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Sample records for 6-diamidino-2-phenylindole dihydrochloride dapi

  1. Behavior of mesenchymal stem cells stained with 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) in osteogenic and non osteogenic cultures.

    PubMed

    Ocarino, N M; Bozzi, A; Pereira, R D O; Breyner, N M; Silva, V L; Castanheira, P; Goes, A M; Serakides, R

    2008-08-01

    4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) is a DNA dye widely used to mark and trace stem cells in therapy. We here studied the effect of DAPI staining on the behavior of mesenchymal stem cells cultured in either a control, non-osteogenic medium or in an osteogenic differentiation medium. In the control medium, the number of stem cells/field, as well as the number of fluorescent cells/field increased up to the sixth day in both control and DAPI-treated cultures. Afterwards, both the number of fluorescent cells and their fluorescence intensity decreased. Control cells were fusiform and with some long extensions that apparently linked them to neighboring cells, while DAPI-treated cells were mostly round cells with fine and short extensions. The trypan-blue exclusion method showed 99% cell viability in both groups, however, both alkaline phosphatase activity and the thiazolyl blue formazan assay (indicative of mitochondrial metabolism) gave significantly lower values in DAPI-marked cells. The mitochondrial mass, as indicated by specific staining and flow cytometry, showed no differences between groups. Mesenchymal stem cells gave origin to mineralized nodules in the osteogenic differentiation medium and there were not DAPI-marked cells on the ninth day of culture. Alkaline phosphatase activity, viability assay and number of cells/field and of mineralized nodules/field were similar in both groups. So, DAPI treatment did not change cell viability and proliferation during osteogenic differentiation of mesenchymal stem cells. However, since these cells loose DAPI marking after 9 days in osteogenic cultures suggests that DAPI may not be an effective marker for mesenchymal stem cells implanted in bone tissue for long periods.

  2. 4',6-diamidino-2-phenylindole (DAPI) interacts with rare structures of GC polymers.

    PubMed

    Barcellona, M L; Chen, Y; Müller, J D; Gratton, E

    2001-01-01

    The binding of 4',6-diamidino-2-phenylindole (DAPI) to double-stranded GC polymers either in the alternating or in homopolymer sequence was investigated using fluorescence techniques. We employed fluctuation correlation spectroscopy, which measures the diffusion coefficient of fluorescent particles, to demonstrate that the fluorescence was originating from relatively slowly diffusing entities. These entities display a very large heterogeneity of diffusing coefficients, indicating that molecular aggregation is extensive in our samples. We used frequency domain fluorometry to characterize the fluorescence lifetime of the species, while varying the GC polymer-dye coverage systematically. At very low coverage we observed a relatively bright fluorescent component with a lifetime value of approximately 4 ns. The stoichiometry of binding of this bright species was such that it can only arise from rare molecular structures, either unusual loops or large molecular aggregates. The amount and characteristics of this bright fluorescent component were different between the homo and the alternating polymer, indicating that the difference in sequence of the two polymers is responsible for the different aggregates which are then detected in the fluorescence experiment. At large GC polymer coverage we observed a relatively wide distribution of fluorescent species with short lifetime values, in the range between 0.12 and 0.2 ns. Given the stoichiometry of binding of this fluorescent component, we concluded that it could arise either from intercalative and/or non-specific binding to the DNA double-stranded molecules. We comment on the origin of the rare but brightly fluorescent binding sites and discuss the potential to detect such unusual DNA structures.

  3. The fluorophore 4',6-diamidino-2-phenylindole (DAPI) induces DNA folding in long double-stranded DNA.

    PubMed

    Beccia, Maria Rosa; Biver, Tarita; Pardini, Alberto; Spinelli, Jacopo; Secco, Fernando; Venturini, Marcella; Busto Vázquez, Natalia; Lopez Cornejo, Maria Pilar; Martin Herrera, Victoria Isabel; Prado Gotor, Rafael

    2012-08-01

    DAPI (4',6-diamidino-2-phenylindole) is a widely used fluorescent dye, whose complicated binding features to DNAs and RNAs have been the object of debates and are still not fully understood. In this study, different approaches were employed, including binding equilibrium measurements (spectrofluorometry), melting experiments (spectrophotometry), viscometric measurements, circular dichroism, and T-jump kinetic analyses; all data concur in shedding light on the complex mechanistic aspects of the binding mode of DAPI to natural DNA. Conditions are found that induce the mode of the DAPI/DNA interaction to change from groove binding to intercalation. Moreover, it is observed, for the first time, that DAPI is able to induce the formation of a rather compact polymer-dye adduct under particular conditions. The results suggest that this form is a folded or coiled DNA structure stabilized by DAPI dye bridges.

