding to control antigens. Administration of IVIG during pregnancy appeared to reduce T cell reactivity to HPA-1 peptides. Postnatal anti-HPA-1a T cell responses from women who had a severe history of NAIT (an intracranial haemorrhage in a previous fetus) were greater than those from women with a mild history. This assay may have the potential to predict disease severity if performed prior to or early in pregnancy. PMID:16178861

  11. 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.

  12. Extracts of various species of Epilobium inhibit proliferation of human prostate cells.

    PubMed

    Vitalone, Annabella; Guizzetti, Marina; Costa, Lucio G; Tita, Beatrice

    2003-05-01

    This study examined whether various species of Epilobium, a phytotherapeutic agent used in folk medicine as a treatment for benign prostatic hyperplasia, may have an antiproliferative effect in PZ-HPV-7 human prostatic epithelial cells in-vitro. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) test, [methyl-(3)H]thymidine incorporation into DNA and flow cytometry analysis were used to evaluate cell proliferation. Ethanolic extracts of E. spicatum, E. rosmarinifolium and E. tetragonum inhibited DNA synthesis in PZ-HPV-7 cells. While at high concentrations all extracts were cytotoxic, DNA synthesis was also decreased at levels that caused no or little cytotoxicity. Treatment of cells with Epilobium extracts did not result in a formation of DNA fragments (evaluated by the TUNEL assay) or chromatin condensation (assessed by Hoechst staining). Flow cytometry analysis indicated that Epilobium extracts inhibit the progression of the cell cycle from the G(0)/G(1) phase. These results suggest that extracts of Epilobium inhibit proliferation of human PZ-HPV-7 cells in-vitro by affecting progression of the cell cycle. This study provides some initial biological plausibility for the use of Epilobium extracts in benign prostatic hyperplasia. PMID:12831512

  13. Decrease of intracellular fluorescein fluorescence polarization (IFFP) in human peripheral blood lymphocytes undergoing stimulation with phytohaemagglutinin (PHA), concanavalin A (ConA), pokeweed mitogen (PWM) and anti-CD3 antibody.

    PubMed

    Eisenthal, A; Marder, O; Dotan, D; Baron, S; Lifschitz-Mercer, B; Chaitchik, S; Tirosh, R; Weinreb, A; Deutsch, M

    1996-01-01

    In the present study we describe the induction of changes in intracellular fluorescein fluorescence polarization (IFFP) in lymphocytes undergoing activation with a variety of stimulants. These stimulants included the lectins phytohaemagglutinin (PHA), concanavalin (ConA), pokeweed mitogen (PWM) and anti-CD3 antibody. Changes in IFFP were detected in individual cells using the Cellscan apparatus. Our results show that by employing mitogenic concentrations of PHA, as revealed in a [3H]-thymidine incorporation assay, a decrease in the IFFP in human peripheral blood lymphocytes (PBL) occurred within 40 min. ConA and anti-CD3 affected similarly IFFP, whereas PWM, a B lymphocyte lectin, had no effect on IFFP at the concentrations employed. Kinetic analysis revealed that changes in IFFP occurred within 20-40 min after exposure to the stimulants and lasted for 24 h. Our results show that stimulants which activate CD3+ lymphocytes caused immediate changes in IFFP, in an enriched population of human PBL. The possible mechanisms involved in IFFP modulation following exposure to selected stimulants are discussed. PMID:8893505

  14. Effects of adenine arabinoside on lymphocytes infected with Epstein-Barr virus.

    PubMed Central

    Benz, W C; Siegel, P J; Baer, J

    1978-01-01

    Low concentrations of adenine arabinoside inhibited growth of two Epstein-Barr virus producer cell lines in culture, while not significantly affecting a nonproducer cell line and a B-cell-negative line. These observations were extended to include freshly infected cells. Mitogen-stimulated human umbilical cord blood lymphocytes were unaffected by the drug at concentration levels that inhibited [3H]thymidine incorporation into the DNA of Epstein-Barr virus-stimulated cells. DNA synthesis in Epstein-Barr virus-superinfected Raji cells was also adversely affected by adenine arabinoside. However, these same low concentrations of adenine arabinoside in the triphosphate form produced less effect on DNA synthesis in nuclear systems and DNA polymerase assays than on growth or DNA synthesis in whole cells. Therefore the effects reported here of low concentrations of the drug on whole cells may be only in part related to DNA polymerase inhibition. The work reported here suggests that adenine arabinoside has multiple sites of action in infected cells. PMID:212577

  15. Inhibition of thrombin-mediated cellular effects by triabin, a highly potent anion-binding exosite thrombin inhibitor.

    PubMed

    Glusa, E; Bretschneider, E; Daum, J; Noeske-Jungblut, C

    1997-06-01

    Triabin, a 17 kDa protein from the saliva of the assassin bug Triatoma pallidipennis is a potent thrombin inhibitor interfering with the anion-binding exosite of the enzyme. The recombinant protein, produced by the baculovirus/insect cell system, was used to study the inhibitory effect on thrombin-mediated cellular responses. The thrombin (1 nM)-stimulated aggregation of washed human platelets and the rise in cytoplasmic calcium in platelets were inhibited by triabin at nanomolar concentrations. In contrast, the rise in calcium induced by the thrombin receptor-activating peptide (10 microM) was not suppressed by triabin. In isolated porcine pulmonary arteries, preconstricted with PGF 2 alpha thrombin (2 nM) elicited an endothelium-dependent relaxation which was inhibited by triabin in the same concentration range as found for the inhibition of platelet aggregation. Higher concentrations of triabin were required to diminish the contractile response of endotheliumdenuded pulmonary vessels to thrombin (10 nM). In cultured bovine coronary smooth muscle cells, the mitogenic activity of thrombin (3 nM), measured by [3H]thymidine incorporation, was also suppressed by triabin. In all these assays, the inhibitory effect of triabin was dependent on the thrombin concentration used. These studies suggest that the new anion-binding exosite thrombin inhibitor triabin is one of the most potent inhibitors of thrombin-mediated cellular effects. PMID:9241757

  16. Acinetobacter and E. coli lipopolysaccharide preparations comparative mitogenicity and induction in vitro of immunoglobulin synthesis in adult and neonatal pig lymphocytes.

    PubMed Central

    Symons, D B; Clarkson, C A

    1979-01-01

    Lipopolysaccharide (LPS) was prepared by phenol/water extraction of bacterial membranes prepared from Acinetobacter and Escherichia coli. The mitogenicity of laboratory-prepared LPS was significantly greater than that of commercial E. coli LPS for pig, sheep, calf and rat lymphocytes, assayed as [3H]-thymidine incorporation. Mouse lymphocytes responded well to commercial LPS and no greater response was obtained with other LPS preparations. A small proportion (14%) of the Acinetobacter LPS preparations was soluble in aqueous medium, the remainder comprising membraneous fragments of variable form and size. It is suggested that the insoluble presentation of LPS to cells may contribute to the improved mitogenicity compared with wholly soluble LPS. Acinetobacter LPS preparations were used to induce synthesis and secretion in vitro of immunoglobulin by adult blood lymphocytes and pre-suckled, neonatal spleen cells of the pig. IgM was the dominant class of immunoglobulin secreted. This work thus demonstrated that virgin, unprimed B cells could be induced into immunoglobulin secretion by mitogen stimulation. PMID:391702

  17. 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.

  18. 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.

  19. Changes in the biochemical composition of testes during spermatogenesis in Asterias vulgaris, with emphasis on the role of polyamines in regulating proliferation

    SciTech Connect

    Smith, F.F.

    1985-01-01

    Testes of Asterias vulgaris are potentially useful for investigating mechanisms regulating spermatogenic events because of their structural simplicity and annual spermatogenic cycle. Examination of major biochemical classes during the spermatogenic cycle provides a definition of the changing chemical microenvironment influencing germinal cells and also suggests temporal relationships among successive spermatogenic events. Testes from seastars collected throughout the year were homogenized and lyophilized and aliquots assayed for DNA, RNA, total protein, free amino acids, total lipids, glycogen, and other carbohydrates; spermatogenic stage was determined by examination of paraffin sections. Activity of ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine synthesis, increases during the proliferative phase of spermatogenesis. Testicular ODC activity correlates well with DNA synthetic rate. To test the possible role of polyamines in regulating initiation of spermatogonial mitoses, intact testes near the beginning of the proliferative phase were incubated in vitro with exogenous polyamines. They subsequently showed a significant increase in incorporation of /sup 3/H-thymidine into DNA. These results suggest a direct role for polyamines in the regulation of spermatogenic proliferation in a. vulgaris. Evidence for a regulatory role of polyamines in the initiation of proliferation, together with existing information on the environmental, hormonal, and cytological interactions, facilitates development of a preliminary model for regulation and entrainment of spermatogenesis.

  20. Interaction of putative estrogens and the estrogen receptor system in Leydig cells in the BALB/c mouse testis resulting in the initiation of DNA synthesis

    SciTech Connect

    Juriansz, R.L.

    1986-01-01

    Continuous administration of estrogens for 7-9 months, both steroidal and nonsteroidal, to male BALB/c mice, leads to the formation of testicular Leydig cell tumors. Three days following the subcutaneous implantation of a pellet of estrogen in cholesterol, there is a peak in the incorporation of /sup 3/H-thymidine into the DNA of the interstitial cells. These effects are hypothesized to be mediated by the estrogen receptor system in the Leydig cell. Common experimental techniques for the measurement of hormone binding, such as dextran coated charcoal treatment, proved to be impossible to employ in this system, therefore a procedure was developed using hydroxyapatite to obtain binding data. The cytosolic estrogen receptor was found to have a dissociation constant for estradiol-17..beta.. of 6.5 x 10/sup -8/ M, while that of the nuclear estrogen receptor was 1.25 x 10/sup -8/ M. Competition assays were utilized to determine the cytosolic estrogen receptor's affinity for nonsteroidal estrogens, steroidal estrogens, and triphenylethylene.

  1. In vitro sensitivity to methyl-prednisolone is associated with clinical response in pediatric idiopathic nephrotic syndrome.

    PubMed

    Cuzzoni, E; De Iudicibus, S; Stocco, G; Favretto, D; Pelin, M; Messina, G; Ghio, L; Monti, E; Pasini, A; Montini, G; Decorti, G

    2016-09-01

    The aim of this study was to evaluate the in vitro steroid sensitivity as a predictor of clinical response to glucocorticoids in childhood idiopathic nephrotic syndrome (INS). Seventy-four patients (median age 4.33, interquartile range [IQR] 2.82-7.23; 63.5% male) were enrolled in a prospective multicenter study: in vitro steroid inhibition of patients' peripheral blood mononuclear cell proliferation was evaluated by [methyl-(3) H] thymidine incorporation assay at disease onset (T0) and after 4 weeks (T4) of treatment. Steroid dependence was associated with increased in vitro sensitivity at T4 assessed both as drug concentration inducing 50% of inhibition (IC50 ; odds ratio [OR] = 0.48, 95% confidence interval [CI] = 0.24-0.85; P = 0.0094) and maximum inhibition at the highest drug concentration (Imax ; OR = 1.13, 95% CI = 1.02-1.31; P = 0.017). IC50 > 4.4 nM and Imax < 92% at T4 were good predictors for optimal clinical response. These results suggest that this test may be useful for predicting the response to glucocorticoid therapy in pediatric INS. PMID:27007551

  2. 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.

  3. 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.

  4. Telomere erosion and senescence in human articular cartilage chondrocytes.

    PubMed

    Martin, J A; Buckwalter, J A

    2001-04-01

    Aging and the degeneration of articular cartilage in osteoarthritis are distinct processes, but a strong association exists between age and the incidence and prevalence of osteoarthritis. We hypothesized that this association is due to in vivo replicative senescence, which causes age-related declines in the ability of chondrocytes to maintain articular cartilage. For this hypothesis to be tested, senescence-associated markers were measured in human articular chondrocytes from donors ranging in age from 1 to 87 years. These measures included in situ staining for senescence-associated beta-galactosidase activity, (3)H-thymidine incorporation assays for mitotic activity, and Southern blots for telomere length determinations. We found that senescence-associated beta-galactosidase activity increased with age, whereas both mitotic activity and mean telomere length declined. These findings indicate that chondrocyte replicative senescence occurs in vivo and support the hypothesis that the association between osteoarthritis and aging is due in part to replicative senescence. The data also imply that transplantation procedures performed to restore damaged articular surfaces could be limited by the inability of older chondrocytes to form new cartilage after transplantation. PMID:11283188

  5. T3-mediated activation of human T cells involves translocation of Ca/sup 2 +//phospholipid-dependent C-kinase

    SciTech Connect

    Galbraith, R.M.; Nel, A.E.; Dirienzo, W.; Canonica, W.; Goldschmidt-Clermont, P.J.

    1986-03-05

    Activation of human T cells with anti-73 involves increased intracellular (Ca/sup 2 +/) and phosphatidylinositol turnover. Quantitative assays of Ca/sup 2 +//phospholipid-dependent C-kinase showed high levels (40-60 pmol/..mu..g protein/min) in the cytosol of unstimulated T cells. After stimulation with anti-T3 (100 ng/ml), cytosol activity was rapidly reduced and membrane activity increased, consistent with translocation and activation of C-kinase. With the known C-kinase activator phorbol myristate acetate (PMA), translocation was also seen, but was more sustained and complete (>99% versus 40-60%). Qualitative analysis of endogenous phosphoproteins showed translocation of enzyme, and established the major substrates at MW 56, 42, 33, 24-25 and 20K. Labeling reactions were reduced in dose-dependent fashion by the C-kinase inhibitor polymyxin B (IC 14 ..mu..M), and addition to cultures caused >90% inhibition of proliferation (/sup 3/H-thymidine incorporation), even in the presence of optimal concentrations of IL-2 (4 ..mu../ml). Thus, C-kinase may be involved in T cell responses induced by modulation of the T3-Ti complex.

  6. Host Defense Mechanisms Against Infectious Bovine Rhinotracheitis Virus: In Vitro Stimulation of Sensitized Lymphocytes by Virus Antigen

    PubMed Central

    Rouse, Barry T.; Babiuk, Lorne A.

    1974-01-01

    Isolated peripheral blood lymphocytes (PBL) from cattle immunized or infected with infectious bovine rhinotracheitis (IBR) virus were cultured in vitro with ultraviolet light-inactivated IBR virus, and the degree of lymphocyte blastogenesis was quantitated by measurement of the uptake of [3H]thymidine into acid-insoluble material. Lymphocyte blastogenesis only occurred with PBL from immunized or infected animals. The optimal conditions for lymphocyte blastogenesis were defined. Blastogenesis was specific since cells from animals immunized against IBR failed to react with two other herpesvirus antigens tested, herpes simplex and equine rhinopneumonitis viruses. Blastogenesis could be prevented by reacting IBR antigen with IBR-specific antibody before adding to cultures, but incorporating IBR-specific antibody in the culture medium after adding free antigen failed to inhibit blastogenesis. With intranasally infected animals, lymphocyte blastogenesis was detectable after 5 days, reached peak levels between days 7 and 10, and then declined to low levels by day 19. In contrast, levels of neutralizing antibody were barely detectable on day 7 and reached maximal concentrations on day 19. The lymphocyte blastogenesis assay was emphasized as a convenient and useful in vitro correlate of cell-mediated immunity that should help define the role of cell-mediated immunity in immunity to herpesviruses. PMID:4426702

  7. Angiogenic, mitogenic, and chemotactic activity in human follicular fluid (HFF)

    SciTech Connect

    Bryant, S.M.; Frederick, J.L.; Gale, J.A.; Campeau, J.D.; diZerega, G.S.

    1986-03-01

    The capacity of human follicular fluid to induce neovascularization was investigated. Three parameters were employed to assess the extent of angiogenic activity: (1) new vessel formation on the chick chorioallantoic membrane (CAM); (2) mitogenesis and (3) chemotaxis of bovine aortic endothelial cells. HFF resuspended in hydron induced new blood vessel formation on the CAM, as manifested by a spoke-wheel pattern of vessels radiating from the locus of application after two to six days. Endothelial cells cultured with a 1:10 dilution of HFF for two days demonstrated an enhanced incorporation of /sup 3/H-thymidine into acid-precipitable material when compared to control cells. The ratio of counts-per-minute for HFF stimulated cells versus control cells was 3.02 +/- 0.53 (anti S.E.M., n = 5). Endothelial cells also exhibited a directional migration towards HFF through a polycarbonate membrane with 8..mu..m pores. The ratio of the number of cells migrating completely through the filter towards a 1:10 dilution of HFF compared to those migrating towards medium alone was 5.61 +/- 0.61 (anti +/- S.E.M., n = 3). Human serum at an equivalent protein concentration as HFF demonstrated no activity in the CAM, mitogenic, and chemotaxis assays. These results demonstrate specific angiogenic, mitogenic and chemotactic activity in human follicular fluid.

  8. 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. PMID:17643984

  9. Constraints on Noun Incorporation in Korean.

    ERIC Educational Resources Information Center

    Khym, Hangyoo

    1997-01-01

    A study of the noun incorporation phenomenon in Korean suggests that noun incorporation occurs at D-structure and obeys the Head Movement Constraint syntactically, and the Theme-Only Constraint semantically. First, the structure of "sunrise"-type words is identified, showing that before derivation through nominalization of the affix "-i,"…

  10. 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 Transportation Other Regulations Relating to Transportation (Continued) NATIONAL HIGHWAY TRAFFIC SAFETY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) ANTHROPOMORPHIC TEST DEVICES Hybrid III Test Dummy § 572.30 Incorporated materials. (a)...

  11. 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...

  12. 49 CFR 572.180 - Incorporated materials.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... CFR part 51. Copies of the materials may be inspected at the Department of Transportation, Docket... 49 Transportation 7 2011-10-01 2011-10-01 false Incorporated materials. 572.180 Section 572.180... Dummy, 50th Percentile Adult Male § 572.180 Incorporated materials. (a) The following materials...

  13. 10 CFR 218.31 - Incorporated procedures.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 3 2010-01-01 2010-01-01 false Incorporated procedures. 218.31 Section 218.31 Energy DEPARTMENT OF ENERGY OIL STANDBY MANDATORY INTERNATIONAL OIL ALLOCATION Procedures § 218.31 Incorporated procedures. The following subparts of part 205 of this chapter are, as appropriate, hereby made applicable...

  14. 49 CFR 572.180 - Incorporated materials.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... CFR part 51. Copies of the materials may be inspected at the Department of Transportation, Docket... 49 Transportation 7 2010-10-01 2010-10-01 false Incorporated materials. 572.180 Section 572.180... Dummy, 50th Percentile Adult Male § 572.180 Incorporated materials. (a) The following materials...

  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, 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...

  17. 77 FR 16761 - Incorporation by Reference

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-03-22

    .... We published an announcement of the petition and a request for comments on February 27, 2012. 77 FR...; ] OFFICE OF THE FEDERAL REGISTER 1 CFR Part 51 Incorporation by Reference AGENCY: Office of the Federal... regulations governing the approval of agency requests to incorporate material by reference into the Code...

  18. 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…

  19. 49 CFR 572.190 - Incorporated materials.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ...) SAE J1733 of 1994-12, “Sign Convention for Vehicle Crash Testing.” (b) The Director of the Federal Register approved the materials incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part... Impact Crash Test Dummy, July 1, 2008,” incorporated by reference in § 572.191; (4) SAE...

  20. 49 CFR 572.190 - Incorporated materials.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ...) SAE J1733 of 1994-12, “Sign Convention for Vehicle Crash Testing.” (b) The Director of the Federal Register approved the materials incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part... Impact Crash Test Dummy, July 1, 2008,” incorporated by reference in § 572.191; (4) SAE...

  1. 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... 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...

  2. Predictive assays in radiation therapy

    SciTech Connect

    West, C.M.L.

    1994-12-31

    There are reports of promising correlations between patient response to radiotherapy and laboratory measurements of tumor radiosensitivity, fibroblast radiosensitivity, tumor proliferation, and tumor oxygenation status. These all need to be substantiated in large clinical studies. The development of rapid, reliable assays, in particular for determining intrinsic radiosensitivity, would greatly facilitate this work. If the results illustrated in the figures in the chapter can be combined and shown to be feasible on a routine clinical basis, then radiobiologists would be able to provide radiotherapists with a useful aid for the individualization of patient treatment. 162 refs., 6 figs., 6 tabs.

  3. Automated cytopathic effect (CPE) assays.

    PubMed

    McAleer, W J; Miller, W J; Hurni, W M; Machlowitz, R A; Hilleman, M R

    1983-07-01

    An automated CPE procedure has been developed that increases the precision and ease of performing titrations of measles, mumps and rubella viruses in vaccine materials. By this procedure, additions of cell suspensions and reagents and the dilution of samples are performed automatically by a modified Dynatiter instrument, using 96-well microtitre plates. Cell monolayers are stained with carbolfuchsin dye to eliminate the need for microscopic examination. Finally, the trays are read in an optical scanner and the end points calculated automatically by a programmable calculator. The increased accuracy and precision attained by performing greater numbers of replicate assays at reasonable cost will be of particular value to vaccine manufacturers. PMID:6885830

  4. Immunohistochemical assay for epidermal growth factor receptor on paraffin-embedded sections: validation against ligand-binding assay and clinical relevance in breast cancer.