  4. Spectroscopic studies on ligand-enzyme interactions: complexation of alpha-chymotrypsin with 4',6-diamidino-2-phenylindole (DAPI).

    PubMed

    Banerjee, Debapriya; Srivastava, Sachin Kumar; Pal, Samir Kumar

    2008-02-14

    In the present study, the interaction of two structurally related proteolytic enzymes trypsin and alpha-chymotrypsin (CHT) with 4',6-Diamidino-2-phenylindole (DAPI) has been addressed. The binding of DAPI to CHT has been characterized by steady-state and picosecond time-resolved spectroscopic techniques. Enzymatic activity of CHT and simultaneous binding of the well-known inhibitor proflavin (PF) in the presence of DAPI clearly rule out the possibility of DAPI binding at the catalytic site of the enzyme. The spectral overlap between the emission of DAPI and absorption of PF offers the opportunity to explore the binding site of DAPI using Förster resonance energy transfer (FRET). FRET studies between DAPI and PF indicate that DAPI is bound to CHT with its transition dipole nearly perpendicular to that of PF. Competitive binding of DAPI with another fluorescent probe 2,6-p-toluidinonaphthalene sulfonate (TNS), having a well-defined binding site, indicates that DAPI and TNS bind at the same hydrophobic site of the enzyme CHT. The difference in the interactions of two well-studied, structurally similar enzymes with the same molecule may find its application in the design of specific substrate mimics or inhibitors of the enzymes.

  5. Fluorescence resonance energy transfer and molecular modeling studies on 4',6-diamidino-2-phenylindole (DAPI) complexes with tubulin.

    PubMed

    Arbildua, José J; Brunet, Juan E; Jameson, David M; López, Maribel; Nova, Esteban; Lagos, Rosalba; Monasterio, Octavio

    2006-03-01

    The goal of this work was to determine the binding properties and location of 4',6-diamidino-2-phenylindole (DAPI) complexed with tubulin. Using fluorescence anisotropy, a dissociation constant of 5.2+/-0.4 microM for the DAPI-tubulin complex was determined, slightly lower than that for the tubulin S complex. The influence of the C-terminal region on the binding of DAPI to tubulin was also characterized. Using FRET experiments, and assuming a kappa2 value of 2/3, distances between Co2+ bound to its high-affinity binding site and the DAPI-binding site and 2',3'-O-(trinitrophenyl)guanosine 5'-triphosphate bound to the exchangeable nucleotide and the DAPI-binding site were found to be 20+/-2 A and 43+/-2 A, respectively. To locate potential DAPI-binding sites on tubulin, a molecular modeling study was carried out using the tubulin crystal structure and energy minimization calculations. The results from the FRET measurements were used to limit the possible location of DAPI in the tubulin structure. Several candidate binding sites were found and these are discussed in the context of the various properties of bound DAPI.

  6. Fluorescence resonance energy transfer and molecular modeling studies on 4′,6-diamidino-2-phenylindole (DAPI) complexes with tubulin

    PubMed Central

    Arbildua, José J.; Brunet, Juan E.; Jameson, David M.; López, Maribel; Nova, Esteban; Lagos, Rosalba; Monasterio, Octavio

    2006-01-01

    The goal of this work was to determine the binding properties and location of 4′,6-diamidino-2-phenylindole (DAPI) complexed with tubulin. Using fluorescence anisotropy, a dissociation constant of 5.2 ± 0.4 μM for the DAPI–tubulin complex was determined, slightly lower than that for the tubulin S complex. The influence of the C-terminal region on the binding of DAPI to tubulin was also characterized. Using FRET experiments, and assuming a κ2 value of 2/3, distances between Co2+ bound to its high-affinity binding site and the DAPI-binding site and 2′,3′-O-(trinitrophenyl)guanosine 5′-triphosphate bound to the exchangeable nucleotide and the DAPI-binding site were found to be 20 ± 2 Å and 43 ± 2 Å, respectively. To locate potential DAPI-binding sites on tubulin, a molecular modeling study was carried out using the tubulin crystal structure and energy minimization calculations. The results from the FRET measurements were used to limit the possible location of DAPI in the tubulin structure. Several candidate binding sites were found and these are discussed in the context of the various properties of bound DAPI. PMID:16452620

  7. Influence of water chlorination on the counting of bacteria with DAPI (4',6-diamidino-2-phenylindole).

    PubMed Central

    Saby, S; Sibille, I; Mathieu, L; Paquin, J L; Block, J C

    1997-01-01

    Counting bacteria in drinking water samples by the epifluorescence technique after 4',6-diamidino-2-phenylindole (DAPI) staining is complicated by the fact that bacterial fluorescence varies with exposure of the cells to sodium hypochlorite. An Escherichia coli laboratory-grown suspension treated with sodium hypochlorite (5 to 15 mg of chlorine liter-1) for 90 min was highly fluorescent after DAPI staining probably due to cell membrane permeation and better and DAPI diffusion. At chlorine concentrations greater than 25 mg liter-1, DAPI-stained bacteria had only a low fluorescence. Stronger chlorine doses altered the DNA structure, preventing the DAPI from complexing with the DNA. When calf thymus DNA was exposed to sodium hypochlorite (from 15 to 50 mg of chlorine liter-1 for 90 min), the DNA lost the ability to complex with DAPI. Exposure to monochloramine did not have a similar effect. Treatment of drinking water with sodium hypochlorite (about 0.5 mg of chlorine liter-1) caused a significant increase in the percentage of poorly fluorescent bacteria, from 5% in unchlorinated waters (40 samples), to 35 to 39% in chlorinated waters (40 samples). The presence of the poorly fluorescent bacteria could explain the underestimation of the real number of bacteria after DAPI staining. Microscopic counting of both poorly and highly fluorescent bacteria is essential under these conditions to obtain the total number of bacteria. A similar effect of chlorination on acridine orange-stained bacteria was observed in treated drinking waters. The presence of the poorly fluorescent bacteria after DAPI staining could be interpreted as a sign of dead cells. PMID:9097452

  8. Direct quantification of inorganic polyphosphate in microbial cells using 4'-6-diamidino-2-phenylindole (DAPI).

    PubMed

    Kulakova, Anna N; Hobbs, Darragh; Smithen, Matthew; Pavlov, Evgeny; Gilbert, Jack A; Quinn, John P; McGrath, John W

    2011-09-15

    Inorganic polyphosphate (polyP) is increasingly being recognized as an important phosphorus sink within the environment, playing a central role in phosphorus exchange and phosphogenesis. Yet despite the significant advances made in polyP research there is a lack of rapid and efficient analytical approaches for the quantification of polyP accumulation in microbial cultures and environmental samples. A major drawback is the need to extract polyP from cells prior to analysis. Due to extraction inefficiencies this can lead to an underestimation of both intracellular polyP levels and its environmental pool size: we observed 23-58% loss of polyP using standard solutions and current protocols. Here we report a direct fluorescence based DAPI assay system which removes the requirement for prior polyP extraction before quantification. This increased the efficiency of polyP detection by 28-55% in microbial cultures suggesting quantitative measurement of the intracellular polyP pool. It provides a direct polyP assay which combines quantification capability with technical simplicity. This is an important step forward in our ability to explore the role of polyP in cellular biology and biogeochemical nutrient cycling.

  9. New Insights into the in situ Microscopic Visualization and Quantification of Inorganic Polyphosphate Stores by 4’,6-Diamidino-2-Phenylindole (DAPI)-Staining

    PubMed Central

    Gomes, F.M.; Ramos, I.B.; Wendt, C.; Girard-Dias, W.; De Souza, W.; Machado, E.A.; K. Miranda, E.A.