    PubMed Central

    Newby, J. C.; A'Hern, R. P.; Leek, R. D.; Smith, I. E.; Harris, A. L.; Dowsett, M.

    1995-01-01

    Epidermal growth factor receptor (EGFR) has been the subject of much research since it was first described as a prognostic factor in breast cancer. The assay methods used and results obtained vary widely between studies. In this study 88 primary breast cancers were assayed for EGFR using a novel immunohistochemical assay performed on paraffin-embedded sections. The monoclonal antibody used was raised against purified, denatured EGFR, reacts with an epitope on the external domain and does not interfere with ligand binding. Twenty-two per cent of the tumours were EGFR positive using this assay. The results obtained were significantly correlated with those obtained by ligand-binding assay (r = 0.621, P = 0.011). The concordance rate was 82% (P < 0.001). The majority of discordant results could be explained by the presence of benign breast tissue and other non-malignant elements which could be seen to express EGFR on the immunohistochemical assay and were excluded from the score for this, but would be incorporated into ligand-binding assay results. The well-established inverse relationship between EGFR (as measured by this assay) and oestrogen receptor (ER) was seen (chi 2 = 24.9, P < 0.0001). In addition, in this exploratory study on a limited tumour set, EGFR was a significant adverse prognostic factor (on univariate but not multivariate analysis) for both relapse-free survival (P = 0.02) and overall survival (P = 0.03) when measured by this immunohistochemical assay, but was not significant when measured by ligand-binding assay. Images Figure 1 Figure 2 PMID:7779717

  5. Indirect conductimetric assay of antibacterial activities.

    PubMed

    Sawai, J; Doi, R; Maekawa, Y; Yoshikawa, T; Kojima, H

    2002-11-01

    The applicability of indirect conductimetric assays for evaluation of antibacterial activity was examined. The minimal inhibitory concentration (MIC) obtained by the indirect method was consistent with that by the direct conductimetric assay and the turbidity method. The indirect assay allows use of growth media, which cannot be used in the direct conductimetric assay, making it possible to evaluate the antibacterial activity of insoluble or slightly soluble materials with high turbidity, such as antibacterial ceramic powders. PMID:12407467

  6. The chemistry behind antioxidant capacity assays.

    PubMed

    Huang, Dejian; Ou, Boxin; Prior, Ronald L

    2005-03-23

    This review summarizes the multifaceted aspects of antioxidants and the basic kinetic models of inhibited autoxidation and analyzes the chemical principles of antioxidant capacity assays. Depending upon the reactions involved, these assays can roughly be classified into two types: assays based on hydrogen atom transfer (HAT) reactions and assays based on electron transfer (ET). The majority of HAT-based assays apply a competitive reaction scheme, in which antioxidant and substrate compete for thermally generated peroxyl radicals through the decomposition of azo compounds. These assays include inhibition of induced low-density lipoprotein autoxidation, oxygen radical absorbance capacity (ORAC), total radical trapping antioxidant parameter (TRAP), and crocin bleaching assays. ET-based assays measure the capacity of an antioxidant in the reduction of an oxidant, which changes color when reduced. The degree of color change is correlated with the sample's antioxidant concentrations. ET-based assays include the total phenols assay by Folin-Ciocalteu reagent (FCR), Trolox equivalence antioxidant capacity (TEAC), ferric ion reducing antioxidant power (FRAP), "total antioxidant potential" assay using a Cu(II) complex as an oxidant, and DPPH. In addition, other assays intended to measure a sample's scavenging capacity of biologically relevant oxidants such as singlet oxygen, superoxide anion, peroxynitrite, and hydroxyl radical are also summarized. On the basis of this analysis, it is suggested that the total phenols assay by FCR be used to quantify an antioxidant's reducing capacity and the ORAC assay to quantify peroxyl radical scavenging capacity. To comprehensively study different aspects of antioxidants, validated and specific assays are needed in addition to these two commonly accepted assays. PMID:15769103

  7. Serum indices: managing assay interference.

    PubMed

    Farrell, Christopher-John L; Carter, Andrew C

    2016-09-01

    Clinical laboratories frequently encounter samples showing significant haemolysis, icterus or lipaemia. Technical advances, utilizing spectrophotometric measurements on automated chemistry analysers, allow rapid and accurate identification of such samples. However, accurate quantification of haemolysis, icterus and lipaemia interference is of limited value if laboratories do not set rational alert limits, based on sound interference testing experiments. Furthermore, in the context of increasing consolidation of laboratories and the formation of laboratory networks, there is an increasing requirement for harmonization of the handling of haemolysis, icterus and lipaemia-affected samples across different analytical platforms. Harmonization may be best achieved by considering both the analytical aspects of index measurement and the possible variations in the effects of haemolysis, icterus and lipaemia interferences on assays from different manufacturers. Initial verification studies, followed up with ongoing quality control testing, can help a laboratory ensure the accuracy of haemolysis, icterus and lipaemia index results, as well as assist in managing any biases in index results from analysers from different manufacturers. Similarities, and variations, in the effect of haemolysis, icterus and lipaemia interference in assays from different manufacturers can often be predicted from the mechanism of interference. Nevertheless, interference testing is required to confirm expected similarities or to quantify differences. It is important that laboratories are familiar with a number of interference testing protocols and the particular strengths and weaknesses of each. A rigorous approach to all aspects of haemolysis, icterus and lipaemia interference testing allows the analytical progress in index measurement to be translated into improved patient care. PMID:27147624

  8. 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.

  9. 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.

  10. The validity of androgen assays

    PubMed Central

    Carruthers, Malcolm; Trinick, Tom R.; Wheeler, Michael J.

    2007-01-01

    Problems in the measurement of androgens and in interpreting results have been reviewed and classified as follows: Preanalytical factors The exact sampling conditions in relation to circadian and seasonal variations, diet, alcohol, physical activity and posture. Physiological and medical factors Androgen levels vary according to the patient's general health, stress, sexual activity and smoking habits. Analytical variables Sample preservation and storage variables are often unknown. The different androgen assays used have widely differing accuracy and precision and are subject to large inter-laboratory variation, which especially in women and children can render the results of routinely available direct immunoassays meaningless. Interpretation of results Laboratory reference ranges vary widely, largely independent of methodology, and fail to take into account the log-normal distribution of androgen values, causing errors in clinical diagnosis and treatment. Other unknowns are antagonists such as SHBG, estrogens, catecholamines, cortisol, and anti-androgens. As well as age, androgen receptor polymorphisms play a major role in regulating androgen levels and resistance to their action. Conclusions Though laboratory assays can support a diagnosis of androgen deficiency in men, they should not be used to exclude it. It is suggested that there needs to be greater reliance on the history and clinical features, together with careful evaluation of the symptomatology, and where necessary a therapeutic trial of androgen treatment given. PMID:17701661

  11. In vitro Tumorsphere Formation Assays

    PubMed Central

    Johnson, Sara; Chen, Hexin; Lo, Pang-Kuo

    2016-01-01

    A tumorsphere is a solid, spherical formation developed from the proliferation of one cancer stem/progenitor cell. These tumorspheres (Figure 1a) are easily distinguishable from single or aggregated cells (Figure 1b) as the cells appear to become fused together and individual cells cannot be identified. Cells are grown in serum-free, non-adherent conditions in order to enrich the cancer stem/progenitor cell population as only cancer stem/progenitor cells can survive and proliferate in this environment. This assay can be used to estimate the percentage of cancer stem/progenitor cells present in a population of tumor cells. The size, which can vary from less than 50 micrometers to 250 micrometers, and number of tumorspheres formed can be used to characterize the cancer stem/progenitor cell population within a population of in vitro cultured cancer cells and within in vivo tumors (Lo et al., 2012; Liu et al., 2009). While several cell lines can be used for tumorsphere formation assay (e.g. primary mammary tumor cells from Her2/neu-transgenic mice, MCF7, BT474 and HCC1954), some cell lines may not form typical tumorsphere structures and may be difficult to count or classify definitively as tumorspheres.

  12. 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.

  13. Proteasome Assay in Cell Lysates

    PubMed Central

    Maher, Pamela

    2016-01-01

    The ubiquitin-proteasome system (UPS) mediates the majority of the proteolysis seen in the cytoplasm and nucleus of mammalian cells. As such it plays an important role in the regulation of a variety of physiological and pathophysiological processes including tumorigenesis, inflammation and cell death (Ciechanover, 2005; Kisselev and Goldberg, 2001). A number of recent studies have shown that proteasome activity is decreased in a variety of neurological disorders including Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis and stroke as well as during normal aging (Chung et al., 2001; Ciechanover and Brundin, 2003; Betarbet et al., 2005). This decrease in proteasome activity is thought to play a critical role in the accumulation of abnormal and oxidized proteins. Protein clearance by the UPS involves two sequential reactions. The first is the tagging of protein lysine residues with ubiquitin (Ub) and the second is the subsequent degradation of the tagged proteins by the proteasome. We herein describe an assay for the second of these two reactions (Valera et al., 2013). This assay uses fluorogenic substrates for each of the three activities of the proteasome: chymotrypsin-like activity, trypsin-like activity and caspase-like activity. Cleavage of the fluorophore from the substrate by the proteasome results in fluorescence that can be detected with a fluorescent plate reader.

  14. 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.

  15. 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.

  16. Growth and osteogenic differentiation of alveolar human bone marrow-derived mesenchymal stem cells on chitosan/hydroxyapatite composite fabric.

    PubMed

    Kim, Beom-Su; Kim, Jin Seong; Chung, Yong Sik; Sin, Yeon-Woo; Ryu, Kyung-Ho; Lee, Jun; You, Hyung-Keun

    2013-06-01

    Scaffolds can be used for tissue engineering because they can serve as templates for cell adhesion and proliferation for tissue repair. In this study, chitosan/hydroxyapatite (CS/HAp) composites were prepared by coprecipitation synthesis. Then, CS and CS/HAp fabrics were prepared by wet spinning. CS fibers with a diameter of 15 ± 1.3 μm and CS/HAp fibers with a diameter of 22 ± 1.2 μm were successfully produced; incorporation of HAp into the CS/HAp fibers was confirmed by X-ray diffraction analysis. Biological in vitro evaluations showed that human mesenchymal stem cells (hMSCs) cultured on CS/HAp fabric showed increased proliferation compared to those cultured on pure CS fabric, which was observed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, DNA content assay, and [(3) H] thymidine incorporation assay. Neither the CS nor CS/HAp scaffold exhibited any cytotoxicity to hMSCs, as shown by viability staining and cytotoxicity fluorescence image assays. After 10 days of culturing, the attachment of cells onto the scaffold was observed by scanning electron microscopy. Furthermore, under osteogenic differentiation conditions, alkaline phosphatase (ALP) activity and calcium accumulation was higher in cells cultured on the CS/HAp scaffold than in cells cultured on the CS scaffold. The mRNA expression of osteoblast markers, including ALP, osteocalcin, Co1Ia1, and runt-related transcription factor 2, was higher in cells cultured on CS/HAp than in cells cultured on the CS fabric. The results of this study indicate that the CS/HAp composite fabric may serve as a good scaffold for bone tissue engineering applications. PMID:23135904

  17. 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.

  18. Toxicity analysis of ocular prosthesis acrylic resin with or without pigment incorporation in human conjunctival cell line.

    PubMed

    da Silva, Emily Vivianne Freitas; Goiato, Marcelo Coelho; Bonatto, Liliane da Rocha; de Medeiros, Rodrigo Antonio; Santos, Daniela Micheline Dos; Rangel, Elidiane Cipriano; Oliveira, Sandra Helena Penha de

    2016-10-01

    The aim of this study was to evaluate the influence of pigment incorporation on the cytotoxicity of ocular prosthesis N1 color acrylic resin. Nine samples were manufactured by heat-polymerization in water bath and divided into 3 groups: acrylic resin without pigment incorporation (group R), acrylic resin with pigment incorporation (group RP), and acrylic pigment (group P). Eluates formed after 72h of sample immersion in medium were incubated with conjunctival cell line (Chang conjunctival cells) for 72h. The negative control group consisted in medium without samples (group C). The cytotoxic effect from the eluates was evaluated using MTT assay (cell proliferation), ELISA assay (quantification of IL1β, IL6, TNF α and CCL3/MIP1α) and RT-PCR assay (mRNA expression of COL IV, TGF β and MMP9). Data were submitted to ANOVA with Bonferroni post-tests (p<0.05). All groups were considered non-cytotoxic based on cell proliferation. However, resin with pigment incorporation showed significant IL6 quantity increase. Resin without pigment incorporation exhibited higher mRNA expression of COL IV, MMP9 and TGF β, however it was also observed for the negative control group. The materials exhibited divergent biological behavior. Despite the pigment incorporation that resulted in an increase of IL6, no cytotoxicity was observed based on cell proliferation. PMID:27521695

  19. Comparison of Established and Emerging Biodosimetry Assays

    PubMed Central

    Rothkamm, K.; Beinke, C.; Romm, H.; Badie, C.; Balagurunathan, Y.; Barnard, S.; Bernard, N.; Boulay-Greene, H.; Brengues, M.; De Amicis, A.; De Sanctis, S.; Greither, R.; Herodin, F.; Jones, A.; Kabacik, S.; Knie, T.; Kulka, U.; Lista, F.; Martigne, P.; Missel, A.; Moquet, J.; Oestreicher, U.; Peinnequin, A.; Poyot, T.; Roessler, U.; Scherthan, H.; Terbrueggen, B.; Thierens, H.; Valente, M.; Vral, A.; Zenhausern, F.; Meineke, V.; Braselmann, H.; Abend, M.

    2014-01-01

    Rapid biodosimetry tools are required to assist with triage in the case of a large-scale radiation incident. Here, we aimed to determine the dose-assessment accuracy of the well-established dicentric chromosome assay (DCA) and cytokinesis-block micronucleus assay (CBMN) in comparison to the emerging γ-H2AX foci and gene expression assays for triage mode biodosimetry and radiation injury assessment. Coded blood samples exposed to 10 X-ray doses (240 kVp, 1 Gy/min) of up to 6.4 Gy were sent to participants for dose estimation. Report times were documented for each laboratory and assay. The mean absolute difference (MAD) of estimated doses relative to the true doses was calculated. We also merged doses into binary dose categories of clinical relevance and examined accuracy, sensitivity and specificity of the assays. Dose estimates were reported by the first laboratories within 0.3–0.4 days of receipt of samples for the γ-H2AX and gene expression assays compared to 2.4 and 4 days for the DCA and CBMN assays, respectively. Irrespective of the assay we found a 2.5–4-fold variation of interlaboratory accuracy per assay and lowest MAD values for the DCA assay (0.16 Gy) followed by CBMN (0.34 Gy), gene expression (0.34 Gy) and γ-H2AX (0.45 Gy) foci assay. Binary categories of dose estimates could be discriminated with equal efficiency for all assays, but at doses ≥1.5 Gy a 10% decrease in efficiency was observed for the foci assay, which was still comparable to the CBMN assay. In conclusion, the DCA has been confirmed as the gold standard biodosimetry method, but in situations where speed and throughput are more important than ultimate accuracy, the emerging rapid molecular assays have the potential to become useful triage tools. PMID:23862692

  20. 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

    2007-02-01

    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. PMID:17263472

  1. 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

  2. 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. PMID:22518030

  3. Northwest Australia's Saladin crude assayed

    SciTech Connect

    Rhodes, A.K.

    1993-10-18

    High-quality Saladin crude oil from offshore Western Australia has been assayed. The 48.2[degree] API, 0.02 wt % sulfur crude's characteristics--determined in 1990--are presented here for the first time. The estimated 30--40 million bbl field, south of Barrow Island, is produced from two platforms in 58 ft of water in block TP 3. Production began in late 1989 from three platforms with three wells each and from two wells drilled directionally from Thevenard Island. The paper lists data on the following properties: API gravity, density, sulfur content, pour point, flash point, viscosity, salinity, heat of combustion, ash content, asphaltene content, wax content, and metal content for the whole crude and various fractions.

  4. 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

  5. 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. PMID:24978020

  6. Toxicity and immunogenicity of Neisseria meningitidis lipopolysaccharide incorporated into liposomes.

    PubMed Central

    Petrov, A B; Semenov, B F; Vartanyan, Y P; Zakirov, M M; Torchilin, V P; Trubetskoy, V S; Koshkina, N V; L'Vov, V L; Verner, I K; Lopyrev, I V

    1992-01-01

    To obtain nontoxic and highly immunogenic lipopolysaccharide (LPS) for immunization, we incorporated Neisseria meningitidis LPS into liposomes. Native LPS and its salts were incorporated by the method of dehydration-rehydration of vesicles or prolonged cosonication. The most complete incorporation of LPS into liposomes and a decrease in toxicity were achieved by the method of dehydration-rehydration of vesicles. Three forms of LPS (H+ form, Mg2+ salt, and triethanolamine salt) showed different solubilities in water, the acidic form of LPS, with the most pronounced hydrophobic properties, being capable of practically complete association with liposomal membranes. An evaluation of the activity of liposomal LPS in vitro (by the Limulus amoebocyte test) and in vivo (by monitoring the pyrogenic reaction in rabbits) revealed a decrease in endotoxin activity of up to 1,000-fold. In addition, the pyrogenic activity of liposomal LPS was comparable to that of a meningococcal polysaccharide vaccine. Liposomes had a pronounced adjuvant effect on the immune response to LPS. Thus, the level of anti-LPS plaque-forming cells in the spleens of mice immunized with liposomal LPS was 1 order of magnitude higher and could be observed for a longer time (until day 21, i.e., the term of observation) than in mice immunized with free LPS. The same regularity was revealed in a study done with an enzyme-linked immunosorbent assay. This study also established that antibodies induced by immunization belonged to the immunoglobulin M and G classes, which are capable of prolonged circulation. Moreover, liposomal LPS induced a pronounced immune response in CBA/N mice (defective in B lymphocytes of the LyB-5+ subpopulation). The latter results indicate that the immunogenic action of liposomal LPS occurs at an early age. PMID:1500196

  7. Toxicity and immunogenicity of Neisseria meningitidis lipopolysaccharide incorporated into liposomes.

    PubMed

    Petrov, A B; Semenov, B F; Vartanyan, Y P; Zakirov, M M; Torchilin, V P; Trubetskoy, V S; Koshkina, N V; L'Vov, V L; Verner, I K; Lopyrev, I V

    1992-09-01

    To obtain nontoxic and highly immunogenic lipopolysaccharide (LPS) for immunization, we incorporated Neisseria meningitidis LPS into liposomes. Native LPS and its salts were incorporated by the method of dehydration-rehydration of vesicles or prolonged cosonication. The most complete incorporation of LPS into liposomes and a decrease in toxicity were achieved by the method of dehydration-rehydration of vesicles. Three forms of LPS (H+ form, Mg2+ salt, and triethanolamine salt) showed different solubilities in water, the acidic form of LPS, with the most pronounced hydrophobic properties, being capable of practically complete association with liposomal membranes. An evaluation of the activity of liposomal LPS in vitro (by the Limulus amoebocyte test) and in vivo (by monitoring the pyrogenic reaction in rabbits) revealed a decrease in endotoxin activity of up to 1,000-fold. In addition, the pyrogenic activity of liposomal LPS was comparable to that of a meningococcal polysaccharide vaccine. Liposomes had a pronounced adjuvant effect on the immune response to LPS. Thus, the level of anti-LPS plaque-forming cells in the spleens of mice immunized with liposomal LPS was 1 order of magnitude higher and could be observed for a longer time (until day 21, i.e., the term of observation) than in mice immunized with free LPS. The same regularity was revealed in a study done with an enzyme-linked immunosorbent assay. This study also established that antibodies induced by immunization belonged to the immunoglobulin M and G classes, which are capable of prolonged circulation. Moreover, liposomal LPS induced a pronounced immune response in CBA/N mice (defective in B lymphocytes of the LyB-5+ subpopulation). The latter results indicate that the immunogenic action of liposomal LPS occurs at an early age. PMID:1500196

  8. APTIMA® Trichomonas vaginalis, a transcription-mediated amplification assay for detection of Trichomonas vaginalis in urogenital specimens.

    PubMed

    Chapin, Kimberle; Andrea, Sarah

    2011-09-01

    The APTIMA(®) Trichomonas vaginalis (APTIMA TV; Gen-Probe Inc.) assay is the only amplification-based assay for T. vaginalis (TV) currently cleared by the US FDA. The assay was cleared in April 2011. APTIMA TV utilizes target capture specimen processing, transcription-mediated amplification and chemiluminescent probe hybridization for the qualitative detection of TV ribosomal RNA. The assay is used for the screening/diagnosis of trichomoniasis in women. Specimen types that can be used include physician-collected endocervical swabs, vaginal swabs, endocervical specimens collected in PreservCyt(®) (Thin Prep, Hologic Incorporated, MA, USA) solution and female urine specimens. The APTIMA TV assay has shown superior performance in side-by-side comparisons with other diagnostic methods in all patient populations and specimen types tested. Clinical sensitivity and specificity are >95 and 98%, respectively. The APTIMA TV assay fills a significant void in sexually transmitted infection diagnostics. PMID:21902528

  9. 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.