    2013-01-01

    Inorganic polyphosphate (PolyP) is a biological polymer that plays important roles in the cell physiology of both prokaryotic and eukaryotic organisms. Among the available methods for PolyP localization and quantification, a 4’,6-diamidino-2-phenylindole(DAPI)-based assay has been used for visualization of PolyP-rich organelles. Due to differences in DAPI permeability to different compartments and/or PolyP retention after fixation, a general protocol for DAPI-PolyP staining has not yet been established. Here, we tested different protocols for DAPI-PolyP detection in a range of samples with different levels of DAPI permeability, including subcellular fractions, free-living cells and cryosections of fixed tissues. Subcellular fractions of PolyP-rich organelles yielded DAPI-PolyP fluorescence, although those with a complex external layer usually required longer incubation times, previous aldehyde fixation and/or detergent permeabilization. DAPI-PolyP was also detected in cryosections of OCT-embedded tissues analyzed by multiphoton microscopy. In addition, a semi-quantitative fluorimetric analysis of DAPI-stained fractions showed PolyP mobilization in a similar fashion to what has been demonstrated with the use of enzyme-based quantitative protocols. Taken together, our results support the use of DAPI for both PolyP visualization and quantification, although specific steps are suggested as a general guideline for DAPI-PolyP staining in biological samples with different degrees of DAPI and PolyP permeability. PMID:24441187

  10. New insights into the in situ microscopic visualization and quantification of inorganic polyphosphate stores by 4',6-diamidino-2-phenylindole (DAPI)-staining.

    PubMed

    Gomes, F M; Ramos, I B; Wendt, C; Girard-Dias, W; De Souza, W; Machado, E A; Miranda, K

    2013-11-05

    Inorganic polyphosphate (PolyP) is a biological polymer that plays important roles in the cell physiology of both prokaryotic and eukaryotic organisms. Among the available methods for PolyP localization and quantification, a 4',6-diamidino-2-phenylindole(DAPI)-based assay has been used for visualization of PolyP-rich organelles. Due to differences in DAPI permeability to different compartments and/or PolyP retention after fixation, a general protocol for DAPI-PolyP staining has not yet been established. Here, we tested different protocols for DAPI-PolyP detection in a range of samples with different levels of DAPI permeability, including subcellular fractions, free-living cells and cryosections of fixed tissues. Subcellular fractions of PolyP-rich organelles yielded DAPI-PolyP fluorescence, although those with a complex external layer usually required longer incubation times, previous aldehyde fixation and/or detergent permeabilization. DAPI-PolyP was also detected in cryosections of OCT-embedded tissues analyzed by multi-photon microscopy. In addition, a semi-quantitative fluorimetric analysis of DAPI-stained fractions showed PolyP mobilization in a similar fashion to what has been demonstrated with the use of enzyme-based quantitative protocols. Taken together, our results support the use of DAPI for both PolyP visualization and quantification, although specific steps are suggested as a general guideline for DAPI-PolyP staining in biological samples with different degrees of DAPI and PolyP permeability.

  11. 4',6-Diamidino-2-phenylindole (DAPI) induces bundling of Escherichia coli FtsZ polymers inhibiting the GTPase activity.

    PubMed

    Nova, Esteban; Montecinos, Felipe; Brunet, Juan E; Lagos, Rosalba; Monasterio, Octavio

    2007-09-15

    FtsZ (Filamentous temperature sensitivity Z) cell division protein from Escherichia coli binds the fluorescence probe DAPI. Bundling of FtsZ was facilitated in the presence of DAPI, and the polymers in solution remained polymerized longer time than the protofilaments formed in the absence of DAPI. DAPI decreased both the maximal velocity of the GTPase activity and the Michaelis-Menten constant for GTP, indicating that behaves like an uncompetitive inhibitor of the GTPase activity favoring the GTP form of FtsZ in the polymers. The results presented in this work support a cooperative polymerization mechanism in which the binding of DAPI favors protofilament lateral interactions and the stability of the resulting polymers.

  12. Phosphorescence and optically detected magnetic resonance of 4',6-diamidino-2-phenylindole (DAPI) and its complexes with [d(CGACGTCG)]2 and [d(GGCCAATTGG)]2.