  10. Application of HPLC in the 32P-postlabeling assay.

    PubMed

    Gorelick, N J

    1993-07-01

    The postlabeling procedure for the detection of DNA modifications entails enzyme-catalyzed incorporation of 32P into nucleotides and chromatographic separation of radiolabeled products for quantification. Alternate versions of this procedure have been developed which vary in sensitivity and in applicability for the detection of different DNA adducts. Methods that utilize HPLC in either of two steps in the procedure (i.e., the separation of modified and unmodified nucleotides before the labeling reaction or the resolution of 32P-labeled adducts) are applicable for the detection of alkyl adducts as well as bulky, hydrophobic adducts and are discussed in this review. In some cases, postlabeling assays have been tailored for the quantitative detection of specific adducts. Use of multiple optimized postlabeling methods to analyze one DNA sample may enable identification of multiple specific adducts in human DNA. The widest and most promising applications for adduct detection with the postlabeling assay are for previously characterized adducts, where adduct standards are available for optimization and characterization of recovery in the assay. 32P-Postlabeling is a powerful way to measure DNA adducts as it is very sensitive. However, caution should be applied in drawing conclusions from postlabeling studies without appropriate corroborative data using another adduct detection method or without appropriate method development preceding the study. Examples of applications in human, laboratory animal, and environmental studies are available. PMID:7686266

  11. 49 CFR 572.80 - Incorporated materials.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies of the materials may be obtained from Rowley-Scher... 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...

  12. 49 CFR 572.80 - Incorporated materials.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies of the materials may be obtained from Rowley-Scher... 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...

  13. 49 CFR 572.80 - Incorporated materials.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies of the materials may be obtained from Rowley-Scher... 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...

  14. 49 CFR 572.80 - Incorporated materials.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies of the materials may be obtained from Rowley-Scher... 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...

  15. 49 CFR 572.80 - Incorporated materials.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies of the materials may be obtained from Rowley-Scher... 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...

  16. Evaluation of a fluorogenic assay for detection of Escherichia coli in foods.

    PubMed Central

    Robison, B J

    1984-01-01

    A fluorogenic assay procedure with 4-methylumbelliferyl-beta-D-glucuronide incorporated into lauryl sulfate broth was evaluated to detect and confirm the presence of Escherichia coli in foods. Fluorescence is indicative of the presence of E. coli; extensive biochemical confirmation is unnecessary with this assay. The 4-methylumbelliferyl-beta-D-glucuronide assay was tested concurrently with our present methodology for detection of E. coli on 270 samples of raw ingredients and powdered food products. Total agreement between the two methods was 94.8%; there was a false-positive rate of 4.8% and no false-negatives. We found the 4-methylumbelliferyl-beta-D-glucuronide assay to be rapid, accurate, simple to perform, and inexpensive. PMID:6385845

  17. Expert system for transuranic waste assay

    SciTech Connect

    Zoolalian, M.L.; Gibbs, A.; Kuhns, J.D.

    1989-01-01

    Transuranic wastes are generated at the Savannah River Site (SRS) as a result of routine production of nuclear materials. These wastes contain Pu-238 and Pu-239 and are placed into lined 55-gallon waste drums. The drums are placed on monitored storage pads pending shipment to the Waste Isolation Pilot Plant in New Mexico. A passive-active neutron (PAN) assay system is used to determine the mass of the radioactive material within the waste drums. Assay results are used to classify the wastes as either low-level or transuranic (TRU). During assays, the PAN assay system communicates with an IBM-AT computer. A Fortran computer program, called NEUT, controls and performs all data analyses. Unassisted, the NEUT program cannot adequately interpret assay results. To eliminate this limitation, an expert system shell was used to write a new algorithm, called the Transuranic Expert System (TRUX), to drive the NEUT program and add decision making capabilities for analysis of the assay results. The TRUX knowledge base was formulated by consulting with human experts in the field of neutron assay, by direct experimentation on the PAN assay system, and by observing operations on a daily basis. TRUX, with its improved ability to interpret assay results, has eliminated the need for close supervision by a human expert, allowing skilled technicians to operate the PAN assay system. 4 refs., 1 fig., 4 tabs.

  18. Lack of effect of furfural on unscheduled DNA synthesis in the in vivo rat and mouse hepatocyte DNA repair assays and in precision-cut human liver slices.

    PubMed

    Lake, B G; Edwards, A J; Price, R J; Phillips, B J; Renwick, A B; Beamand, J A; Adams, T B

    2001-10-01

    The ability of furfural to induce unscheduled DNA synthesis (UDS) in hepatocytes of male and female B6C3F(1) mice and male F344 rats after in vivo administration and in vitro in precision-cut human liver slices has been studied. Preliminary toxicity studies established the maximum tolerated dose (MTD) of furfural to be 320 and 50 mg/kg in the mouse and rat, respectively. Furfural was dosed by gavage at levels of 0 (control), 50, 175 and 320 mg/kg to male and female mice and 0, 5, 16.7 and 50 mg/kg to male rats. Hepatocytes were isolated by liver perfusion either 2-4 h or 12-16 h after treatment, cultured in medium containing [3H]thymidine for 4 h and assessed for UDS by grain counting of autoradiographs. Furfural treatment did not produce any statistically significant increase or any dose-related effects on UDS in mouse and rat hepatocytes either 2-4 h or 12-16 h after dosing. In contrast, UDS was markedly induced in mice and rats 2-4 h after treatment with 20 mg/kg dimethylnitrosamine and 12-16 h after treatment of mice and rats with 200 mg/kg o-aminoazotoluene and 50 mg/kg 2-acetylaminofluorene (2-AAF), respectively. Precision-cut human liver slices from four donors were cultured for 24 h in medium containing [3H]thymidine and 0-10 mM furfural. Small increases in the net grain count (i.e. nuclear grain count less mean cytoplasmic grain count) observed with 2-10 mM furfural were not due to any increase in the nuclear grain count. Rather, it was the result of concentration-dependent decreases in the mean cytoplasmic grain counts and to a lesser extent in nuclear grain counts, due to furfural-induced cytotoxicity. In contrast, marked increases in UDS (both net grain and nuclear grain counts) were observed in human liver slices treated with 0.02 and 0.05 mM 2-AAF, 0.002 and 0.02 mM aflatoxin B(1) and 0.005 and 0.05 mM 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. This study demonstrates that furfural does not induce UDS in the hepatocytes of male and female B6C3F

  19. Validation and Application of a Dried Blood Spot Ceftriaxone Assay

    PubMed Central

    Page-Sharp, Madhu; Nunn, Troy; Salman, Sam; Moore, Brioni R.; Batty, Kevin T.; Davis, Timothy M. E.

    2015-01-01

    Dried blood spot (DBS) antibiotic assays can facilitate pharmacokinetic/pharmacodynamic (PK/PD) studies in situations where venous blood sampling is logistically and/or ethically problematic. In this study, we aimed to develop, validate, and apply a DBS ceftriaxone assay. A liquid chromatography-tandem mass spectroscopy (LC-MS/MS) DBS ceftriaxone assay was assessed for matrix effects, process efficiency, recovery, variability, and limits of quantification (LOQ) and detection (LOD). The effects of hematocrit, protein binding, red cell partitioning, and chad positioning were evaluated, and thermal stability was assessed. Plasma, DBS, and cell pellet ceftriaxone concentrations in 10 healthy adults were compared, and plasma concentration-time profiles of DBS and plasma ceftriaxone were incorporated into population PK models. The LOQ and LOD for ceftriaxone in DBS were 0.14 mg/liter and 0.05 mg/liter, respectively. Adjusting for hematocrit, red cell partitioning, and relative recovery, DBS-predicted plasma concentrations were comparable to measured plasma concentrations (r > 0.95, P < 0.0001), and Bland-Altman plots showed no significant bias. The final population PK estimates of clearance, volume of distribution, and time above threshold MICs for measured and DBS-predicted plasma concentrations were similar. At 35°C, 21°C, 4°C, −20°C, and −80°C, ceftriaxone retained >95% initial concentrations in DBS for 14 h, 35 h, 30 days, 21 weeks, and >11 months, respectively. The present DBS ceftriaxone assay is robust and can be used as a surrogate for plasma concentrations to provide valid PK and PK/PD data in a variety of clinical situations, including in studies of young children and of those in remote or resource-poor settings. PMID:26438505

  20. 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.

  1. Staphylococcus aureus Cell Extract Transcription-Translation Assay: Firefly Luciferase Reporter System for Evaluating Protein Translation Inhibitors

    PubMed Central

    Murray, Robert W.; Melchior, Earline P.; Hagadorn, Jeanne C.; Marotti, Keith R.

    2001-01-01

    The promoter for the Staphylococcus aureus capsule polysaccharide synthesis gene (cap1A) was cloned in front of the firefly luciferase gene for use in a cell extract S. aureus transcription-translation system. The assay is rapid, reproducible, and sensitive and has a lower background level than the radiolabeled amino acid incorporation translation assays. We present data evaluating a transcription inhibitor and a number of protein translation inhibitors in this system. PMID:11353649

  2. Matrix effects of TRU (transuranic) assays using the SWEPP PAN assay system

    SciTech Connect

    Smith, J.R.

    1990-08-01

    The Drum Assay System (DAS) at the Stored Waste Experimental Pilot Plant (SWEPP) is a second-generation active-passive neutron assay system. It has been used to assay over 5000 208-liter drums of transuranic waste from the Rocky Flats Plant (RFP). Data from these assays have been examined and compared with the assays performed at Rocky Flats, mainly utilize counting of {sup 239}Pu gamma rays. For the most part the passive assays are in very good agreement with the Rocky Flats assays. The active assays are strongly correlated with the results of the other two methods, but require matrix-dependent correction factors beyond those provided by the system itself. A set of matrix-dependent correction factors has been developed from the study of the assay results. 3 refs., 4 figs., 3 tabs.

  3. 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.

  4. Highly Sensitive Multiplex Assay for Detection of Human Immunodeficiency Virus Type 1 and Hepatitis C Virus RNA

    PubMed Central

    Giachetti, C.; Linnen, J. M.; Kolk, D. P.; Dockter, J.; Gillotte-Taylor, K.; Park, M.; Ho-Sing-Loy, M.; McCormick, M. K.; Mimms, L. T.; McDonough, S. H.

    2002-01-01

    Various nucleic acid assays have been developed and implemented for diagnostics and therapeutic monitoring of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) infections. The high-throughput, semiautomated assays described here were developed to provide a method suitable for screening plasma specimens for the presence of HIV-1 and HCV RNAs. Three assays were developed: a multiplex HIV-1/HCV assay for simultaneous detection of HIV-1 and HCV, and discriminatory assays for specific detection of HIV-1 and HCV. The assay systems utilize three proprietary technologies: (i) target capture-based sample preparation, (ii) transcription-mediated amplification (TMA), and (iii) hybridization protection assay (HPA). An internal control is incorporated into each reaction to control for every step of the assay and identify random false-negative reactions. The assays demonstrated a sensitivity of at least 100 copies/ml for each target, and they detected with similar sensitivity all major variants of HCV and HIV-1, including HIV-1 group O strains. Assay sensitivity for one virus was not affected by the presence of the other. The specificity of these TMA-driven assays was ≥99.5% in both normal donor specimens and plasma containing potentially interfering substances or other blood-borne pathogens. Statistical receiver operating characteristic plots of 1 − specificity versus sensitivity data determined very wide analyte cutoff values for each assay at the point at which the assay specificity and sensitivity were both ≥99.5%. The sensitivity, specificity, and throughput capability predict that these assays will be valuable for large-volume plasma screening, either in a blood bank setting or in other diagnostic applications. PMID:12089255

  5. 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.

  6. 233U Assay A Neutron NDA System

    SciTech Connect

    Hensley, D.C.; Lucero, A.J.; Pierce, L.

    1998-11-17

    The assay of highly enriched {sup 233}U material presents some unique challenges. Techniques which apply to the assay of materials of Pu or enriched {sup 235}U do not convert easily over to the assay of {sup 233}U. A specialized neutron assay device is being fabricated to exploit the singles neutron signal, the weak correlated neutron signal, and an active correlated signal. These pieces of information when combined with {gamma} ray isotopics information should give a good overall determination of {sup 233}U material now stored in bldg. 3019 at the Oak Ridge National Laboratory.

  7. A novel cromakalim analogue induces cell cycle arrest and apoptosis in human cervical carcinoma HeLa cells through the caspase- and mitochondria-dependent pathway.

    PubMed

    Zhang, Xin; Zhao, Jing; Kang, Saeromi; Yi, Myeongjin; You, Song; Shin, Dong-Soo; Kim, Dong-Kyoo

    2011-12-01

    In the present study, a series of seven synthetic croma-kalim analogues were prepared and evaluated for cytotoxic effect on human cervical carcinoma HeLa cells using WST-8 assay. A preliminary screening of these cromakalim analogues showed that 1-[(3S,4R)-4-(2-ethoxy-4-methyl-1H-pyrrol-1-yl)-3-hydroxy- 2,2-dimethylchroman-6-yl-3-phenylurea (compound 6) had the highest cytotoxic effect (IC50 of 138 µM) and significantly inhibited HeLa cell proliferation after 36 h. In an effort to understand the cytotoxic mechanism of compound 6, we examined its effect on apoptosis and cell cycle distribution. Our results showed that compound 6 induced marked changes in apoptotic morphology and significantly increased early apoptosis of HeLa cells after 48 h by using Annexin V-FITC/PI dual staining assay. This apoptotic induction was associated with an increase in Bax expression, a decrease in Bcl-2 expression, release of cytochrome c and subsequent activation of caspase-9 and -3, which indicated that compound 6 induced apoptosis via caspase- and mitochondria-dependent pathway. By DNA content analysis and [3H]thymidine incorporation assay, compound 6 was found to induce an increase in the number of cells in G1 phase, accompanied by a decrease in the S phase to prevent DNA synthesis after 24 h of treatment. In addition, compound 6 caused significant DNA damage, as detected by the alkaline comet assay. Taken together, the data demonstrate that compound 6 induces apoptosis in HeLa cells through caspase- and mitochondria-dependent pathway and this apoptotic effect is associated with cell cycle arrest and DNA damage. These findings provide further understanding of the molecular mechanisms of compound 6 in cervical cancer. PMID:21833470

  8. LT-HSC Methylcellulose Assay

    PubMed Central

    Kerenyi, Marc A.

    2016-01-01

    Hematopoietic differentiation is a highly complex process originating from an extraordinary population of cells called long-term repopulating hematopoietic stem cells (LT-HSCs). The unique feature of all stem cells, including HSCs, is their exceptional ability to divide asymmetrically giving rise to two different kinds of offspring. One daughter cell becomes an LT-HSC itself (self-renews) to maintain the LT-HSC pool, whereas the second daughter cell pursues a differentiation fate to ultimately give rise to terminally differentiated mature blood cells (Orkin and Zon, 2008). Quantification of phenotypic LT-HSCs can be performed by multi-color flow cytometry and the gold standard for assessment of LT-HSC self-renewal and function is competitive bone marrow transplantation (Miller et al., 2008). Although these methods are irreplaceable to determine LT-HSC abundance and functionality, they have their disadvantages and limitations. For example, competitive bone marrow transplantation is typically monitored as a function of peripheral blood donor contribution over 12–16 weeks. While reduced peripheral blood donor contribution by itself signifies impairment in the stem/progenitor cells compartment, it cannot unambiguously discriminate between reduced LT-HSC self-renewal, impaired LT-HSC differentiation or compromised progenitor cell differentiation. Here we describe an LT-HSCs methylcellulose colony-forming assay, as a fast complementary in vitro method to directly assess LT-HSC differentiation capacity. As described in Kerenyi et al. (2013), this technique acts as a powerful tool to differentiate between LT-HSC or progenitor cell differentiation defects.

  9. 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.

  10. Incorporating Duration Information in Activity Recognition

    NASA Astrophysics Data System (ADS)

    Chaurasia, Priyanka; Scotney, Bryan; McClean, Sally; Zhang, Shuai; Nugent, Chris

    Activity recognition has become a key issue in smart home environments. The problem involves learning high level activities from low level sensor data. Activity recognition can depend on several variables; one such variable is duration of engagement with sensorised items or duration of intervals between sensor activations that can provide useful information about personal behaviour. In this paper a probabilistic learning algorithm is proposed that incorporates episode, time and duration information to determine inhabitant identity and the activity being undertaken from low level sensor data. Our results verify that incorporating duration information consistently improves the accuracy.

  11. Improvement of Microbial Assays of Vitamins

    PubMed Central

    Heed, Edward J.

    1972-01-01

    A method for the improvement of microbial assays of vitamins, which involves the addition of a surfactant to the incubated test, was developed. This surfactant tends to eliminate bacterial clumping, giving a uniform suspension of single cells, thereby making the turbidity readings less erratic and the actual assay standard curves more closely related to the desired theoretical curve. PMID:4627232

  12. Microtissue Culture Plaque Assay for Herpesvirus saimiri

    PubMed Central

    Chan, Emerson W.; Dunkel, Virginia C.

    1973-01-01

    A microtissue culture method for the plaque assay of Herpesvirus saimiri has been developed. Virus titrations carried out in Microtest II tissue culture plates (Falcon) yielded reproducible results that agreed well with those obtained by employing macrocultures. The described method is quantitative, reproducible, economical, and suitable for routine assay of large numbers of virus samples. Images PMID:4201642

  13. A bioluminescent assay for measuring glucose uptake.

    PubMed

    Valley, Michael P; Karassina, Natasha; Aoyama, Natsuyo; Carlson, Coby; Cali, James J; Vidugiriene, Jolanta

    2016-07-15

    Identifying activators and inhibitors of glucose uptake is critical for both diabetes management and anticancer therapy. To facilitate such studies, easy-to-use nonradioactive assays are desired. Here we describe a bioluminescent glucose uptake assay for measuring glucose transport in cells. The assay is based on the uptake of 2-deoxyglucose and the enzymatic detection of the 2-deoxyglucose-6-phosphate that accumulates. Uptake can be measured from a variety of cell types, it can be inhibited by known glucose transporter inhibitors, and the bioluminescent assay yields similar results when compared with the radioactive method. With HCT 116 cells, glucose uptake can be detected in as little as 5000 cells and remains linear up to 50,000 cells with signal-to-background values ranging from 5 to 45. The assay can be used to screen for glucose transporter inhibitors, or by multiplexing with viability readouts, changes in glucose uptake can be differentiated from overall effects on cell health. The assay also can provide a relevant end point for measuring insulin sensitivity. With adipocytes and myotubes, insulin-dependent increases in glucose uptake have been measured with 10- and 2-fold assay windows, respectively. Significant assay signals of 2-fold or more have also been measured with human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and skeletal myoblasts. PMID:27130501

  14. Development of an upconverting chelate assay

    NASA Astrophysics Data System (ADS)

    Xiao, Xudong; Haushalter, Jeanne P.; Kotz, Kenneth T.; Faris, Gregory W.

    2005-04-01

    We report progress on performing a cell-based assay for the detection of EGFR on cell surfaces by using upconverting chelates. An upconversion microscope has been developed for performing assays and testing optical response. A431 cells are labeled with europium DOTA and imaged using this upconverting microscope.

  15. Fluorescence polarization assays in signal transduction discovery.

    PubMed

    Sportsman, J Richard; Daijo, Janet; Gaudet, Elizabeth A

    2003-05-01

    Fluorescence polarization (FP) has become widely employed for high throughput screening used in pharmaceutical drug discovery. Assays of important signal transduction targets are now adapted to FP. In this review we examine assays for cyclic adenosine monophosphate, phosphodiesterases, and protein kinases and phosphatases using FP competitive immunoassays and a direct enzymatic method called IMAP. PMID:12678698

  16. 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 HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification....

  17. 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....

  18. 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....

  19. 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 HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification....

  20. 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....

  1. 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....

  2. 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....

  3. 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....

  4. 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....

  5. 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....

  6. Fluorometric functional assay for ion channel proteins in lipid nanovesicle membranes

    NASA Astrophysics Data System (ADS)

    Patti, J. T.; Montemagno, C. D.

    2007-08-01

    Voltage-gated membrane proteins function as biomolecular transistors, making them attractive components for biologically based nanodevices. A functional assay for purified channel proteins is described and demonstrated with sodium selective, voltage-gated NaChBac ion channels. Purified NaChBac proteins were incorporated into a nanovesicle system utilizing oxonol VI, a fluorescent indicator of trans-membrane voltage. The ionophore valinomycin was used to trigger a change in membrane potential, allowing the observation of sodium permeability using a fluorometer. This method is suitable for concurrently testing a large population of purified proteins prior to incorporation in nanodevices.

  7. 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…

  8. 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 Transportation Other Regulations Relating to Transportation (Continued) NATIONAL HIGHWAY TRAFFIC SAFETY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) ANTHROPOMORPHIC TEST DEVICES Side Impact Dummy 50th Percentile Male § 572.40...