    PubMed

    Misra, Ajay; Ozarowski, Andrzej; Maki, August H

    2002-05-21

    Phosphorescence and optical detection of magnetic resonance (ODMR) is used to study the excited triplet state of 4',6-diamidino-2-phenyl indole (DAPI) and its complexes with the oligonucleotides [d(CGACGTCG)](2) and [d(GGCCAATTGG)](2), where binding occurs by intercalation between GC base pairs and by minor groove insertion, respectively. Weaker binding of DAPI to phosphate is also detected, and the triplet state of this complex is characterized. Intercalation with [d(CGACGTCG)](2) produces a phosphorescence redshift, while groove binding with [d(GGCCAATTGG)](2) leads to a blueshift. Both binding modes give rise to a small decrease in the zero-field splitting (zfs) of the DAPI triplet state. The largest redshift and zfs decrease are found for the phosphate complex. The phosphorescence lifetimes are shorter by an order of magnitude than that of indole or tryptophan as expected for the lower triplet state energy, E(00), of DAPI. The lifetimes agree well with a correlation with E(00) introduced by Siebrand [Siebrand, W. (1966) J. Chem. Phys. 44, 4055-4057] except for the [d(GGCCAATTGG)](2) minor groove complex with a lifetime that is about 20% too long. The longer lifetime is attributed to distortion of the amidino groups in this complex, resulting in less efficient intersystem crossing.

  13. Comparison of 4', 6'-diamidino-2-phenylindole and Giemsa stainings in preimplantation mouse embryos micronucleus assay including a triple dose study.

    PubMed

    Tian, Ying; Shen, Li; Gao, Yu; Yamauchi, Toru; Shen, Xiao-ming; Ma, Ning

    2007-04-01

    Analysis of micronuclei (MN) in preimplantation embryos is a good method for the evaluation of cytogenetic damage induced by occupational and environmental mutagen during early pregnancy. To examine whether conventional Giemsa staining produced the same accuracy of micronuclei as the DNA-specific 4', 6'-diamidino-2-phenylindole (DAPI) staining in preimplantation embryo induced by maternal exposure to chlorpyrifos, we conducted assays on 469 mouse (3 groups) preimplantation embryos micronucleus. Slides were stained with DAPI. After DAPI staining, the slides were de-stained and restained with Giemsa. Giemsa staining showed similar frequencies in MN to DNA-specific DAPI staining in all three groups. Both staining techniques revealed significant increases in frequency of MN in the treated group in comparison to the control group. Both methods showed a statistically significant correlation between MN frequency and the dose of chlorpyrifos. Compared with DAPI staining, the sensitivity of Giemsa staining was 85.0%, 86.0% and 90.9% for control, 40 mg/kg, and 80 mg/kg chlorpyrifos treated group, respectively. The specificity was 97.9%, 91.4% and 96.5% for control, 40 mg/kg, and 80 mg/kg chlorpyrifos treated group, respectively. Thus, we recommend that Giemsa staining technique be a standard staining method in detecting MN of preimplantation embryos induced by occupational or environmental hazards.

  14. DAPI derivative: a fluorescent DNA dye that can be covalently attached to biomolecules.

    PubMed

    Li, Min; Wu, Robert S; Tsai, Jane S C

    2003-12-15

    The preparation of a DAPI (4',6-diamidino-2-phenylindole) derivative is described. The resulting derivative retains the fluorogenic property upon binding to double-stranded DNA. Its ability for bioconjugation through amide linkage is demonstrated.

  15. DAPI fluorescence in nuclei isolated from tumors.

    PubMed

    Krishan, Awtar; Dandekar, Payal D

    2005-08-01

    In DNA histograms of some human solid tumors stained with nuclear isolation medium--4,6-diamidino-2-phenylindole dihydrochloride (NIM-DAPI), the coefficient of variation (CV) of the G0/G1 peak was broad, and in nuclear volume vs DNA scattergrams, a prominent slope was seen. To determine the cause for this, nuclei from frozen breast tumors were stained with NIM-DAPI and analyzed after dilution or resuspension in PBS. In two-color (blue vs red) analysis, most of the slope and broad CV was due to red fluorescence of nuclei stained with NIM-DAPI, which was reduced on dilution or resuspension in PBS, resulting in elimination of the slope and tightening of the CV.