  9. Incorporating Learning into the Cognitive Assessment Framework

    ERIC Educational Resources Information Center

    Studer, Cassandra; Junker, Brian; Chan, Helen

    2012-01-01

    The authors aimed to incorporate learning into the cognitive assessment framework that exists for static assessment data. In order to accomplish this, they derive a common likelihood function for dynamic models and introduce Parameter Driven Process for Change + Cognitive Diagnosis Model (PDPC + CDM), a dynamic model which tracks learning…

  10. 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…

  11. Beyond Classroom Boundaries: Incorporating Context in Teaching.

    ERIC Educational Resources Information Center

    CATESOL Journal, 1994

    1994-01-01

    This English-as-a-Second-Language (ESL) journal periodically devotes entire issues to specific issues. The theme of this issue is "Incorporating Context in Teaching." Articles include: "Learning Beyond the Classroom: Developing the Community Connection" (Tim Beard); "Smiling through the Turbulence: The Flight Attendant Syndrome and Other Issues of…

  12. 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…

  13. 78 FR 69006 - Incorporation by Reference

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-18

    ... the Code of Federal Regulations (77 FR 11414 (February 27, 2012)). Given the recent government... correcting the docket number and adding a link to the docket. Correction In proposed rule FR Doc. 2013-24217...; ] OFFICE OF THE FEDERAL REGISTER 1 CFR Part 51 RIN 3095-AB78 Incorporation by Reference AGENCY: Office...

  14. 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…

  15. 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…

  16. 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.

  17. 49 CFR 572.180 - Incorporated materials.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... approved the materials incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51... §§ 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”; and, (5) SAE...

  18. 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…

  19. Acquisition of Noun Incorporation in Inuktitut.

    ERIC Educational Resources Information Center

    Allen, Shanley; Crago, Martha

    An investigation of the first language acquisition of productive nouns in Inuktitut (Inupiaq) is presented. This study begins with descriptions of noun incorporation, relevant aspects of the structure of Inuktitut, and working criteria of productivity. Acquisition data from Inuktitut and corroborating data from Greenlandic are outlined and…

  20. Incorporating Active Learning into a Traditional Curriculum.

    ERIC Educational Resources Information Center

    Carroll, Robert G.; Huang, Alice H.

    1997-01-01

    Discusses self-learning exercises (SLEs) incorporated into the Medical Physiology course for first-year students at the Morehouse School of Medicine in Atlanta, GA. Twenty to thirty percent of course material is presented in these exercises instead of in lectures. The exercises develop active learning and problem-solving skills. Formal analysis…

  1. TOXICITY OF SEDIMENT-INCORPORATED DRILLING FLUIDS

    EPA Science Inventory

    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 calcula...

  2. Incorporation of National Universities in Japan

    ERIC Educational Resources Information Center

    Oba, Jun

    2007-01-01

    In April 2004, Japanese national universities were incorporated and became much more autonomous from the government in their operations. Their managerial structure was realigned--placing the president at the centre of the decision-making process, and with the participation of external persons--to be more responsive to the changing needs of…

  3. 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…

  4. Semiconducting compounds and devices incorporating same

    SciTech Connect

    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.

  5. 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…

  6. Incorporating the Internet into Traditional Library Instruction.

    ERIC Educational Resources Information Center

    Fonseca, Tony; King, Monica

    2000-01-01

    Presents a template for teaching traditional library research and one for incorporating the Web. Highlights include the differences between directories and search engines; devising search strategies; creating search terms; how to choose search engines; evaluating online resources; helpful Web sites; and how to read URLs to evaluate a Web site's…

  7. 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.

  8. 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)

  9. Micronucleus assay in aquatic animals.

    PubMed

    Bolognesi, Claudia; Hayashi, Makoto

    2011-01-01

    Aquatic pollutants produce multiple consequences at organism, population, community and ecosystem level, affecting organ function, reproductive status, population size, species survival and thus biodiversity. Among these, carcinogenic and mutagenic compounds are the most dangerous as their effects may exert a damage beyond that of individual and may be active through several generations. The application of genotoxicity biomarkers in sentinel organisms allows for the assessment of mutagenic hazards and/or for the identification of the sources and fate of the contaminants. Micronucleus (MN) test as an index of accumulated genetic damage during the lifespan of the cells is one of the most suitable techniques to identify integrated response to the complex mixture of contaminants. MN assay is today widely applied in a large number of wild and transplanted aquatic species. The large majority of studies or programmes on the genotoxic effect of the polluted water environment have been carried out with the use of bivalves and fish. Haemocytes and gill cells are the target tissues most frequently considered for the MN determination in bivalves. The MN test was widely validated and was successfully applied in a large number of field studies using bivalves from the genera Mytilus. MN in fish can be visualised in different cell types: erythrocytes and gill, kidney, hepatic and fin cells. The use of peripheral erythrocytes is more widely used because it avoids the complex cell preparation and the killing of the animals. The MN test in fish erythrocytes was validated in laboratory with different species after exposure to a large number of genotoxic agents. The erythrocyte MN test in fish was also widely and frequently applied for genotoxicity assessment of freshwater and marine environment in situ using native or caged animals following different periods of exposure. Large interspecies differences in sensitivity for MN induction were observed. Further validation studies are

  10. Assays for Determination of Protein Concentration.

    PubMed

    Olson, Bradley J S C

    2016-01-01

    Biochemical analysis of proteins relies on accurate quantification of protein concentration. Detailed in this appendix are some commonly used methods for protein analysis, e.g., Lowry, Bradford, bicinchoninic acid (BCA), UV spectroscopic, and 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) assays. The primary focus of this report is assay selection, emphasizing sample and buffer compatibility. The fundamentals of generating protein assay standard curves and of data processing are considered, as are high-throughput adaptations of the more commonly used protein assays. Also included is a rapid, inexpensive, and reliable BCA assay of total protein in SDS-PAGE sample buffer that is used for equal loading of SDS-PAGE gels. © 2016 by John Wiley & Sons, Inc. PMID:27248579

  11. 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.

  12. The C. elegans healthspan and stress-resistance assay toolkit.

    PubMed

    Keith, Scott Alexander; Amrit, Francis Raj Gandhi; Ratnappan, Ramesh; Ghazi, Arjumand

    2014-08-01

    A wealth of knowledge on the genetic mechanisms that govern aging has emerged from the study of mutants that exhibit enhanced longevity and exceptional resilience to adverse environmental conditions. In these studies, lifespan has been an excellent proxy for establishing the rate of aging, but it is not always correlated with qualitative measures of healthy aging or 'healthspan'. Although the attributes of healthspan have been challenging to define, they share some universal features that are increasingly being incorporated into aging studies. Here we describe methods used to determine Caenorhabditis elegans healthspan. These include assessments of tissue integrity and functionality and resistance to a variety of biotic and abiotic stressors. We have chosen to include simple, rapid assays in this collection that can be easily undertaken in any C. elegans laboratory, and can be relied on to provide a preliminary but thorough insight into the healthspan of a population. PMID:24727065

  13. Performance Evaluation of the Serum Thyroglobulin Assays With Immunochemiluminometric Assay and Immunoradiometric Assay for Differentiated Thyroid Cancer

    PubMed Central

    Cho, Yoon Young; Chun, Sejong; Lee, Soo-Youn; Chung, Jae Hoon

    2016-01-01

    Background Measurement of postoperative serum thyroglobulin (Tg) is important for detecting persistent or recurrent differentiated thyroid cancer. We evaluated the analytic performance of the DxI 800 assay (Beckman Coulter, USA) for serum Tg and anti-thyroglobulin antibodies (TgAbs) in comparison with that of the GAMMA-10 assay (Shinjin Medics Inc., Korea) for serum Tg and RIA-MAT 280 assay (Stratec, Germany) for TgAb. Methods We prospectively collected blood samples from 99 patients thyroidectomized for thyroid cancer. The functional sensitivity was investigated in standards and human serum. Precision and linearity were evaluated according to the guidelines of the Clinical and Laboratory Standards Institute. The correlation between the two assays was assessed in samples with different Tg ranges. Results The functional sensitivity of the DxI 800 assay for serum Tg was between 0.0313 and 0.0625 ng/mL. The total CV was 3.9–5.6% for serum Tg and 5.3–6.9% for serum TgAb. The coefficient of determination (R2) was 1.0 and 0.99 for serum Tg and TgAb, respectively. The cut-offs for serum TgAb were 4.0 IU/mL (DxI 800) and 60.0 IU/mL (RIA-MAT 280), and the overall agreement was 68.7%. The correlation between the two assays was excellent; the correlation coefficient was 0.99 and 0.88 for serum Tg and TgAb, respectively. Conclusions The DxI 800 is a sensitive assay for serum Tg and TgAb, and the results correlated well with those from the immunoradiometric assays (IRMA). This assay has several advantages over the IRMA and could be considered an alternative test for Tg measurement. PMID:27374705

  14. 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

  15. Prenatal diagnosis of the Hunter syndrome and the introduction of a new fluorimetric enzyme assay.

    PubMed

    Keulemans, J L M; Sinigerska, I; Garritsen, V H; Huijmans, J G M; Voznyi, Y V; van Diggelen, O P; Kleijer, W J

    2002-11-01

    Prenatal diagnosis of the Hunter syndrome (mucopolysaccharidosis type II; MPS II) is preferably achieved by the assay of iduronate-2-sulphate sulphatase (IDS) in uncultured chorionic villi (CV) as this allows early (12th week), rapid (2-3 days) and reliable results. We summarize the results of 174 prenatal analyses in the past 30 years, using various methods such as radiolabelled sulphate incorporation in amniotic fluid (AF) cells, glycosaminoglycan (GAG)-electrophoresis in AF and IDS assay in CV, CV-cells, AF and AF-cells. Twenty-seven fetuses with MPS II were diagnosed after finding clearly abnormal results in pregnancies with a male fetus; very low IDS activity has also been measured in some pregnancies with a (heterozygous) female fetus, emphasizing the need to combine enzyme assay with fetal sex determination. IDS activity has until recently been assessed by a cumbersome radioactive enzyme assay. Here we describe the use of a novel fluorigenic 4-methylumbelliferyl substrate, which allows a sensitive, rapid and convenient assay of IDS activity and reliable early prenatal diagnosis. This novel IDS assay was validated in retrospective analyses of 14 CV, CV-cell, AF and AF-cell samples from affected pregnancies in addition to prospective prenatal diagnosis in eight pregnancies at risk with one MPS II-affected fetus. PMID:12424767

  16. Development of a nested-PCR assay for the detection of Cryptosporidium parvum in finished water.

    PubMed

    Monis, P T; Saint, C P

    2001-05-01

    A nested-PCR assay, incorporating an internal positive control, was developed for Cryptosporidium monitoring in finished water. This assay was capable of reproducibly detecting 8 oocysts in spiked-filtered water samples collected from 5 South Australian water treatment plants. The RT-PCR assay of Kaucner and Stinear (Appl. Environ. Microbiol. 64(5) (1998) 1743) was also evaluated for the detection of Cryptosporidium parvum. Initially, under our experimental conditions, a detection level of 27 oocysts was achieved for spiked reagent water samples. This level was improved to 5 oocysts by modification of the method. Untreated South Australian source waters concentrated by calcium carbonate flocculation were found to be highly inhibitory to the RT-PCR assay. Concentration of similar samples using Envirochek filters appeared to eliminate PCR inhibition. While both methods possessed similar sensitivities the nested-PCR assay was more reproducible, more cost effective, simpler to perform and could detect both viable and non-viable intact Cryptosporidium parvum oocysts, which is an important consideration for plant operators. These factors make the nested-PCR assay the method of choice for screening large numbers of potable water samples, where a reliable low level of detection is essential. PMID:11329665

  17. Scintillation proximity assay (SPA) technology to study biomolecular interactions.

    PubMed

    Cook, Neil; Harris, Alison; Hopkins, Alison; Hughes, Kelvin

    2002-05-01

    Scintillation proximity assay (SPA) is a versatile homogeneous technique for radioactive assays which eliminates the need for separation steps. In SPA, scintillant is incorporated into small fluomicrospheres. These microspheres or "beads" are constructed in such a way as to bind specific molecules. If a radioactive molecule is bound to the bead, it is brought into close enough proximity that it can stimulate the scintillant contained within to emit light. Otherwise, the unbound radioactivity is too distant, the energy released is dissipated before reaching the bead, and these disintegrations are not detected. In this unit, the application of SPA technology to measuring protein-protein interactions, Src Homology 2 (SH2) and 3 (SH3) domain binding to specific peptide sequences, and receptor-ligand interactions are described. Three other protocols discuss the application of SPA technology to cell-adhesion-molecule interactions, protein-DNA interactions, and radioimmunoassays. In addition, protocols are given for preparation of SK-N-MC cells and cell membranes. PMID:18429228

  18. Antimicrobial efficacy of phenanthrenequinone based Schiff base complexes incorporating methionine amino acid: Structural elucidation and in vitro bio assay

    NASA Astrophysics Data System (ADS)

    Arun, Thesingu Rajan; Raman, Natarajan

    2014-06-01

    This work focuses the synthesis and characterization of few novel mixed ligand Schiff base metal complexes and their biological activities. For deriving the structural aspects, spectral techniques such as FT-IR, UV-Vis., 1H NMR, Raman, EPR and the physicochemical characterizations including elemental analysis, molar conductance and magnetic susceptibility method have been involved. All the complexes adopt square planar geometry. DNA binding ability of these complexes has been explored using diverse techniques viz. UV-Vis. absorption, fluorescence spectroscopy, viscometry and cyclic voltammetry. These studies prove that CT-DNA binding of the complexes follows the intercalation mode. Comparative DNA oxidative cleavage ability of the complexes has been done under ultraviolet photo radiation on pUC19 DNA. In addition, the biocidal action of the complexes has been investigated against few pathogenic bacteria and fungi by disc diffusion method. Importantly, the amylase inhibition activity of Cu(II) complex has been explored. The amylase inhibition property has been found to be increased upon increasing the complex concentration.

  19. Celastrol Blocks Interleukin-6 Gene Expression via Downregulation of NF-κB in Prostate Carcinoma Cells

    PubMed Central

    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. PMID:24664372

  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. PMID:24664372

  1. Reversal of P-glycoprotein-mediated multidrug resistance in human hepatoma cells by hedyotiscone A, a compound isolated from Hedyotis corymbosa.

    PubMed

    Yue, Grace Gar-Lee; Kin-Ming Lee, Julia; Cheng, Ling; Chung-Lap Chan, Ben; Jiang, Lei; Fung, Kwok-Pui; Leung, Ping-Chung; Bik-San Lau, Clara

    2012-06-01

    Multidrug resistance is a major problem in hepatocellular carcinoma. Hedyotiscone A, a compound isolated from Chinese herbal medicine Hedyotis corymbosa (HC, family Rubiaceae), was used as the chemical marker to distinguish between HC and an anticancer herb Hedyotis diffusa (HD) in our previous study. The present study aimed to investigate whether HA exhibited antiproliferative activities in multidrug-resistant hepatocellular carcinoma cells R-HepG2 and the parental cells HepG2 using MTT assay and [(3)H]-thymidine incorporation assay. Our results showed that HA could significantly inhibit cell proliferation in R-HepG2 and HepG2 (IC(50) = 43.7 and 56.3 µg/mL, respectively), but not in normal human liver cells WRL-68 (IC(50) > 100 µg/mL) cells, suggesting its selective cytotoxic effects. Besides, HA induced apoptosis in R-HepG2 cells, as confirmed by annexin-V & propidium iodide staining, and DNA fragmentation assay. The caspase cascade was activated as shown by a significant increase of cleaved caspases-3, -7 and -9 in HA-treated R-HepG2 cells. The activities and protein expression of P-glycoprotein as well as mRNA expression of MDR1 were also decreased in HA-treated R-HepG2 cells. Our study demonstrated for the first time the antiproliferative activities of hedyotiscone A in multidrug-resistant R-HepG2 cells. The findings revealed the potential of this compound in treating multidrug-resistant tumor. PMID:22352391

  2. Bioactive 1,4-dihydroisonicotinic acid derivatives prevent oxidative damage of liver cells.

    PubMed

    Borovic, Suzana; Tirzitis, Gunars; Tirzite, Dace; Cipak, Ana; Khoschsorur, Gholam A; Waeg, Georg; Tatzber, Franz; Scukanec-Spoljar, Mira; Zarkovic, Neven

    2006-05-10

    1,4-Dihydroisonicotinic acid derivatives (1,4-DHINA) are compounds closely related to derivatives of 1,4-dihydropyridine, a well-known calcium channel antagonists. 1,4-DHINA we used were derived from a well-known antioxidant Diludin. Although some compounds have neuromodulatory or antimutagenic properties, their activity mechanisms are not well known. This study was performed to obtain data on antioxidant and bioprotective activities of: 2,6-dimethyl-3,5-diethoxycarbonyl-1,4-dihydroisonicotinic acid (Ia); sodium 2-(2,6-dimethyl-3,5-diethoxycarbonyl-1,4-dihydropyridine-4-carboxamido)glutamate (Ib) and sodium 2-(2,6-dimethyl-3,5-diethoxycarbonyl-1,4-dihydropyridine-4-carboxamido)ethane-sulphate (Ic). 1,4-DHINA's activities were studied in comparison to Trolox by: N,N-Diphenyl-N'-picrylhydrazyl (DPPH*), deoxyribose degradation, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) radical scavenging and antioxidative capacity assays; copper-induced lipid peroxidation of cultured rat liver cells (malondialdehyde determination by high performance liquid chromatography and 4-hydroxynonenal-protein conjugates by dot-blot); (3)H-thymidine incorporation and trypan blue assay for liver cells growth and viability. In all assays used Ia was the most potent antioxidant. Ia was also a potent antioxidant at non-toxic concentrations for liver cell cultures. It completely abolished, while Ic only slightly decreased copper-induced lipid peroxidation of liver cells. Thus, antioxidant capacities are important activity principle of Ia, which was even superior to Trolox in the cell cultures used, while activity principles of Ic and Ib remain yet to be determined. PMID:16600211

  3. Use of a Rapid Ethylene Glycol Assay: a 4-Year Retrospective Study at an Academic Medical Center.

    PubMed

    Rooney, Sydney L; Ehlers, Alexandra; Morris, Cory; Drees, Denny; Davis, Scott R; Kulhavy, Jeff; Krasowski, Matthew D

    2016-06-01

    Ethylene glycol (EG) is a common cause of toxic ingestions. Gas chromatography (GC)-based laboratory assays are the gold standard for diagnosing EG intoxication. However, GC requires specialized instrumentation and technical expertise that limits feasibility for many clinical laboratories. The objective of this retrospective study was to determine the utility of incorporating a rapid EG assay for management of cases with suspected EG poisoning. The University of Iowa Hospitals and Clinics core clinical laboratory adapted a veterinary EG assay (Catachem, Inc.) for the Roche Diagnostics cobas 8000 c502 analyzer and incorporated this assay in an osmolal gap-based algorithm for potential toxic alcohol/glycol ingestions. The main limitation is that high concentrations of propylene glycol (PG), while readily identifiable by reaction rate kinetics, can interfere with EG measurement. The clinical laboratory had the ability to perform GC for EG and PG, if needed. A total of 222 rapid EG and 24 EG/PG GC analyses were documented in 106 patient encounters. Of ten confirmed EG ingestions, eight cases were managed entirely with the rapid EG assay. PG interference was evident in 25 samples, leading to 8 GC analyses to rule out the presence of EG. Chart review of cases with negative rapid EG assay results showed no evidence of false negatives. The results of this study highlight the use of incorporating a rapid EG assay for the diagnosis and management of suspected EG toxicity by decreasing the reliance on GC. Future improvements would involve rapid EG assays that completely avoid interference by PG. PMID:26553280

  4. Emissive sensors and devices incorporating these sensors

    DOEpatents

    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.

  5. Implications for incorporating simulation in nursing education.

    PubMed

    Miller, Shelley

    2010-01-01

    The current intricately complex state of health care holds a multitude of challenges for the novice nurse in acquiring, developing, and maintaining standards of proficiency in clinical practice. This paper will explore the implications or reasons for incorporating simulation in nursing education for two primary reasons. First, simulation is an interactive teaching strategy designed for augmenting the learning process. Simulation is an example of active learning that engages the student to think through a situation then take action through the application of knowledge, and finally to self-reflect during the process of debriefing. Secondly, an increasing number of educational institutions have made the commitment to incorporate simulation technology into nursing curricula. PMID:21175101

  6. Ionic liquid incorporating thiosalicylate for metal removal

    NASA Astrophysics Data System (ADS)

    Wilfred, Cecilia Devi; Mustafa, Fadwa Babiker; Romeli, Fatimah Julia

    2012-09-01

    Ionic liquids are a class of organic molten salts "designer solvents" that are composed totally of anions (inorganic and organic polyatomic) and organic cations. The replacement of volatile organic solvents from a separation process is of utmost importance since the use of a large excess of these solvents is hazardous and creates ecological problem. The new method for metal ion extraction is by using task-specific ionic liquids such as ionic liquids which incorporate thiosalicylate functionality. This paper looks at producing a new cluster of ionic liquids which incorporates thiosalicylate with pyridinium cation. Its thermophysical properties such as density and viscosity in single and binary mixtures are studied. The ionic liquids' capability in metal removal processes is evaluated.

  7. Measuring and incorporating vulnerability into conservation planning.

    PubMed

    Wilson, Kerrie; Pressey, Robert L; Newton, Adrian; Burgman, Mark; Possingham, Hugh; Weston, Chris

    2005-05-01

    Conservation planning is the process of locating and designing conservation areas to promote the persistence of biodiversity in situ. To do this, conservation areas must be able to mitigate at least some of the proximate threats to biodiversity. Information on threatening processes and the relative vulnerability of areas and natural features to these processes is therefore crucial for effective conservation planning. However, measuring and incorporating vulnerability into conservation planning have been problematic. We develop a conceptual framework of the role of vulnerability assessments in conservation planning and propose a definition of vulnerability that incorporates three dimensions: exposure, intensity, and impact. We review and categorize methods for assessing the vulnerability of areas and the features they contain and identify the relative strengths and weaknesses of each broad approach. Our review highlights the need for further development and evaluation of approaches to assess vulnerability and for comparisons of their relative effectiveness. PMID:15920667

  8. 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.

  9. Mechanism of incorporation of zinc into hydroxyapatite.

    PubMed

    Matsunaga, Katsuyuki; Murata, Hidenobu; Mizoguchi, Teruyasu; Nakahira, Atsushi

    2010-06-01

    The atomic level mechanism of incorporation of Zn(2+) into hydroxyapatite (HAp), which is a potential dopant to promote bone formation, was investigated, based on first principles total energy calculations and experimental X-ray absorption near edge structure (XANES) analyses. It was found that Zn(2+)-doped HAp tends to have a Ca-deficient chemical composition and substitutional Zn(2+) ions are associated with a defect complex with a Ca(2+) vacancy and two charge compensating protons. Moreover, first principles calculations demonstrated that Zn(2+) incorporation into HAp can take place by occupying the Ca(2+) vacancy of the defect complex. The Ca(2+) vacancy complex is not only the origin of the calcium deficiency in HAp, but also plays a key role in the uptake of trace elements during mineralization. PMID:19944784

  10. Comparative Incorporation of PNA into DNA Nanostructures.

    PubMed

    Pedersen, Ronnie O; Kong, Jing; Achim, Catalina; LaBean, Thomas H

    2015-01-01

    DNA has shown great promise as a building material for self-assembling nanoscale structures. To further develop the potential of this technology, more methods are needed for functionalizing DNA-based nanostructures to increase their chemical diversity. Peptide nucleic acid (PNA) holds great promise for realizing this goal, as it conveniently allows for inclusion of both amino acids and peptides in nucleic acid-based structures. In this work, we explored incorporation of a positively charged PNA within DNA nanostructures. We investigated the efficiency of annealing a lysine-containing PNA probe with complementary, single-stranded DNA sequences within nanostructures, as well as the efficiency of duplex invasion and its dependence on salt concentration. Our results show that PNA allows for toehold-free strand displacement and that incorporation yield depends critically on binding site geometry. These results provide guidance for the design of PNA binding sites on nucleic acid nanostructures with an eye towards optimizing fabrication yield. PMID:26404232

  11. Incorporating Nonchemical Stressors Into Cumulative Risk Assessments

    PubMed Central

    Rider, Cynthia V.; Dourson, Michael L.; Hertzberg, Richard C.; Mumtaz, Moiz M.; Price, Paul S.; Simmons, Jane Ellen

    2012-01-01

    The role of nonchemical stressors in modulating the human health risk associated with chemical exposures is an area of increasing attention. On 9 March 2011, a workshop titled “Approaches for Incorporating Nonchemical Stressors into Cumulative Risk Assessment” took place during the 50th Anniversary Annual Society of Toxicology Meeting in Washington D.C. Objectives of the workshop included describing the current state of the science from various perspectives (i.e., regulatory, exposure, modeling, and risk assessment) and presenting expert opinions on currently available methods for incorporating nonchemical stressors into cumulative risk assessments. Herein, distinct frameworks for characterizing exposure to, joint effects of, and risk associated with chemical and nonchemical stressors are discussed. PMID:22345310

  12. 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.

  13. 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.

  14. Practical assay for nitrite and nitrosothiol as an alternative to the Griess assay or the 2,3-diaminonaphthalene assay.

    PubMed

    Shen, Yanming; Zhang, Quanjuan; Qian, Xuhong; Yang, Youjun

    2015-01-20

    Nitrite is a heavily assayed substrate in the fields of food safety, water quality control, disease diagnosis, and forensic investigation and more recently in basic biological studies on nitric oxide physiology and pathology. The colorimetric Griess assay and the fluorimetric 2,3-diaminonaphthalene (DAN) assay are the current gold standards for nitrite quantification. They are not without limitations, yet have amazingly survived 156 and 44 years, respectively, due to the lack of a practical alternative. Both assays exhibit slow detection kinetics due to inactivation of nucleophiles under strongly acidic media, require an extensive incubation time for reaction to go completion, and hence offer a limited detection throughput. By converting an intermolecular reaction of the Griess assay intramolecularly, we designed a novel probe (NT555) for nitrite detection, which displays superior detection kinetics and sensitivity. NT555 was constructed following our "covalent-assembly" probe design principle. Upon detection, it affords a gigantic bathochromic shift of the absorption spectrum and a sensitive turn-on fluorescence signal from a zero background, both of which are typical of an "assembly" type probe. Overall, NT555 has addressed various difficulties associated with the Griess and the DAN assays and represents an attractive alternative for practical applications. PMID:25519711

  15. 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.

  16. Incorporation of squalene into rod outer segments

    SciTech Connect

    Keller, R.K.; Fliesler, S.J. )

    1990-08-15

    We have reported previously that squalene is the major radiolabeled nonsaponifiable lipid product derived from ({sup 3}H)acetate in short term incubations of frog retinas. In the present study, we demonstrate that newly synthesized squalene is incorporated into rod outer segments under similar in vitro conditions. We show further that squalene is an endogenous constituent of frog rod outer segment membranes; its concentration is approximately 9.5 nmol/mumol of phospholipid or about 9% of the level of cholesterol. Pulse-chase experiments with radiolabeled precursors revealed no metabolism of outer segment squalene to sterols in up to 20 h of chase. Taken together with our previous absolute rate studies, these results suggest that most, if not all, of the squalene synthesized by the frog retina is transported to rod outer segments. Synthesis of protein is not required for squalene transport since puromycin had no effect on squalene incorporation into outer segments. Conversely, inhibition of isoprenoid synthesis with mevinolin had no effect on the incorporation of opsin into the outer segment. These latter results support the conclusion that the de novo synthesis and subsequent intracellular trafficking of opsin and isoprenoid lipids destined for the outer segment occur via independent mechanisms.

  17. 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.

  18. 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...

  19. Antibacterial and antioxidant activity of the secondary metabolites from in vitro cultures of the Alice sundew (Drosera aliciae).

    PubMed

    Krolicka, Aleksandra; Szpitter, Anna; Maciag, Monika; Biskup, Edyta; Gilgenast, Ewelina; Wegrzyn, Grazyna; Lojkowska, Ewa

    2009-07-01

    The objective of the present study was to evaluate the antioxidant as well as the antibacterial properties of secondary metabolites obtained from Drosera aliciae (Alice sundew) plants grown in vitro and to examine the mechanism of their antimicrobial action. Bactericidal activity of extracts from D. aliciae, as well as pure ramentaceone (naphthoquinone), which is present in this plant, were examined against human pathogenic strains of micro-organisms that are both resistant and susceptible to antibiotics. A chloroform extract proved to be more effective than a methanol preparation against all of the tested strains, except for Pseudomonas aeruginosa isolates. The lowest minimal-bactericidal-concentration value was in the case of Staphylococcus aureus (25-50 mg fresh weight·ml(-1)). The influence of D. aliciae extracts and ramentaceone on the synthesis of DNA, RNA or proteins in cultures of Enterococcus faecalis was estimated by measurement of the incorporation of the radioactively labelled precursors [3H]thymidine, [3H]uridine or [3H]leucine respectively. The methanol extract of D. aliciae, except for a moderate effect on DNA synthesis, had no influence on RNA and protein synthesis. The chloroform preparation caused about a 75% decrease in [3H]uridine incorporation in comparison with the control after 60 min and a significant diminution in DNA and protein synthesis (44 and 30% respectively). Ramentaceone also decreased DNA and RNA synthesis, but less efficiently than did the chloroform extract, and it caused no changes in [3H]leucine incorporation. The methanol extract from D. aliciae proved to be an effective antioxidant in both the DPPH (2,2-diphenyl-10-picrylhydrazyl free radical) and the FRAP (ferric reducing antioxidant power) assay, with the activities exceeding those of well-known antioxidants, namely the flavonoids. The chloroform extract and ramentaceone showed no antioxidative properties. PMID:18782083

  20. Cast Tube Assay: A 3-D in vitro assay for visualization and quantification of horizontal chemotaxis and cellular invasion.

    PubMed

    Whitehead, Breffeni Constantine; Bezuidenhout, Deon; Chokoza, Cindy; Davies, Neil Hamer; Goetsch, Kyle Peter

    2016-01-01

    Directed cell motility, as controlled by soluble factors, is crucial for many biological processes, including development, cancer progression, and wound healing. The use of directed cell motility also shows promise for applications in regenerative medicine such as therapeutic angiogenesis. Unfortunately, current in vitro 3-D migration and invasion models limit our understanding and application of these processes. Here, we present a novel and cost-effective 3-D chemotaxis assay for assessing the invasive response of cells to a chemoattractant extracellular matrix (ECM). Our system takes advantage of a custom-casting chamber to set two gels in contact with each other along a defined front, one containing a suitable chemoattractant and the other the cells. Rotation of the chamber allows easy visualization of invasion across the interface. The effectiveness of the assay was demonstrated by studying the invasion of both human dermal fibroblasts (FBs) and smooth muscle cells (SMCs) into a polyethylene glycol (PEG) hydrogel containing basic fibroblast growth factor (bFGF). Incorporation of bFGF resulted in significantly increased and directional invasion for both cell groups. PMID:27528071

  1. Evaluation of Sustained BMP-2 Release Profiles Using a Novel Fluorescence-Based Retention Assay

    PubMed Central

    Jang, Jun-Hyeog

    2015-01-01

    The purpose of this study was to develop and characterize a novel fluorescence-based retention assay for the evaluation of the release profile of bone morphogenetic protein-2 (BMP-2) released from bone graft carrier. In this study, we evaluated the binding, release kinetics, and delivery efficacies of BMP-2 incorporated into hydroxyapatite (HA) bone grafts. The evaluation of the release profile of BMP-2 from HA bone grafts using a fluorescence-based retention assay revealed initial burst releases from the HA bone grafts followed by long sustained releases up to 14 weeks. The sustained biological activity of the released BMP-2 from HA bone grafts over the full 14-week period supports a long sustained mechanism via fluorescence-based retention assay. Thus, the results from this study show that BMP-2 could be incorporated into HA bone grafts for sustained release over a prolonged period of time with retention of bioactivity and our fluorescence-based retention assay, which is principally detecting the retention profile of BMP-2 in HA bone grafts, is more accurate than conventionally collecting the released BMP-2 for evaluation of BMP-2 release profiles. PMID:25901352

  2. Structure-function relationship of new anthralin derivatives assayed for growth inhibition and cytotoxicity in human keratinocyte cultures.

    PubMed

    Bonnekoh, B; Tanzer, H; Seidel, M; Geisel, J; Merk, H F; Mahrle, G; Wiegrebe, W

    1991-11-01

    HaCaT keratinocyte cultures were exposed to twelve hydrophilic anthralin derivatives 1 to 12 with substituents at C-1 and C-8 of the anthrone skeleton, of one H at C-10 and of both H's at C-10 by lacton rings. After 3 microM treatment growth was determined by cellular protein content, 3H-thymidine- and 14C-amino-acid-uptake and cytotoxicity by the release of cytoplasmic LDH into the culture medium. In comparison to acetone control (100%) anthralin suppressed mean protein content, as well as DNA- and protein-synthesis to 33, 28, and 21%, respectively, and the drug revealed an enzyme release of 660%. In relation to the parent drug we found similar cell growth inhibitory effects of compounds 4, 6, 8, 9, 10, and 12. Deriv. 4, 8, and 10 were, however, to some extent less cytotoxic than anthralin, whereas deriv. 6, 9, and 12 were in the same range. An extreme suppression of growth parameters which differed from the anthralin effect by a factor 0.5-0.8 was caused by deriv. 11, showing the same cytotoxicity. Deriv. 1, 2, 3, 5, and 7 did not demonstrate any cytotoxicity. Concerning growth parameters, deriv. 2 induced a slight stimulation, deriv. 3 and 7 were completely ineffective, deriv. 1 and 5 induced slightly to moderately inhibited proliferation but both being much less effective than anthralin. These data indicate that the "minimum structure" concept by Krebs and Schaltegger--claiming 1-hydroxy-9-anthrone as a precondition for clinical antipsoriatic potency--is not valid at least in cell-biological tests and point toward possible usefulness of some experimental model compounds as alternative antipsoriatics. PMID:1804068

  3. 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

  4. Precision estimates for tomographic nondestructive assay

    SciTech Connect

    Prettyman, T.H.

    1995-12-31

    One technique being applied to improve the accuracy of assays of waste in large containers is computerized tomography (CT). Research on the application of CT to improve both neutron and gamma-ray assays of waste is being carried out at LANL. For example, tomographic gamma scanning (TGS) is a single-photon emission CT technique that corrects for the attenuation of gamma rays emitted from the sample using attenuation images from transmission CT. By accounting for the distribution of emitting material and correcting for the attenuation of the emitted gamma rays, TGS is able to achieve highly accurate assays of radionuclides in medium-density wastes. It is important to develope methods to estimate the precision of such assays, and this paper explores this problem by examining the precision estimators for TGS.

  5. Proximity assays for sensitive quantification of proteins.

    PubMed

    Greenwood, Christina; Ruff, David; Kirvell, Sara; Johnson, Gemma; Dhillon, Harvinder S; Bustin, Stephen A

    2015-06-01

    Proximity assays are immunohistochemical tools that utilise two or more DNA-tagged aptamers or antibodies binding in close proximity to the same protein or protein complex. Amplification by PCR or isothermal methods and hybridisation of a labelled probe to its DNA target generates a signal that enables sensitive and robust detection of proteins, protein modifications or protein-protein interactions. Assays can be carried out in homogeneous or solid phase formats and in situ assays can visualise single protein molecules or complexes with high spatial accuracy. These properties highlight the potential of proximity assays in research, diagnostic, pharmacological and many other applications that require sensitive, specific and accurate assessments of protein expression. PMID:27077033

  6. LIMITATIONS OF THE FLUORESCENT PROBE VIABILITY ASSAY

    EPA Science Inventory

    Cell viability commonly is determined flow cytometrically by the carboxyfluorescein diacetate (CFDA)/propidium iodide (PI) assay. FDA is taken up by the viable cell and converted via cytoplasmic esterase-catalyzed hydrolysis to carboxyfluorescein (CF). F fluorescence intensity is...

  7. Variables Affecting Two Electron Transport System Assays

    PubMed Central

    Burton, G. Allen; Lanza, Guy R.

    1986-01-01

    Several methodological variables were critical in two commonly used electron transport activity assays. The dehydrogenase assay based on triphenyl formazan production exhibited a nonlinear relationship between formazan production (dehydrogenase activity) and sediment dilution, and linear formazan production occurred for 1 h in sediment slurries. Activity decreased with increased time of sediment storage at 4°C. Extraction efficiencies of formazan from sediment varied with alcohol type; methanol was unsatisfactory. Phosphate buffer (0.06 M) produced higher activity than did either U.S. Environmental Protection Agency reconstituted hard water or Tris buffer sediment diluents. Intracellular formazan crystals were dissolved within minutes when in contact with immersion oil. Greater crystal production (respiration) detected by a tetrazolium salt assay occurred at increased substrate concentrations. Test diluents containing macrophyte exudates produced greater activity than did phosphate buffer, U.S. Environmental Protection Agency water, or ultrapure water diluents. Both assays showed decreases in sediment or bacterial activity through time. PMID:16347067

  8. Scrape Loading/Dye Transfer Assay.

    PubMed

    Babica, Pavel; Sovadinová, Iva; Upham, Brad L

    2016-01-01

    The scrape loading/dye transfer (SL/DT) technique is a simple functional assay for the simultaneous assessment of gap junctional intercellular communication (GJIC) in a large population of cells. The equipment needs are minimal and are typically met in standard cell biology labs, and SL/DT is the simplest and quickest of all the assays that measure GJIC. This assay has also been adapted for in vivo studies. The SL/DT assay is also conducive to a high-throughput setup with automated fluorescence microscopy imaging and analysis to elucidate more samples in shorter time, and hence can serve a broad range of in vitro pharmacological and toxicological needs. PMID:27207291

  9. 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. ...

  10. Proximity assays for sensitive quantification of proteins

    PubMed Central

    Greenwood, Christina; Ruff, David; Kirvell, Sara; Johnson, Gemma; Dhillon, Harvinder S.; Bustin, Stephen A.

    2015-01-01

    Proximity assays are immunohistochemical tools that utilise two or more DNA-tagged aptamers or antibodies binding in close proximity to the same protein or protein complex. Amplification by PCR or isothermal methods and hybridisation of a labelled probe to its DNA target generates a signal that enables sensitive and robust detection of proteins, protein modifications or protein–protein interactions. Assays can be carried out in homogeneous or solid phase formats and in situ assays can visualise single protein molecules or complexes with high spatial accuracy. These properties highlight the potential of proximity assays in research, diagnostic, pharmacological and many other applications that require sensitive, specific and accurate assessments of protein expression. PMID:27077033

  11. 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

  12. CONTROL ASSAY DEVELOPMENT: METHODOLOGY AND LABORATORY VERIFICATION

    EPA Science Inventory

    The report describes Control Assay Development (CAD), a data acquisition program designed to evaluate the potential applicability of various treatment processes for the control of solid, liquid, and gaseous emissions from coal conversion plants. The CAD program described could be...

  13. Current and future application of genetic toxicity assays: the role and value of in vitro mammalian assays.

    PubMed

    Elespuru, Rosalie K; Agarwal, Rajiv; Atrakchi, Aisar H; Bigger, C Anita H; Heflich, Robert H; Jagannath, Devaraya R; Levy, Dan D; Moore, Martha M; Ouyang, Yanli; Robison, Timothy W; Sotomayor, Rene E; Cimino, Michael C; Dearfield, Kerry L

    2009-06-01

    With the advent of new technologies (e.g., genomics, automated analyses, and in vivo monitoring), new regulations (e.g., the reduction of animal tests by the European REACH), and new approaches to toxicology (e.g., Toxicity Testing in the 21st Century, National Research Council), the field of regulatory genetic toxicology is undergoing a serious re-examination. Within this context, Toxicological Sciences has published a series of articles in its Forum Section on the theme, "Genetic Toxicity Assessment: Employing the Best Science for Human Safety Evaluation" (beginning with Goodman et al.). As a contribution to the Forum discussions, we present current methods for evaluating mutagenic/genotoxic risk using standard genotoxicity test batteries, and suggest ways to address and incorporate new technologies. We recognize that the occurrence of positive results in relation to cancer prediction has led to criticism of in vitro mammalian cell genetic toxicity assays. We address criticism of test results related to weak positives, associated only with considerable toxicity, only seen at high concentrations, not accompanied by positive results in the other tests of standard test batteries, and/or not correlating well with rodent carcinogenicity tests. We suggest that the problems pointed out by others with these assays already have been resolved, to a large extent, by international groups working to update assay protocols, and by changes in data interpretation at regulatory agencies. New guidances at the U.S. Environmental Protection Agency and the U.S. Food and Drug Administration improve data evaluation and help refocus risk assessment. We discuss the results of international groups working together to integrate new technologies and evaluate new tests, including human monitoring. We suggest that strategies for identifying human health risks should naturally change to integrate new technologies; however, changes should be made only when justified by strong scientific

  14. Electrochemical Assay of Gold-Plating Solutions

    NASA Technical Reports Server (NTRS)

    Chiodo, R.

    1982-01-01

    Gold content of plating solution is assayed by simple method that required only ordinary electrochemical laboratory equipment and materials. Technique involves electrodeposition of gold from solution onto electrode, the weight gain of which is measured. Suitable fast assay methods are economically and practically necessary in electronics and decorative-plating industries. If gold content in plating bath is too low, poor plating may result, with consequent economic loss to user.