  16. Dynamics of polyphosphate-accumulating bacteria in wastewater treatment plant microbial communities detected via DAPI (4',6'-diamidino-2-phenylindole) and tetracycline labeling.

    PubMed

    Günther, S; Trutnau, M; Kleinsteuber, S; Hause, G; Bley, T; Röske, I; Harms, H; Müller, S

    2009-04-01

    Wastewater treatment plants with enhanced biological phosphorus removal represent a state-of-the-art technology. Nevertheless, the process of phosphate removal is prone to occasional failure. One reason is the lack of knowledge about the structure and function of the bacterial communities involved. Most of the bacteria are still not cultivable, and their functions during the wastewater treatment process are therefore unknown or subject of speculation. Here, flow cytometry was used to identify bacteria capable of polyphosphate accumulation within highly diverse communities. A novel fluorescent staining technique for the quantitative detection of polyphosphate granules on the cellular level was developed. It uses the bright green fluorescence of the antibiotic tetracycline when it complexes the divalent cations acting as a countercharge in polyphosphate granules. The dynamics of cellular DNA contents and cell sizes as growth indicators were determined in parallel to detect the most active polyphosphate-accumulating individuals/subcommunities and to determine their phylogenetic affiliation upon cell sorting. Phylotypes known as polyphosphate-accumulating organisms, such as a "Candidatus Accumulibacter"-like phylotype, were found, as well as members of the genera Pseudomonas and Tetrasphaera. The new method allows fast and convenient monitoring of the growth and polyphosphate accumulation dynamics of not-yet-cultivated bacteria in wastewater bacterial communities.

  17. Analysis of DAPI and SYBR Green I as Alternatives to Ethidium Bromide for Nucleic Acid Staining in Agarose Gel Electrophoresis

    NASA Astrophysics Data System (ADS)

    Bourzac, Kevin M.; Lavine, Lori J.; Rice, Margaret S.

    2003-11-01

    DNA electrophoresis and staining is a common procedure in biochemistry laboratories, but the use of ethidium bromide (EB) for DNA detection is worrisome as EB is a mutagen and probable carcinogen. Five alternative stains were evaluated for DNA detection, safety, cost, and ease of use: BlueView, methylene blue, Carolina Blu, DAPI (4',6-diamidino-2-phenylindole dihydrochloride:hydrate), and SYBR Green I. BlueView, Carolina Blu, and methylene blue are not sensitive enough to detect the microgram amounts of DNA used in many procedures. However, DAPI and SYBR Green I are good staining alternatives to ethidium bromide in that they have similar sensitivity and are both easy to use. SYBR Green I is more expensive than EB or DAPI; however, the limited safety data suggest that SYBR Green I is the safest stain.

  18. DAPI Staining of Drosophila Embryos.

    PubMed

    Rothwell, Wendy F; Sullivan, William

    2007-10-01

    INTRODUCTIONDrosophila embryos can be stained with specific fluorescent probes or antibodies through either direct or indirect immunofluorescence. In particular, several effective probes exist for visualizing DNA. 4',6-diamidino-2-phenylindole (DAPI) is a commonly used DNA-binding dye. Because it is specific for double-stranded DNA, no prior RNase treatment is required. While the embryo staining method described here uses DAPI, other fluorescent DNA probes can be processed similarly.

  19. The hazards of DAPI photoconversion: effects of dye, mounting media and fixative, and how to minimize the problem.

    PubMed

    Jež, Mojca; Bas, Tuba; Veber, Matija; Košir, Andrej; Dominko, Tanja; Page, Raymond; Rožman, Primož