  15. Rapid assays for environmental and biological monitoring.

    PubMed

    Szurdoki, F; Jaeger, L; Harris, A; Kido, H; Wengatz, I; Goodrow, M H; Székács, A; Wortberg, M; Zheng, J; Stoutamire, D W; Sanborn, J R; Gilman, S D; Jones, A D; Gee, S J; Choudary, P V; Hammock, B D

    1996-05-01

    Rapid, inexpensive, sensitive, and selective enzyme-linked immunosorbent assays (ELISAs) now are utilized in environmental science. In this laboratory, many ELISAs have been developed for pesticides and other toxic substances and also for their metabolites. Compounds for which ELISAs have recently been devised include insecticides (organophosphates, carbaryl, pyrethroids, and fenoxycarb), herbicides (s-triazines, arylureas, triclopyr, and bromacil), fungicides (myclobutanil), TCDD, and metabolites of naphthalene and toluene. New rapid assays have been developed for mercury. PMID:8642182

  16. Automated optical sensing system for biochemical assays

    NASA Astrophysics Data System (ADS)

    Oroszlan, Peter; Duveneck, Gert L.; Ehrat, Markus; Widmer, H. M.

    1994-03-01

    In this paper, we present a new system called FOBIA that was developed and optimized with respect to automated operation of repetitive assay cycles with regenerable bioaffinity sensors. The reliability and precision of the new system is demonstrated by an application in a competitive assay for the detection of the triazine herbicide Atrazine. Using one sensor in more than 300 repetitive cycles, a signal precision better than 5% was achieved.

  17. Lipid-sensing high-throughput ApoA-I assays.

    PubMed

    Niedziela-Majka, Anita; Lad, Latesh; Chisholm, Jeffrey W; Lagpacan, Leanna; Schwartz, Karen; Hung, Magdeleine; Jin, Debi; Fung, Wanchi; Brendza, Katherine M; Liu, Xiaohong; Pagratis, Nikos; Sakowicz, Roman

    2012-09-01

    Apolipoprotein A-I (ApoA-I), a primary protein component of high-density lipoprotein (HDL), plays an important role in cholesterol metabolism mediating the formation of HDL and the efflux of cellular cholesterol from macrophage foam cells in arterial walls. Lipidation of ApoA-I is mediated by adenosine triphosphate (ATP) binding cassette A1 (ABCA1). Insufficient ABCA1 activity may lead to increased risk of atherosclerosis due to reduced HDL formation and cholesterol efflux. The standard radioactive assay for measuring cholesterol transport to ApoA-I has low throughput and poor dynamic range, and it fails to measure phospholipid transfer. We describe the development of two sensitive, nonradioactive high-throughput assays that report on the lipidation of ApoA-I: a homogeneous assay based on time-resolved fluorescence resonance energy transfer (TR-FRET) and a discontinuous assay that uses the label-free Epic platform. The TR-FRET assay employs HiLyte Fluor 647-labeled ApoA-I with N-terminal biotin bound to streptavidin-terbium. When fluorescent ApoA-I was incorporated into HDL, TR-FRET decreased proportionally to the increase in the ratio of lipids to ApoA-I, demonstrating that the assay was sensitive to the amount of lipid bound to ApoA-I. In the Epic assay, biotinylated ApoA-I was captured on a streptavidin-coated biosensor. Measured resonant wavelength shift was proportional to the amount of lipids associated with ApoA-I, indicating that the assay senses ApoA-I lipidation. PMID:22811478

  18. Protein immobilization techniques for microfluidic assays

    PubMed Central

    Kim, Dohyun; Herr, Amy E.

    2013-01-01

    Microfluidic systems have shown unequivocal performance improvements over conventional bench-top assays across a range of performance metrics. For example, specific advances have been made in reagent consumption, throughput, integration of multiple assay steps, assay automation, and multiplexing capability. For heterogeneous systems, controlled immobilization of reactants is essential for reliable, sensitive detection of analytes. In most cases, protein immobilization densities are maximized, while native activity and conformation are maintained. Immobilization methods and chemistries vary significantly depending on immobilization surface, protein properties, and specific assay goals. In this review, we present trade-offs considerations for common immobilization surface materials. We overview immobilization methods and chemistries, and discuss studies exemplar of key approaches—here with a specific emphasis on immunoassays and enzymatic reactors. Recent “smart immobilization” methods including the use of light, electrochemical, thermal, and chemical stimuli to attach and detach proteins on demand with precise spatial control are highlighted. Spatially encoded protein immobilization using DNA hybridization for multiplexed assays and reversible protein immobilization surfaces for repeatable assay are introduced as immobilization methods. We also describe multifunctional surface coatings that can perform tasks that were, until recently, relegated to multiple functional coatings. We consider the microfluidics literature from 1997 to present and close with a perspective on future approaches to protein immobilization. PMID:24003344

  19. Radioimmune assay of human platelet prostaglandin synthetase

    SciTech Connect

    Roth, G.J.; Machuga, E.T.

    1982-02-01

    Normal platelet function depends, in part, on platelet PG synthesis. PG synthetase (cyclo-oxygenase) catalyzes the first step in PG synthesis, the formation of PGH/sub 2/ from arachidonic acid. Inhibition of the enzyme by ASA results in an abnormality in the platelet release reaction. Patients with pparent congenital abnormalities in the enzyme have been described, and the effects have been referred to as ''aspirin-like'' defects of the platelet function. These patients lack platelet PG synthetase activity, but the actual content of PG synthetase protein in these individuals' platelets is unknown. Therefore an RIA for human platelet PG synthetase would provide new information, useful in assessing the aspirin-like defects of platelet function. An RIA for human platelet PG synthetase is described. The assay utilizes a rabbit antibody directed against the enzyme and (/sup 125/I)-labelled sheep PG synthetase as antigen. The human platelet enzyme is assayed by its ability to inhibit precipitation of the (/sup 125/I)antigen. The assay is sensitive to 1 ng of enzyme. By the immune assay, human platelets contain approximately 1200 ng of PG synethetase protein per 1.5 mg of platelet protein (approximately 10/sup 9/ platelets). This content corresponds to 10,000 enzyme molecules per platelet. The assay provides a rapid and convenient assay for the human platelet enzyme, and it can be applied to the assessment of patients with apparent platelet PG synthetase (cyclo-oxygenase) deficiency.

  20. Immunoperoxidase inhibition assay for rabies antibody detection.

    PubMed

    Batista, H B C R; Lima, F E S; Maletich, D; Silva, A C R; Vicentini, F K; Roehe, L R; Spilki, F R; Franco, A C; Roehe, P M

    2011-06-01

    An immunoperoxidase inhibition assay (IIA) for detection of rabies antibodies in human sera is described. Diluted test sera are added to microplates with paraformaldehyde-fixed, CER cells infected with rabies virus. Antibodies in test sera compete with a rabies polyclonal rabbit antiserum which was added subsequently. Next, an anti-rabbit IgG-peroxidase conjugate is added and the reaction developed by the addition of the substrate 3-amino-9-ethylcarbazole (AEC). The performance of the assay was compared to that of the "simplified fluorescence inhibition microtest" (SFIMT), an established virus neutralization assay, by testing 422 human sera. The IIA displayed 97.6% sensitivity, 98% specificity and 97.6% accuracy (Kappa correlation coefficient=0.9). The IIA results can be read by standard light microscopy, where the clearly identifiable specific staining is visible in antibody-negative sera, in contrast to the absence of staining in antibody-positive samples. The assay does not require monoclonal antibodies or production of large amounts of virus; furthermore, protein purification steps or specialized equipment are not necessary for its performance. The IIA was shown to be suitable for detection of rabies antibodies in human sera, with sensitivity, specificity and accuracy comparable to that of a neutralization-based assay. This assay may be advantageous over other similar methods designed to detect rabies-specific binding antibodies, in that it can be easily introduced into laboratories, provided basic cell culture facilities are available. PMID:21458492

  1. An ultrafiltration assay for lysyl oxidase.

    PubMed

    Shackleton, D R; Hulmes, D J

    1990-03-01

    A modification of the original microdistillation assay for lysyl oxidase is described in which Amicon C-10 microconcentrators are used to separate, by ultrafiltration, the 3H-labeled products released from a [4,5-3H]-lysine-labeled elastin substrate. Enzyme activity is determined by scintillation counting of the ultrafiltrate, after subtraction of radioactivity released in the presence of beta-aminopropionitrile, a specific inhibitor of the enzyme. Conditions are described which optimize both the sensitivity and the efficient use of substrate. The assay shows linear inhibition of activity in up to 1 M urea; hence, as the enzyme is normally diluted in the assay, samples in 6 M urea can be assayed directly, without prior dialysis, and corrected for partial inhibition. Comparable results are obtained when enzyme activity is assayed by ultrafiltration or microdistillation. The assay is simple and convenient and, by using disposable containers throughout, it eliminates the need for time-consuming decontamination of radioactive glassware. PMID:1971160

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. Incorporation of radioactive wastes into styrenated polyester

    SciTech Connect

    Ikladious, N.E.; Ghattas, N.K.; Eskander, S.B.

    1986-01-01

    Styrenated polyester (poly(oxydiethylene maleate)) is examined as a medium for immobilization of simulated spent-ion exchange resin used at Inshas Reactor (Egypt). Compressive strength and hardness values illustrated the stability of the final products towards radiation. TG, DTG, and DTA diagrams showed the thermal instability of the final waste form at about 375/sup 0/C. Leaching experiment on incorporated blocks of active resin labelled with /sup 137/Cs, /sup 144/Ce, and /sup 106/Ru showed that the cumulative leaching rate for Ce is lower than those for Ru and Cs.

  7. Incorporation of aluminum nanopowder into NTO.

    SciTech Connect

    Lee, K. Y.; Kennedy, J. E.; Peterson, P. D.; Asay, B. W.

    2004-01-01

    Most insensitive high explosives have relatively low output and the user is forced to choose between enhancing operational safety and meeting ordnance performance requirements. The goals of this project are to investigate ways to incorporate Al nanopowder in NTO and to devise formulations that are insensitive and that exhibit high performance. In this paper, the novel method for the preparation of nanoNTO and its formulation with Al nanopowder will be presented, together with the characterization test results. Data from minisandwich velocity test on NTO and NTO/Al mix and the techniques used to verify the potential encapsulation of Al in NTO will also be presented.

  8. 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.

  9. 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.

  10. 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.

  11. Incorporating interfacial phenomena in solidification models

    NASA Technical Reports Server (NTRS)

    Beckermann, Christoph; Wang, Chao Yang

    1994-01-01

    A general methodology is available for the incorporation of microscopic interfacial phenomena in macroscopic solidification models that include diffusion and convection. The method is derived from a formal averaging procedure and a multiphase approach, and relies on the presence of interfacial integrals in the macroscopic transport equations. In a wider engineering context, these techniques are not new, but their application in the analysis and modeling of solidification processes has largely been overlooked. This article describes the techniques and demonstrates their utility in two examples in which microscopic interfacial phenomena are of great importance.

  12. 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

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

    PubMed

    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

  14. Effect of incorporation of nanoscale bioactive glass and hydroxyapatite in PCL/chitosan nanofibers for bone and periodontal tissue engineering.

    PubMed

    Shalumon, K T; Sowmya, S; Sathish, D; Chennazhi, K P; Nair, Shantikumar V; Jayakumar, R

    2013-03-01

    A biomimetic scaffold which can very closely mimic the extracellular matrix of the bone was fabricated by incorporating nano-bioceramic particles such as nano bioglass (nBG) and nano hydroxyapatite (nHAp) within electrospun nanofibrous scaffold. A comparative study between nHAp incorporated poly(caprolactone) (PCL)-chitosan (CS) and nBG incorporated PCL-CS nanofibrous scaffolds was carried out and their feasibility in tissue engineering was investigated. All the samples were optimized to obtain fibers of similar diameter from 100-200 nm for the ease of comparison between the samples. Protein adsorption studies showed that PCL-CS incorporated with 3 wt% nHAp and 3 wt% nBG adsorbed more proteins on their surface than other samples. Cell attachment and proliferation studies using human periodontal ligament fibroblast cells (hPLFs) and osteoblast like cells (MG-63 cell lines) showed that nBG incorporated samples are slightly superior to nHAp incorporated counterparts. Cell viability test using alamar blue assay and live/dead staining confirms that the scaffolds are cytocompatible. ALP activity confirmed the osteoblastic behavior of hPDLFs. Also the presence of nHAp and nBG enhanced the ALP activity of hPDLF on the PCH3 and PCB3 scaffolds. These studies indicate that nBG incorporated electrospun scaffolds are comparatively better candidates for orthopedic and periodontal tissue engineering applications. PMID:23620999

  15. Cell Culture Assay for Human Noroviruses [response

    SciTech Connect

    Straub, Tim M.; Honer Zu Bentrup, Kerstin; Orosz Coghlan, Patricia; Dohnalkova, Alice; Mayer, Brooke K.; Bartholomew, Rachel A.; Valdez, Catherine O.; Bruckner-Lea, Cindy J.; Gerba, Charles P.; Abbaszadegan, Morteza A.; Nickerson, Cheryl A.

    2007-07-01

    We appreciate the comments provided by Leung et al., in response to our recently published article “In Vitro Cell Culture Infectivity Assay for Human Noroviruses” by Straub et al. (1). The specific aim of our project was to develop an in vitro cell culture infectivity assay for human noroviruses (hNoV) to enhance risk assessments when they are detected in water supplies. Reverse transcription (RT) qualitative or quantitative PCR are the primary assays for waterborne NoV monitoring. However, these assays cannot distinguish between infectious vs. non-infectious virions. When hNoV is detected in water supplies, information provided by our infectivity assay will significantly improve risk assessment models and protect human health, regardless of whether we are propagating NoV. Indeed, in vitro cell culture infectivity assays for the waterborne pathogen Cryptosporidium parvum that supplement approved fluorescent microscopy assays, do not result in amplification of the environmentally resistant hard-walled oocysts (2). However, identification of life cycle stages in cell culture provides evidence of infectious oocysts in a water supply. Nonetheless, Leung et al.’s assertion regarding the suitability of our method for the in vitro propagation of high titers of NoV is valid for the medical research community. In this case, well-characterized challenge pools of virus would be useful for developing and testing diagnostics, therapeutics, and vaccines. As further validation of our published findings, we have now optimized RT quantitative PCR to assess the level of viral production in cell culture, where we are indeed finding significant increases in viral titer. The magnitude and time course of these increases is dependent on both virus strain and multiplicity of infection. We are currently preparing a manuscript that will discuss these findings in greater detail, and the implications this may have for creating viral challenge pools

  16. Controlling variation in the comet assay

    PubMed Central

    Collins, Andrew R.; El Yamani, Naouale; Lorenzo, Yolanda; Shaposhnikov, Sergey; Brunborg, Gunnar; Azqueta, Amaya

    2014-01-01

    Variability of the comet assay is a serious issue, whether it occurs from experiment to experiment in the same laboratory, or between different laboratories analysing identical samples. Do we have to live with high variability, just because the comet assay is a biological assay rather than analytical chemistry? Numerous attempts have been made to limit variability by standardizing the assay protocol, and the critical steps in the assay have been identified; agarose concentration, duration of alkaline incubation, and electrophoresis conditions (time, temperature, and voltage gradient) are particularly important. Even when these are controlled, variation seems to be inevitable. It is helpful to include in experiments reference standards, i.e., cells with a known amount of specific damage to the DNA. They can be aliquots frozen from a single large batch of cells, either untreated (negative controls) or treated with, for example, H2O2 or X-rays to induce strand breaks (positive control for the basic assay), or photosensitiser plus light to oxidize guanine (positive control for Fpg- or OGG1-sensitive sites). Reference standards are especially valuable when performing a series of experiments over a long period—for example, analysing samples of white blood cells from a large human biomonitoring trial—to check that the assay is performing consistently, and to identify anomalous results necessitating a repeat experiment. The reference values of tail intensity can also be used to iron out small variations occurring from day to day. We present examples of the use of reference standards in human trials, both within one laboratory and between different laboratories, and describe procedures that can be used to control variation. PMID:25368630

  17. An in vitro method which assesses corneal epithelial toxicity due to antineoplastic, preservative and antimicrobial agents.

    PubMed

    Lazarus, H M; Imperia, P S; Botti, R E; Mack, R J; Lass, J H

    1989-01-01

    We developed an in vitro model for studying the cytotoxicity of pharmacologic agents on corneal epithelium employing 3H-thymidine incorporation. Primary rabbit corneal epithelial cell cultures were established, and the cells plated prior to each experiment. 3H-thymidine incorporation was measured after the addition of drug or vehicle to these confluent cells, and dose-response curves were generated. Marked inhibition of 3H-thymidine incorporation was reached at chemotherapeutic concentrations achieved clinically for cytosine arabinoside (10(-7) M), methotrexate (10(-3) M), and 5-fluorouracil (10(-6) M). A 10(-4) M concentration of 2-deoxycytidine, a naturally occurring competitive inhibitor of cytosine arabinoside, protected cells up to a concentration of 10(-5) M. We utilized these data to undertake an in vivo prophylaxis study in 13 leukemia patients receiving high-dose iv cytosine arabinoside. Topical deoxycytidine 10(-4) M and 1% prednisolone phosphate, given 12 hours prior to the start of antileukemic therapy, were effective in reducing symptoms and signs of keratitis; both were better than historical placebo-treated eyes. Ophthalmic preservatives were studied in vitro at concentrations used clinically: benzalkonium chloride (BAC) (0.004-0.02%) was the most toxic, thimerosal (TMS) (0.001-0.004%) intermediate, and chlorobutanol (CHB) (0.2-0.5%) the least toxic. Antiviral agents (final concentration) included: trifluridine (TFT) (1.0%), ethyldeoxuridine (EDU) (2.0%), and idoxuridine (IDU) (0.1%). Dose but not time-dependent concentrations of these 3 agents were noted to cause toxicity; however, (E)-5(2-bromovinyl)-2'-deoxyuridine (BVDU) (0.1%) was non-toxic. Similarly, tobramycin and amikacin were significantly less toxic than gentamicin and neomycin in this system. These in vitro cytotoxicity data correlate well with previous in vivo and pre-clinical corneal epithelial toxicity studies. Our model may be useful in the toxicologic study of future topical

  18. 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.

  19. Estrogen receptor beta (ERβ) mediates expression of β-catenin and proliferation in prostate cancer cell line PC-3.

    PubMed

    Lombardi, Ana Paola G; Pisolato, Raisa; Vicente, Carolina M; Lazari, Maria Fatima M; Lucas, Thaís F G; Porto, Catarina S

    2016-07-15

    The aim of the present study was to characterize the mechanism underlying estrogen effects on the androgen-independent prostate cancer cell line PC-3. 17β-estradiol and the ERβ-selective agonist DPN, but not the ERα-selective agonist PPT, increased the incorporation of [methyl-(3)H]thymidine and the expression of Cyclin D2, suggesting that ERβ mediates the proliferative effect of estrogen on PC-3 cells. In addition, upregulation of Cyclin D2 and incorporation of [methyl-(3)H]thymidine induced by 17β-estradiol and DPN were blocked by the ERβ-selective antagonist PHTPP in PC-3 cells. Upregulation of Cyclin D2 and incorporation of [methyl-(3)H]thymidine induced by DPN were also blocked by PKF118-310, a compound that disrupts β-catenin-TCF (T-cell-specific transcription factor) complex, suggesting the involvement of β-catenin in the estradiol effects in PC-3 cells. A diffuse immunostaining for non-phosphorylated β-catenin was detected in the cytoplasm of PC-3 cells. Low levels of non-phosphorylated β-catenin immunostaining were also detected near the plasma membrane and in nuclei. Treatment of PC-3 cells with 17β-estradiol or DPN markedly increased non-phosphorylated β-catenin expression. These effects were blocked by pretreatment with the ERβ-selective antagonist PHTPP, PI3K inhibitor Wortmannin or AKT inhibitor MK-2206, indicating that ERβ-PI3K/AKT mediates non-phosphorylated β-catenin expression. Cycloheximide blocked the DPN-induced upregulation of non-phosphorylated β-catenin, suggesting de novo synthesis of this protein. In conclusion, these results suggest that estrogen may play a role in androgen-independent prostate cancer cell proliferation through a novel pathway, involving ERβ-mediated activation of β-catenin. PMID:27107935

  20. Incorporation of a Michael acceptor enhances the antitumor activity of triterpenoic acids.

    PubMed

    Heller, Lucie; Schwarz, Stefan; Perl, Vincent; Köwitsch, Alexander; Siewert, Bianka; Csuk, René

    2015-08-28

    Finding and developing drugs for the treatment of cancer has been challenging scientists for many decades, and using compounds of natural origin represents one of several strategies. Triterpenoic acids are a very promising class of secondary metabolites being able to induce apoptosis while their cytotoxicity is low. Therefore, derivatizations have to be conducted to improve cytotoxicity while retaining their ability to induce programmed cell death. The incorporation of a Michael acceptor into molecules resulted very often in drugs of improved cytotoxicity. Thus, in this study we synthesized and evaluated several Michael acceptor substituted compounds derived from glycyrrhetinic, ursolic, oleanolic and platanic acid. The influence of the presence of such a functional group onto the cytotoxicity was investigated in colorimetric sulforhodamine B assays employing several human cancer cell lines. EC50 values in the single-digit micromolar range were measured. Thus, the incorporation of a Michael acceptor unit into triterpenoic acids enhances the cytotoxicity of these compounds significantly. PMID:26177446

  1. 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.

  2. Flexible ultrasonic transducers incorporating piezoelectric fibres.

    PubMed

    Harvey, Gerald; Gachagan, Anthony; Mackersie, John W; McCunnie, Thomas; Banks, Robert

    2009-09-01

    It is possible to produce a high-performance, flexible 1-3 connectivity piezoelectric ceramic composite with conventional methods but the process is difficult and time-consuming. Extensive finite element modeling was used to design a piezocomposite structure which incorporated randomly positioned piezoceramic fibers in a polymer matrix. Simple manufacturing techniques were developed which resulted in the production of large numbers of fully populated fiber composites that offered performance comparable with a conventional 1-3 piezocomposite. A modified process facilitated the production of efficient fiber piezocomposite elements separated by polymer channels which conformed to a highly flexible (13 mm radius of curvature), 2-D matrix array configuration. This arrangement has been termed a Composite Element Composite Array Transducer, or CECAT. These devices were evaluated in terms of their impedance spectra, pulse-echo response, and surface displacement characteristics. The random piezoceramic fiber arrangements showed comparable sensitivity and bandwidth to periodic devices while minimizing the parasitic interpillar modes associated with periodic structures. Investigations have indicated that CECAT arrays constructed with 250 microm diameter fibers can be operated at frequencies of up to 3 MHz and transducers incorporating 10 microm diameter fibers can extend the frequency range above 6 MHz. Conversely, improved low-frequency devices can be produced with taller pillars than possible with conventional manufacturing techniques. PMID:19812003

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. Evaluation of three gentamicin serum assay techniques

    SciTech Connect

    Matzke, G.R.; Gwizdala, C.; Wery, J.; Ferry, D.; Starnes, R.

    1982-01-01

    This investigation was designed to compare the enzyme-modified immunoassay (Syva--EMIT) with a radioimmunoassay (New England Nuclear--RIA) and the radiometric assay (Johnston--BACTEC) to determine the optimal assay for use in our aminoglycoside dosing service. The serum concentration determinations obtained via the three assay methods were analyzed by linear regression analysis. Significant positive correlations were noted between the three assay techniques (p less than 0.005) during both sample collection phases. The coefficients of determination for EMIT vs BACTEC and RIA vs BACTEC were 0.73 and 0.83 during phase 1, respectively, and 0.65 and 0.68 during phase 2, respectively. The slope of the regression lines also varied markedly during the two phases; 0.49 and 0.42 for EMIT and for RIA vs BACTEC, respectively, during phase 1 compound with 1.12 and 0.77, respectively, during phase 2. The differences noted in these relationships during phase 1 and 2 may be related to the alteration of the pH of the control sera utilized in the BACTEC assay. In contrast, RIA vs EMIT regression analysis indicated that existence of a highly significant relationship (p less than 0.0005 and r2 . 0.90). The EMIT technique was the easiest and most accurate for determination of serum gentamicin concentrations, whereas the BACTEC method was judged unacceptable for clinical use.

  9. Comet assay to sense neutron 'fingerprint'.

    PubMed

    Gajendiran, N; Tanaka, K; Kamada, N

    2000-09-18

    The suitability of comet assay to identify DNA damage induced by neutrons of varying energy was tested. For this purpose, monoenergetic neutrons from Hiroshima University Radiobiological Research Accelerator (HIRRAC) were used to induce DNA damage in irradiated human peripheral blood lymphocytes. The level of damage was computed as tail moment for different doses (0.125-1 Gy) and compared with the effects resulting from irradiation with (60)Co gamma. The neutron-irradiated cells exhibited longer comet tails consisting of tiny pieces of broken DNA in contrast to the streaking tails generated by (60)Co gamma. The peak biological effectiveness occurred at 0.37 and 0.57 MeV; a further increase or decrease in neutron energy led to a reduced RBE value. The RBE values, as measured by the comet assay, were 6.3, 5.4, 4.7, 4.3, 2.6, and 1.7 for 0.37, 0.57, 0.79, 0.186, 1, and 2.3 MeV neutrons. The lower RBE value obtained by the comet assay when compared to that for other biological end points is discussed. This study reports the usefulness of the alkaline comet assay for identifying DNA damage induced by neutrons of the same radiation weighting factor. The comet assay is a potential tool for use in neutron therapy, as well as a method for the rapid screening of samples from individuals accidentally exposed to radiation. PMID:11024477

  10. 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.

  11. Enhanced circulation time and antitumor activity of doxorubicin by comblike polymer-incorporated liposomes.

    PubMed

    Han, Hee Dong; Lee, Aeri; Hwang, Taewon; Song, Chung Kil; Seong, Hasoo; Hyun, Jinho; Shin, Byung Cheol

    2007-07-31

    Polymer incorporation on liposomal membranes has been extensively studied as a method of enhancing the circulation time of liposomes in the bloodstream. In this study, we investigated the in vitro and in vivo characteristics of liposomes whose surface was modified using a comblike polymer comprised of a poly(methyl methacrylate) (PMMA) backbone and short poly(ethylene oxide) (PEO) side chains. Doxorubicin (DOX)-loaded liposomes incorporating with the comblike polymer were prepared and their circulation time, biodistribution and antitumor activity were evaluated in B16F10 melanoma tumor-bearing mice. The circulation half-life time in the bloodstream of the comblike polymer-incorporated liposomes (CPILs) was approximately 14- or 2-fold higher than those of the conventional or polyethyleneglycol-fixed liposomes (PEG-liposomes), respectively. Additionally, in the biodistribution assay, the accumulation of the CPILs in the tumor was higher than those of the other liposomes. Based on this result, the antitumor activities of the CPILs were higher than those of conventional liposome formulation of DOX or free DOX due to the higher passive targeting efficiency of the long-circulating CPILs to tumor. This study suggests that the incorporation of the comblike polymer on the liposomal membrane is a promising tool to further improve circulation time of liposomes in tumor-bearing mice. PMID:17524514

  12. Liposomal incorporation of Artemisia arborescens L. essential oil and in vitro antiviral activity.

    PubMed

    Sinico, Chiara; De Logu, Alessandro; Lai, Francesco; Valenti, Donatella; Manconi, Maria; Loy, Giuseppe; Bonsignore, Leonardo; Fadda, Anna Maria

    2005-01-01

    The effect of liposomal inclusion on the in vitro antiherpetic activity of Artemisia arborescens L. essential oil was investigated. In order to study the influence of vesicle structure and composition on the antiviral activity of the vesicle-incorporated oil, multilamellar (MLV) and unilamellar (SUV) positively charged liposomes were prepared by the film method and sonication. Liposomes were obtained from hydrogenated (P90H) and non-hydrogenated (P90) soy phosphatidylcholine. Formulations were examined for their stability for over one year, monitoring the oil leakage from vesicles and the average size distribution. The antiviral activity was studied against Herpes simplex virus type 1 (HSV-1) by a quantitative tetrazolium-based colorimetric method. Results showed that Artemisia essential oil can be incorporated in good amounts in the prepared vesicular dispersions. Stability studies pointed out that vesicle dispersions were very stable for at least six months and neither oil leakage nor vesicle size alteration occurred during this period. After one year of storage oil retention was still good, but vesicle fusion was present. Antiviral assays demonstrated that the liposomal incorporation of A. arborescens essential oil enhanced its in vitro antiherpetic activity especially when vesicles were made with P90H. On the contrary, no significant difference in antiviral activity was observed between the free and SUV-incorporated oil. PMID:15567314

  13. Incorporation of chitosan nanospheres into thin mineralized collagen coatings for improving the antibacterial effect.

    PubMed

    Kong, Ziqiang; Yu, Mengfei; Cheng, Kui; Weng, Wenjian; Wang, Huiming; Lin, Jun; Du, Piyi; Han, Gaorong

    2013-11-01

    It is desired that the coatings on metallic implants have both excellent biological responses and good loading-release capacities of biological factors or drugs. So far, the challenge still remains, because the morphology and composition of the bioactive coatings are usually not favorable for accommodating drug molecules. In this study, we adopted an approach of incorporating chitosan nanospheres into a thin mineralized collagen coating; this approach is based on the good loading-release behavior of the nanospheres and the good cytocompatibility of the thin coating. The incorporation of chitosan nanospheres into the mineralized collagen coatings was realized by electrolytic co-deposition. The morphologies and microstructures of the resulting coatings were characterized by SEM, and the phase and chemical compositions of the coatings were measured by XRD and FTIR. The loading-release capacity for vancomycin hydrochloride (VH) was determined by ultraviolet spectrophotometry. MTS assay was used to evaluate cytocompatibility, and in vitro bacterial adhesion was tested for assessing the antibacterial effects of the VH-loaded coatings. The chitosan nanospheres adhered tightly to collagen fibrils. The incorporated coatings facilitated the sustained release of VH, and had a clear antibacterial effect. The incorporation of chitosan nanospheres into mineralized collagen coatings demonstrates an effective way to improve the drug loading-release capacity for the thin coatings. This formulation had a highly effective biological response. PMID:23893027

  14. Incorporation of different bridge length linkers in enzyme and its use in the preparation of enzyme conjugates for immunoassay.

    PubMed

    Shrivastav, Tulsidas G

    2004-01-01

    An enzyme horseradish peroxidase (HRP), as a starting material, has been used to introduce different bridge length linkers, and its use in the preparation of enzyme conjugates for immunoassay is described. HRP was conjugated to adipic acid dihydrazide (ADH), gamma amino butyric acid (GABA), followed by ADH and 6-amino caproic acid (6ACA) followed by ADH. The different bridge length linkers-incorporated enzyme was coupled to a carboxylic derivative of cortisol. Four enzyme conjugates with different bridge length were prepared, such as cortisol-21-hemisuccinate-HRP (cortisol-21-HS-HRP), cortisol-21-HS-ADH-HRP, cortisol-21-HS-ADH-GABA-HRP, and cortisol-21-HS-ADH-6ACA-HRP. The influence of linker on sensitivity and specificity of the cortisol assay was studied. The study revealed that incorporation of a linker between hapten and enzyme increases the sensitivity and specificity of the assay. PMID:15461384

  15. Quantitative Molecular Assay for Fingerprinting Microbial Communities of Wastewater and Estrogen-Degrading Consortia

    PubMed Central

    Yu, Chang-Ping; Ahuja, Rajiv; Sayler, Gary; Chu, Kung-Hui

    2005-01-01

    A quantitative fingerprinting method, called the real-time terminal restriction fragment length polymorphism (real-time-t-RFLP) assay, was developed for simultaneous determination of microbial diversity and abundance within a complex community. The real-time-t-RFLP assay was developed by incorporating the quantitative feature of real-time PCR and the fingerprinting feature of t-RFLP analysis. The assay was validated by using a model microbial community containing three pure strains, an Escherichia coli strain (gram negative), a Pseudomonas fluorescens strain (gram negative), and a Bacillus thuringiensis strain (gram positive). Subsequently, the real-time-t-RFLP assay was applied to and proven to be useful for environmental samples; the richness and abundance of species in microbial communities (expressed as the number of 16S rRNA gene copies of each ribotype per milliliter) of wastewater and estrogen-degrading consortia (enriched with 17α-estradiol, 17β-estradiol, or estrone) were successfully characterized. The results of this study strongly suggested that the real-time-t-RFLP assay can be a powerful molecular tool for gaining insight into microbial communities in various engineered systems and natural habitats. PMID:15746346

  16. Ultrasensitive isolation, identification and quantification of DNA-protein adducts by ELISA-based RADAR assay.

    PubMed

    Kiianitsa, Kostantin; Maizels, Nancy

    2014-07-01

    Enzymes that form transient DNA-protein covalent complexes are targets for several potent classes of drugs used to treat infectious disease and cancer, making it important to establish robust and rapid procedures for analysis of these complexes. We report a method for isolation of DNA-protein adducts and their identification and quantification, using techniques compatible with high-throughput screening. This method is based on the RADAR assay for DNA adducts that we previously developed (Kiianitsa and Maizels (2013) A rapid and sensitive assay for DNA-protein covalent complexes in living cells. Nucleic Acids Res., 41:e104), but incorporates three key new steps of broad applicability. (i) Silica-assisted ethanol/isopropanol precipitation ensures reproducible and efficient recovery of DNA and DNA-protein adducts at low centrifugal forces, enabling cell culture and DNA precipitation to be carried out in a single microtiter plate. (ii) Rigorous purification of DNA-protein adducts by a procedure that eliminates free proteins and free nucleic acids, generating samples suitable for detection of novel protein adducts (e.g. by mass spectroscopy). (iii) Identification and quantification of DNA-protein adducts by direct ELISA assay. The ELISA-based RADAR assay can detect Top1-DNA and Top2a-DNA adducts in human cells, and gyrase-DNA adducts in Escherichia coli. This approach will be useful for discovery and characterization of new drugs to treat infectious disease and cancer, and for development of companion diagnostics assays for individualized medicine. PMID:24914050

  17. Attempts to detect cyclic adenosine 3':5'-monophosphate in higher plants by three assay methods.

    PubMed

    Bressan, R A; Ross, C W

    1976-01-01

    Endogenous levels of cyclic adenosine-3':5'-monophosphate in coleoptile first leaf segments of oat (Avena sativa L.), potato (Solanum tuberosum L.) tubers, tobacco (Nicotiana tabacum L.) callus, and germinating seeds of lettuce (Lactuca sativa L.) were measured with a modified Gilman binding assay and a protein kinase activation assay. The incorporation of adenosine-8-(14)C into compounds with properties similar to those of cyclic AMP was also measured in studies with germinating lettuce seeds. The binding assay proved reliable for mouse and rat liver analyses, but was nonspecific for plant tissues. It responded to various components from lettuce and potato tissues chromatographically similar to but not identical with cyclic AMP. The protein kinase activation assay was much more specific, but it also exhibited positive responses in the presence of compounds not chromatographically identical to cyclic AMP. The concentrations of cyclic AMP in the plant tissues tested were at the lower limits of detection and characterization obtainable with these assays. The estimates of maximal levels were much lower than reported in many previous studies. PMID:16659419

  18. The AR/NCOA1 axis regulates prostate cancer migration by involvement of PRKD1.

    PubMed

    Luef, Birgit; Handle, Florian; Kharaishvili, Gvantsa; Hager, Martina; Rainer, Johannes; Janetschek, Günter; Hruby, Stephan; Englberger, Christine; Bouchal, Jan; Santer, Frédéric R; Culig, Zoran

    2016-06-01

    Due to the urgent need for new prostate cancer (PCa) therapies, the role of androgen receptor (AR)-interacting proteins should be investigated. In this study we aimed to address whether the AR coactivator nuclear receptor coactivator 1 (NCOA1) is involved in PCa progression. Therefore, we tested the effect of long-term NCOA1 knockdown on processes relevant to metastasis formation. [(3)H]-thymidine incorporation assays revealed a reduced proliferation rate in AR-positive MDA PCa 2b and LNCaP cells upon knockdown of NCOA1, whereas AR-negative PC3 cells were not affected. Furthermore, Boyden chamber assays showed a strong decrease in migration and invasion upon NCOA1 knockdown, independently of the cell line's AR status. In order to understand the mechanistic reasons for these changes, transcriptome analysis using cDNA microarrays was performed. Protein kinase D1 (PRKD1) was found to be prominently up-regulated by NCOA1 knockdown in MDA PCa 2b, but not in PC3 cells. Inhibition of PRKD1 reverted the reduced migratory potential caused by NCOA1 knockdown. Furthermore, PRKD1 was negatively regulated by AR. Immunohistochemical staining of PCa patient samples revealed a strong increase in NCOA1 expression in primary tumors compared with normal prostate tissue, while no final conclusion could be drawn for PRKD1 expression in tumor specimens. Thus, our findings directly associate the AR/NCOA1 complex with PRKD1 regulation and cellular migration and support the concept of therapeutic inhibition of NCOA1 in PCa. PMID:27255895

  19. Upregulation of TRPM7 augments cell proliferation and interleukin-8 release in airway smooth muscle cells of rats exposed to cigarette smoke.

    PubMed

    Lin, Xiaoling; Yang, Cheng; Huang, Linjie; Chen, Ming; Shi, Jianting; Ouyang, Lihua; Tang, Tiantian; Zhang, Wei; Li, Yiqun; Liang, Ruiyun; Jiang, Shanping

    2016-06-01

    Proliferation and synthetic function (i.e. the capacity to release numerous chemokines and cytokines) of airway smooth muscle cells (ASMCs) are important in airway remodeling induced by cigarette smoke exposure. However, the molecular mechanism has not been clarified. Transient receptor potential cation channel subfamily M member 7 (TRPM7) is expressed ubiquitously and is crucial for the cellular physiological function of many cell types. The present study aimed to detect the expression of TRPM7 in ASMCs from smoke‑exposed rats and determine the importance of TRPM7 in proliferation and interleukin‑8 (IL‑8) release. ASMCs were isolated and cultured from smoke‑exposed rats. Expression levels of TRPM7 were determined by reverse transcription‑polymerase chain reaction, western blot analysis and immunofluorescence. TRPM7 was silenced with TRPM7‑short hairpin RNA lentivirus vector. DNA synthesis, cell number and IL‑8 release of ASMCs induced by cigarette smoke extract (CSE) and tumor necrosis factor‑α (TNF‑α) were assessed using [3H]-thymidine incorporation assay, hemocytometer and enzyme‑linked immunosorbent assay, respectively. It was determined that mRNA and protein expression levels of TRPM7 were increased in ASMCs from smoke‑exposed rats. Stimulation with CSE or TNF‑α elevated DNA synthesis, cell number and IL‑8 release were more marked in ASMCs from smoke‑exposed rats. Silencing of TRPM7 reduced DNA synthesis, cell number and IL‑8 release induced by CSE or TNF‑α in ASMCs from smoke-exposed rats. In conclusion, expression of TRPM7 increased significantly in ASMCs from smoke‑exposed rats and the upregulation of TRPM7 led to augmented cell proliferation and IL-8 release in ASMCs from rats exposed to cigarette smoke. PMID:27108806

  20. Upregulation of TRPM7 augments cell proliferation and interleukin-8 release in airway smooth muscle cells of rats exposed to cigarette smoke

    PubMed Central

    LIN, XIAOLING; YANG, CHENG; HUANG, LINJIE; CHEN, MING; SHI, JIANTING; OUYANG, LIHUA; TANG, TIANTIAN; ZHANG, WEI; LI, YIQUN; LIANG, RUIYUN; JIANG, SHANPING

    2016-01-01

    Proliferation and synthetic function (i.e. the capacity to release numerous chemokines and cytokines) of airway smooth muscle cells (ASMCs) are important in airway remodeling induced by cigarette smoke exposure. However, the molecular mechanism has not been clarified. Transient receptor potential cation channel subfamily M member 7 (TRPM7) is expressed ubiquitously and is crucial for the cellular physiological function of many cell types. The present study aimed to detect the expression of TRPM7 in ASMCs from smoke-exposed rats and determine the importance of TRPM7 in proliferation and interleukin-8 (IL-8) release. ASMCs were isolated and cultured from smoke-exposed rats. Expression levels of TRPM7 were determined by reverse transcription-polymerase chain reaction, western blot analysis and immunofluorescence. TRPM7 was silenced with TRPM7-short hairpin RNA lentivirus vector. DNA synthesis, cell number and IL-8 release of ASMCs induced by cigarette smoke extract (CSE) and tumor necrosis factor-α (TNF-α) were assessed using [3H]-thymidine incorporation assay, hemocytometer and enzyme-linked immunosorbent assay, respectively. It was determined that mRNA and protein expression levels of TRPM7 were increased in ASMCs from smoke-exposed rats. Stimulation with CSE or TNF-α elevated DNA synthesis, cell number and IL-8 release were more marked in ASMCs from smoke-exposed rats. Silencing of TRPM7 reduced DNA synthesis, cell number and IL-8 release induced by CSE or TNF-α in ASMCs from smoke-exposed rats. In conclusion, expression of TRPM7 increased significantly in ASMCs from smoke-exposed rats and the upregulation of TRPM7 led to augmented cell proliferation and IL-8 release in ASMCs from rats exposed to cigarette smoke. PMID:27108806

  1. Chemical heterogeneity as a result of hydroxylamine cleavage of a fusion protein of human insulin-like growth factor I.