    2013-01-01

    Immunocytochemistry is a powerful tool for detection and visualization of specific molecules in living or fixed cells, their localization and their relative abundance. One of the most commonly used fluorescent DNA dyes in immunocytochemistry applications is 4',6-diamidino-2-phenylindole dihydrochloride, known as DAPI. DAPI binds strongly to DNA and is used extensively for visualizing cell nuclei. It is excited by UV light and emits characteristic blue fluorescence. Here, we report a phenomenon based on an apparent photoconversion of DAPI that results in detection of a DAPI signal using a standard filter set for detection of green emission due to blue excitation. When a sample stained with DAPI only was first imaged with the green filter set (FITC/GFP), only a weak cytoplasmic autofluorescence was observed. Next, we imaged the sample with a DAPI filter set, obtaining a strong nuclear DAPI signal as expected. Upon reimaging the same samples with a FITC/GFP filter set, robust nuclear fluorescence was observed. We conclude that excitation with UV results in a photoconversion of DAPI that leads to detection of DAPI due to excitation and emission in the FITC/GFP channel. This phenomenon can affect data interpretation and lead to false-positive results when used together with fluorochrome-labeled nuclear proteins detected with blue excitation and green emission. In order to avoid misinterpretations, extra precaution should be taken to prepare staining solutions with low DAPI concentration and DAPI (UV excitation) images should be acquired after all other higher wavelength images. Of various DNA dyes tested, Hoechst 33342 exhibited the lowest photoconversion while that for DAPI and Hoechst 33258 was much stronger. Different fixation methods did not substantially affect the strength of photoconversion. We also suggest avoiding the use of mounting medium with high glycerol concentrations since glycerol showed the strongest impact on photoconversion. This photoconversion

  20. DAPI: a DNA-specific fluorescent probe.

    PubMed

    Kapuscinski, J

    1995-09-01

    DAPI (4',6-diamidino-2-phenylindole) is a DNA-specific probe which forms a fluorescent complex by attaching in the minor grove of A-T rich sequences of DNA. It also forms nonfluorescent intercalative complexes with double-stranded nucleic acids. The physicochemical properties of the dye and its complexes with nucleic acids and history of the development of this dye as a biological stain are described. The application of DAPI as a DNA-specific probe for flow cytometry, chromosome staining, DNA visualization and quantitation in histochemistry and biochemistry is reviewed. The mechanisms of DAPI-nucleic acid complex formation including minor groove binding, intercalation and condensation are discussed.

  1. Labeling nuclear DNA using DAPI.

    PubMed

    Chazotte, Brad

    2011-01-01

    A number of fluorescent stains are available that label DNA and allow easy visualization of the nucleus in interphase cells and chromosomes in mitotic cells, including Hoechst, 4',6-diamidino-2-phenylindole (DAPI), ethidium bromide, propidium iodide, and acridine orange. Although not as bright as the vital Hoechst stains for DNA, DAPI has greater photostability. It is believed that DAPI associates with the minor groove of double-stranded DNA, with a preference for the adenine-thymine clusters. Cells must be permeabilized and/or fixed for DAPI to enter the cell and to bind DNA. Fluorescence increases approximately 20-fold when DAPI is bound to double-stranded DNA. This protocol describes the use of DAPI to label nuclear DNA of cells grown in culture.

  2. DAPI staining and fluorescence microscopy techniques for phytoplasmas.

    PubMed

    Andrade, Nancy M; Arismendi, Nolberto L

    2013-01-01

    The 4',6-diamidino-2-phenylindole (DAPI) stain technique is a simple method that was developed for confirming the presence of phytoplasmas in hand-cut or freezing microtome sections of infected tissues. DAPI binds AT-rich DNA preferentially, so that phytoplasmas, localized among phloem cells, can be visualized in a fluorescence microscope. The procedure is quick, easy to use, inexpensive, and can be used as a preliminary or quantitative method to detect or quantify phytoplasma-like bodies in infected plants.

  3. Effects of N-methyl pyrrolidone on the uptake of hypericin in human bladder carcinoma and co-staining with DAPI investigated by confocal microscopy.

    PubMed

    Saw, Constance Lay Lay; Olivo, Malini; Wohland, Thorsten; Fu, Chit Yaw; Kho, Kiang Wei; Soo, Khee Chee; Sia Heng, Paul Wan