    PubMed Central

    Canova-Davis, E; Eng, M; Mukku, V; Reifsnyder, D H; Olson, C V; Ling, V T

    1992-01-01

    Recombinant DNA techniques were used to biosynthesize human insulin-like growth factor I (hIGF-I) as a fusion protein wherein the fusion polypeptide is an IgG-binding moiety derived from staphylococcal protein A. This fusion protein is produced in Escherichia coli and secreted into the fermentation broth. In order to release mature recombinant-derived hIGF-I (rhIGF-I), the fusion protein is treated with hydroxylamine, which cleaves a susceptible Asn-Gly bond that has been engineered into the fusion protein gene. Reversed-phase h.p.l.c. was used to estimate the purity of the rhIGF-I preparations, especially for the quantification of the methionine sulphoxide-containing variant. It was determined that hydroxylamine cleavage of the fusion protein produced, as a side reaction, hydroxamates of the asparagine and glutamine residues in rhIGF-I. Although isoelectric focusing was effective in detecting, and reversed-phase h.p.l.c. for producing enriched fractions of the hydroxamate variants, ion-exchange chromatography was a more definitive procedure, as it allowed quantification and facile removal of these variants. The identity of the variants as hydroxamates was established by Staphylococcus aureus V8 proteinase digestion, followed by m.s., as the modification was transparent to amino acid and N-terminal sequence analyses. The biological activity of rhIGF-I was established by its ability to incorporate [3H]thymidine into the DNA of BALB/c373 cells and by a radioreceptor assay utilizing human placental membranes. Both assays demonstrate that the native, recombinant and methionine sulphoxide and hydroxamate IGF-I variants are essentially equipotent. Images Fig. 2. PMID:1637301

  2. Restricted Heterochromatin Formation Links NFATc2 Repressor Activity With Growth Promotion in Pancreatic Cancer

    PubMed Central

    BAUMGART, SANDRA; GLESEL, ELISABETH; SINGH, GARIMA; CHEN, NAI-MING; REUTLINGER, KRISTINA; ZHANG, JINSAN; BILLADEAU, DANIEL D.; FERNANDEZ-ZAPICO, MARTIN E.; GRESS, THOMAS M.; SINGH, SHIV K.; ELLENRIEDER, VOLKER

    2012-01-01

    BACKGROUND & AIMS Transcriptional silencing of the p15INK4b tumor suppressor pathway overcomes cellular protection against unrestrained proliferation in cancer. Here we show a novel pathway involving the oncogenic transcription factor nuclear factor of activated T cells (NFAT) c2 targeting a p15INK4b-mediated failsafe mechanism to promote pancreatic cancer tumor growth. METHODS Immunohistochemistry, real-time polymerase chain reaction, immunoblotting, and immunofluorescence microscopy were used for expression studies. Cancer growth was assessed in vitro by [3H]thymidine incorporation, colony formation assays, and in vivo using xenograft tumor models. Protein-protein interactions, promoter regulation, and local histone modifications were analyzed by immunoprecipitation, DNA pull-down, reporter, and chromatin immunoprecipitation assays. RESULTS Our study uncovered induction of NFATc2 in late-stage pancreatic intraepithelial neoplasia lesions with increased expression in tumor cell nuclei of advanced cancers. In the nucleus, NFATc2 targets the p15INK4b promoter for inducible heterochromatin formation and silencing. NFATc2 binding to its cognate promoter site induces stepwise recruitment of the histone methyltransferase Suv39H1, causes local H3K9 trimethylation, and allows docking of heterochromatin protein HP1γ to the repressor complex. Conversely, inactivation of NFATc2 disrupts this repressor complex assembly and local heterochromatin formation, resulting in restoration of p15INK4b expression and inhibition of pancreatic cancer growth in vitro and in vivo. CONCLUSIONS Here we describe a novel mechanism for NFATc2-mediated gene regulation and identify a functional link among its repressor activity, the silencing of the suppressor pathway p15INK4b, and its pancreatic cancer growth regulatory functions. Thus, we provide evidence that inactivation of oncogenic NFATc2 might be an attractive strategy in treatment of pancreatic cancer. PMID:22079596

  3. 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. PMID:27174747

  4. 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.

  5. T cells recognizing leukemic CD34+ progenitor cells mediate the antileukemic effect of donor lymphocyte infusions for relapsed chronic myeloid leukemia after allogeneic stem cell transplantation

    PubMed Central

    Smit, Willem M.; Rijnbeek, Marion; van Bergen, Cornelis A. M.; Fibbe, Willem E.; Willemze, Roel; Falkenburg, J. H. Frederik

    1998-01-01

    Adoptive immunotherapy with donor lymphocyte infusions (DLI) is an effective treatment for relapsed chronic myeloid leukemia (CML) after allogeneic stem cell transplantation. To identify the effector and target cell populations responsible for the elimination of the leukemic cells in vivo we developed an assay to measure the frequency of T lymphocyte precursor cells capable of suppressing leukemic progenitor cells. Target cells in this assay were CML cells that were cultured in the presence of stem cell factor, interleukin 3, granulocyte–macrophage colony-stimulating factor, granulocyte colony-stimulating factor, and erythropoietin. [3H]thymidine incorporation at day 7 represented the proliferation of the progeny of the CD34+ CML progenitor cells, and not of the more mature CD34− CML cells. Effector cells were mononuclear cells, which were used in a limiting dilution analysis to measure the frequencies of CML progenitor cell-inhibitory lymphocyte precursors (PCILp) in peripheral blood of seven patients before and after DLI for relapsed CML. In the six patients who entered complete remission, a 5- to 100-fold increase of PCILp was found during the clinical response. In the patient with resistant relapse the frequency of PCILp was <10 per ml before and after DLI. Leukemia-reactive helper T lymphocyte precursor frequencies remained unchanged after DLI. A significant increase in cytotoxic T lymphocyte precursor frequency against more mature leukemic cells was found in only two responding patients. These results indicate that T cells specifically directed against CD34+ CML progenitor cells mediate the antileukemic effect of DLI. PMID:9707616

  6. Nitidine chloride inhibits proliferation, induces apoptosis via the Akt pathway and exhibits a synergistic effect with doxorubicin in ovarian cancer cells.

    PubMed

    Ding, Feng; Liu, Tianfeng; Yu, Nina; Li, Shihong; Zhang, Xiaofei; Zheng, Guanghong; Lv, Chunming; Mou, Kai; Xu, Jia; Li, Bo; Wang, Surong; Song, Haibo

    2016-09-01

    Nitidine chloride (NC) exhibits anti-tumor properties in various types of tumor. However, to the best of our knowledge there is no previous evidence of NC involvement in the apoptosis or proliferation of ovarian cancer cells and the underlying molecular mechanisms. The present study aimed to investigate the influence of NC on the viability and apoptosis of ovarian cancer cells and the synergistic effect NC and doxorubicin (DOX) may have on ovarian cancer cells. The viability and proliferation of ovarian cancer cells were examined using a methyl thiazolyl tetrazolium assay and 3H-thymidine incorporation assay. The apoptotic rate of ovarian cancer cells was detected by flow cytometry. The expression of apoptosis‑associated proteins and Akt serine/threonine kinase 1 (Akt) were determined by western blot analysis following NC treatment. The inhibitory effect of NC on the proliferation of ovarian cancer cells was demonstrated in a time and dose‑dependent manner. The pro-apoptotic effect of NC on ovarian cancer cells was also observed. It was determined that NC significantly downregulated the protein expression levels of B‑cell CLL/lymphoma 2 (Bcl-2) and upregulated the expression of Bcl‑2‑associated X protein, p53, caspase‑3 and ‑9. NC suppressed Akt phosphorylation. Additionally, the present study demonstrated that the effect of NC on the proliferation and apoptosis of ovarian cancer cells was Akt‑dependent by using the phosphatidylinositol-4,5-bisphosphate 3-kinase/Akt signaling pathway inhibitor, LY294002. NC exhibited a synergistic inhibitory effect on the viability of ovarian cancer cells when combined with DOX. The current study demonstrated that NC inhibited the proliferation and induced the apoptosis of ovarian cancer cells via the Akt signaling pathway and highlighted its potential clinical application for the treatment of ovarian cancer. PMID:27485415

  7. Over-expression of heme oxygenase-1 promotes oxidative mitochondrial damage in rat astroglia.

    PubMed

    Song, Wei; Su, Haixiang; Song, Sisi; Paudel, Hemant K; Schipper, Hyman M

    2006-03-01

    Glial heme oxygenase-1 is over-expressed in the CNS of subjects with Alzheimer disease (AD), Parkinson disease (PD) and multiple sclerosis (MS). Up-regulation of HO-1 in rat astroglia has been shown to facilitate iron sequestration by the mitochondrial compartment. To determine whether HO-1 induction promotes mitochondrial oxidative stress, assays for 8-epiPGF(2alpha) (ELISA), protein carbonyls (ELISA) and 8-OHdG (HPLC-EC) were used to quantify oxidative damage to lipids, proteins, and nucleic acids, respectively, in mitochondrial fractions and whole-cell compartments derived from cultured rat astroglia engineered to over-express human (h) HO-1 by transient transfection. Cell viability was assessed by trypan blue exclusion and the MTT assay, and cell proliferation was determined by [3H] thymidine incorporation and total cell counts. In rat astrocytes, hHO-1 over-expression (x 3 days) resulted in significant oxidative damage to mitochondrial lipids, proteins, and nucleic acids, partial growth arrest, and increased cell death. These effects were attenuated by incubation with 1 microM tin mesoporphyrin, a competitive HO inhibitor, or the iron chelator, deferoxamine. Up-regulation of HO-1 engenders oxidative mitochondrial injury in cultured rat astroglia. Heme-derived ferrous iron and carbon monoxide (CO) may mediate the oxidative modification of mitochondrial lipids, proteins and nucleic acids in these cells. Glial HO-1 hyperactivity may contribute to cellular oxidative stress, pathological iron deposition, and bioenergetic failure characteristic of degenerating and inflamed neural tissues and may constitute a rational target for therapeutic intervention in these conditions. PMID:16222706

  8. Microplate Assay for Colletotrichum Spore Production

    PubMed Central

    Slade, S. J.; Harris, R. F.; Smith, C. S.; Andrews, J. H.; Nordheim, E. V.

    1987-01-01

    A simple microplate method was devised to assay spore production by Colletotrichum gloeosporioides by growing the fungus on 1 ml of solid media in the wells of tissue culture plates. Growth and sporulation on microplates were compared at days 4 and 8 with growth and sporulation in 100-ml liquid batch cultures that involved 11 common media. Spore production per unit volume of medium was the same for solid and liquid forms of the media. Qualitative assessment of mycelial growth measured on microplates agreed with that of growth measured in liquid cultures. The microplate assay indicated that V8 juice was the best medium and that an organic content of about 6 mg/ml was optimal for high sporulation and low mycelium production. The assay provides a convenient, rapid, and inexpensive means of screening media for the production of fungal conidia in large numbers, to be used, for example, in biological control programs. PMID:16347310

  9. Assays of Transendothelial Migration in vitro

    PubMed Central

    Luscinskas, F. William

    2009-01-01

    The inflammatory response is critical for our ability to heal wounds and fight off foreign microorganisms. Uncontrolled inflammation is also at the root of most pathology. Recruitment of leukocytes to the site of inflammation plays a defining role in the inflammatory response, and migration of leukocytes across endothelium is arguably the point of no return of the inflammatory response. Assays to study the transmigration of leukocytes have and will continue to shed light on the regulation of this vital response. Assays of transendothelial migration in vitro allow the controlled observation of this phenomenon as well as experiments to study its regulation. In this chapter we describe in vitro assays of transendothelial migration that have been used successfully in the authors’ laboratories for decades and have proven to be reproducible, reliable, and predictive of the behavior of leukocytes and endothelial cells in models of inflammation in vivo. PMID:18772016

  10. Miniaturization of hydrolase assays in thermocyclers.

    PubMed

    Lucena, Severino A; Moraes, Caroline S; Costa, Samara G; de Souza, Wanderley; Azambuja, Patrícia; Garcia, Eloi S; Genta, Fernando A

    2013-03-01

    We adapted the protocols of reducing sugar measurements with dinitrosalicylic acid and bicinchoninic acid for thermocyclers and their use in enzymatic assays for hydrolases such as amylase and β-1,3-glucanase. The use of thermocyclers for these enzymatic assays resulted in a 10 times reduction in the amount of reagent and volume of the sample needed when compared with conventional microplate protocols. We standardized absorbance readings from the polymerase chain reaction plates, which allowed us to make direct readings of the techniques above, and a β-glycosidase assay was also established under the same conditions. Standardization of the enzymatic reaction in thermocyclers resulted in less time-consuming temperature calibrations and without loss of volume through leakage or evaporation from the microplate. Kinetic parameters were successfully obtained, and the use of the thermocycler allowed the measurement of enzymatic activities in biological samples from the field with a limited amount of protein. PMID:23123426

  11. Nuclear Resonance Fluorescence for Materials Assay

    SciTech Connect

    Quiter, Brian J.; Ludewigt, Bernhard; Mozin, Vladimir; Prussin, Stanley

    2009-06-29

    This paper discusses the use of nuclear resonance fluorescence (NRF) techniques for the isotopic and quantitative assaying of radioactive material. Potential applications include age-dating of an unknown radioactive source, pre- and post-detonation nuclear forensics, and safeguards for nuclear fuel cycles Examples of age-dating a strong radioactive source and assaying a spent fuel pin are discussed. The modeling work has ben performed with the Monte Carlo radiation transport computer code MCNPX, and the capability to simulate NRF has bee added to the code. Discussed are the limitations in MCNPX?s photon transport physics for accurately describing photon scattering processes that are important contributions to the background and impact the applicability of the NRF assay technique.

  12. Nuclear Resonance Fluorescence for Materials Assay

    SciTech Connect

    Quiter, Brian; Ludewigt, Bernhard; Mozin, Vladimir; Prussin, Stanley

    2009-06-05

    This paper discusses the use of nuclear resonance fluorescence (NRF) techniques for the isotopic and quantitative assaying of radioactive material. Potential applications include age-dating of an unknown radioactive source, pre- and post-detonation nuclear forensics, and safeguards for nuclear fuel cycles Examples of age-dating a strong radioactive source and assaying a spent fuel pin are discussed. The modeling work has ben performed with the Monte Carlo radiation transport computer code MCNPX, and the capability to simulate NRF has bee added to the code. Discussed are the limitations in MCNPX's photon transport physics for accurately describing photon scattering processes that are important contributions to the background and impact the applicability of the NRF assay technique.

  13. Competitive protein binding assay for piritrexim

    SciTech Connect

    Woolley, J.L. Jr.; Ringstad, J.L.; Sigel, C.W. )

    1989-09-01

    A competitive protein binding assay for piritrexim (PTX, 1) that makes use of a commercially available radioassay kit for methotrexate has been developed. After it is selectively extracted from plasma, PTX competes with ({sup 125}I)methotrexate for binding to dihydrofolate reductase isolated from Lactobacillus casei. Free drug is separated from bound drug by adsorption to dextran-coated charcoal. Piritrexim is measurable over a range of 0.01 to 10.0 micrograms/mL in plasma with a coefficient of variation less than 15%. The limit of sensitivity of the assay is approximately 2 ng/mL. An excellent correlation between this assay and a previously published HPLC method was found.

  14. A High-Throughput Radiometric Kinase Assay.

    PubMed

    Duong-Ly, Krisna C; Peterson, Jeffrey R

    2016-01-01

    Aberrant kinase signaling has been implicated in a number of diseases. While kinases have become attractive drug targets, only a small fraction of human protein kinases have validated inhibitors. Screening of libraries of compounds against a kinase or kinases of interest is routinely performed during kinase inhibitor development to identify promising scaffolds for a particular target and to identify kinase targets for compounds of interest. Screening of more focused compound libraries may also be conducted in the later stages of inhibitor development to improve potency and optimize selectivity. The dot blot kinase assay is a robust, high-throughput kinase assay that can be used to screen a number of small-molecule compounds against one kinase of interest or several kinases. Here, a protocol for a dot blot kinase assay used for measuring insulin receptor kinase activity is presented. This protocol can be readily adapted for use with other protein kinases. PMID:26501904

  15. Flow cytometer measurement of binding assays

    DOEpatents

    Saunders, George C.

    1987-01-01

    A method of measuring the result of a binding assay that does not require separation of fluorescent smaller particles is disclosed. In a competitive binding assay the smaller fluorescent particles coated with antigen compete with antigen in the sample being analyzed for available binding sites on larger particles. In a sandwich assay, the smaller, fluorescent spheres coated with antibody attach themselves to molecules containing antigen that are attached to larger spheres coated with the same antibody. The separation of unattached, fluorescent smaller particles is made unnecessary by only counting the fluorescent events triggered by the laser of a flow cytometer when the event is caused by a particle with a light scatter measurement within a certain range corresponding to the presence of larger particles.

  16. Nanoparticles for Use in Enzyme Assays.

    PubMed

    Kim, Young-Pil; Kim, Hak-Sung

    2016-02-01

    Nanoparticles (NPs) have created new ways to enhance the performance of classical biosensors in analytical sciences. NPs with unprecedented physiochemical properties can serve both as excellent carriers of bioreceptors and as signal enhancers, leading to improved assay platforms with high sensitivity and selectivity. Because enzymes play central roles in many cellular functions, specific and precise assays of their functions are of great significance in medical science and biotechnology. Here we review recent advances in NP-based biosensors and their use in enzyme assays. With fast and specific responses to enzyme-mediated reactions, NPs transduce and amplify the initial responses into various types of signals, such as electrochemical, optical and magnetic ones. Translation of their potential should lead to functionalized NPs finding wide applications in diagnostics, drug development and biotechnology. PMID:26662229

  17. Robot speeds assays and enhances safeguards

    SciTech Connect

    Phelan, P.F.; Powell, W.D.; Blankenship, R.W.

    1990-01-01

    At the Los Alamos National Laboratory Plutonium Facility, a robotics system utilizing a gantry robot and an automated inventory system operates five calorimeters and two gamma isotopic assay instruments. This system has significantly improved safeguards, because the opportunity for diversion has been greatly reduced. Not only is the accountability much more timely because throughput has doubled but the special nuclear material has been made physically more secure in several ways. First, items awaiting assay are kept in the inventory system, whose doors remain locked whenever the robot is unattended. An alarm sounds if the doors are unlocked without authorization. Second, light curtains surround the robot's work envelope and pressure-sensitive pads cover the floor to detect entry into the assay area. Third, the robot weighs each item whenever it is moved, and the result is compared with the weight that was measured when the item was first put into inventory. 2 refs., 3 figs.

  18. 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

  19. The activity of deoxyribonucleic acid polymerase and deoxyribonucleic acid synthesis in nuclei from brain fractionated by zonal centrifugation

    PubMed Central

    Stambolova, M. A.; Cox, D.; Mathias, A. P.

    1973-01-01

    1. The DNA polymerase (EC 2.7.7.7) activity in purified intact brain nuclei from infant rats was investigated. The effects of pH, Mg2+, glycerol, sonication and storage of the nuclei under different conditions were examined and a suitable assay system was established. 2. The nuclei from infant brain cells were fractionated by zonal centrifugation in a discontinuous sucrose gradient into five zones: zone (I) contained neuronal nuclei (59%) and astrocytic nuclei (41%); zone (II) contained astrocytic nuclei (81%) and neuronal nuclei (19%); zone (III) contained astrocytic nuclei (82%) and oligodendrocytic nuclei (18%); zone (IV) contained oligodendrocytic nuclei (92%) and zone (V) contained oligodendrocytic nuclei (100%). 3. The content of DNA, RNA and protein for each fraction was measured. 4. The distribution of DNA polymerase activity in the fractionated infant and adult rat brain nuclei was determined. The highest activity was found in the neuronal nuclei from zone (I) and the following zones exhibited a progressive decline. In contrast with the nuclei from infant rats those from adults had a much higher activity and expressed a preference for native DNA as template. 5. The deoxyribonuclease activity in all classes of nuclei was measured with [3H]DNA as substrate. A general correspondence in the pattern of the relative activities in the nuclear fractions with the distribution of DNA polymerase was found. 6. The incorporation of [3H]thymidine into nuclear DNA in infant and adult rat brain was investigated. The specific radioactivity of the DNA in the 10-day-old rats was highest in zone (V) whereas in the nuclei of adult rats, which exhibited a comparatively low incorporation, the highest specific radioactivity was associated with zones (I) and (V). PMID:4780694

  20. Radiofrequency (microwave) radiation exposure of mammalian cells during UV-induced DNA repair synthesis

    SciTech Connect

    Meltz, M.L.; Walker, K.A.; Erwin, D.N.

    1987-05-01

    The effect of continuous-wave (CW) and pulsed-wave (PW) radiofrequency radiation (RFR) in the microwave range on UV-induced DNA repair has been investigated in MRC-5 normal human diploid fibroblasts. RFR exposure at power densities of 1 (or 5) and 10 mW/cm2 gave a maximum specific absorption rate (SAR) (at 10 mW/cm2) of 0.39 +/- 0.15 W/kg for 350 MHz RFR, 4.5 +/- 3.0 W/kg for 850 MHz RFR, and 2.7 +/- 1.6 W/kg for 1.2 GHz RFR. RFR exposures for 1 to 3 h at 37 degrees C, in either continuous-wave or pulsed-wave modes, had no effect on the rate of repair replication label incorporated into preexisting UV-damaged DNA. RFR exposures (PW), with a constant medium temperature of 39 degrees C at 350 and 850 MHz during the repair period after UV damage, also had no effect. Assay for induction of repair synthesis by RFR exposure alone in non-UV irradiated cells was negative for the 350-, 850-, and 1200-MHz CW and PW RFR at 37 degrees C and the 350- and 850-MHz PW RFR at 39 degrees C. RFR does not induce DNA repair under these exposure conditions. In preliminary experiments--with the tissue culture medium maintained at 39 degrees C and RFR exposures (PW) at the frequencies of 350, 850, and 1200 MHz--no effect on incorporation of (/sup 3/H)thymidine into DNA undergoing semiconservative synthesis was observed.