    2007-10-01

    Photodynamic diagnosis (PDD) using hypericin (HY), a natural photosensitizer, detects bladder cancer significantly better than white light endoscopy. However, the lipophilicity of HY complicates its administration for clinical applications. Currently, pharmaceutical preparations for HY without plasma protein are being developed. Formulations containing a biocompatible solvent, N-methyl pyrrolidone (NMP) have been shown to enhance the photodynamic therapeutic effects of HY. It was recently reported that, NMP formulations of HY were able to produce significantly higher contrast for fluorescence detection of tumors than albumin-containing HY formulations. This present work hypothesizes that NMP acts both as a solvent and penetration enhancer to improve the delivery of HY into cells by increasing the permeability of cell membranes. This paper reports the use of 3-D confocal microscopy to monitor real-time uptake of HY in human carcinoma. 3-D confocal microscopy was used to investigate the possibility of nuclear localization of HY in MGH cells. The fluorescence of HY was confirmed to be emitted from HY containing cells using spectrometry. The localization of a DNA fluorescent probe 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) was used to confirm the possibility of colocalization of DAPI and HY. The colocalization analysis in the present study suggests that it was very unlikely that HY colocalized in the nucleus that was stained by DAPI. Fluorescein leakage tests showed that 1% NMP changes the permeability of cell membranes, and enhanced the delivery of HY into cells resulting in lower cell survival ratios. Thus, NMP was able to enhance the photodynamic therapeutic effects of HY on cancer cells.

  4. Two-photon excited surface plasmon enhanced energy transfer between DAPI and gold nanoparticles: Opportunities in intra-cellular imaging and sensing

    NASA Astrophysics Data System (ADS)

    Zhang, Yinan; Birch, David J. S.; Chen, Yu

    2011-09-01

    We have demonstrated energy transfer between 4'-6-Diamidino-2-phenylindole (DAPI), a commonly used DNA label, and gold nanoparticles under two-photon excitation in solution using fluorescence lifetime imaging microscopy (FLIM). With comparable size and concentration, gold nanorods (GNRs) are shown to provide more efficient energy transfer than gold nanospheres (GNSs). We attribute this transfer enhancement effect to the longitudinal surface plasmon mode of GNRs overlapping with the excitation wavelength. Energy transfer under two-photon excitation between GNRs and DAPI has also been observed in cell culture and found to be in accord with the solution phase results.

  5. [Chromosome CPD(PI/DAPI)- and CMA/DAPI-banding patterns in Allium cepa L].

    PubMed

    Kim, E S; Punina, E O; Rodionov, A V

    2002-04-01

    Chromosome banding patterns of Allium cepa L. were obtained by using fluorochrome combinations chromomycin A3 (CMA) + 4',6-diamidino-2-phenylindole (DAPI), DAPI + actinomycin D (AMD) and propidium iodide (PI) + DAPI. In A. cepa, telomeric heterochromatin displayed dull fluorescence after staining with DAPI and DAPI/AMD. After staining with the GC-specific CMA and AT-specific DAPI, the CMA-positive fluorescence of the NOR region and the telomeric bands of C-heterochromatin was observed. In combination with DAPI, PI, a dye with low AT/GC specificity, produced almost uniform fluorescence of chromosomal arms and heterochromatin, whereas the NOR-adjoining regions displayed bright fluorescence. Denaturation of chromosomal DNA (95 degrees C for 1-3 min) followed by renaturation in the 2 x SSC buffer (37 degrees C, 12 h) altered the chromosome fluorescence patterns: specific PI-positive bands appeared and the contrast of CMA-banding increased. Bright fluorescence of the NOR and adjoining regions was also observed in the case. Three-minute denaturation led also to a bright PI-positive fluorescence of telomeric heterochromatin. The denaturation of chromosomal DNA before staining results in changes of the DAPI fluorescence pattern and in the appearance of DAPI fluorescence in GR-rich NOP regions. The mechanisms underlying the effects of denaturation/renaturation procedures on chromosome banding patterns obtained with different fluorochromes are discussed.

  6. Excited-state solvation and proton transfer dynamics of DAPI in biomimetics and genomic DNA.

    PubMed

    Banerjee, Debapriya; Pal, Samir Kumar

    2008-08-14

    The fluorescent probe DAPI (4',6-diamidino-2-phenylindole) is an efficient DNA binder. Studies on the DAPI-DNA complexes show that the probe exhibits a wide variety of interactions of different strengths and specificities with DNA. Recently the probe has been used to report the environmental dynamics of a DNA minor groove. However, the use of the probe as a solvation reporter in restricted environments is not straightforward. This is due to the presence of two competing relaxation processes (intramolecular proton transfer and solvation stabilization) in the excited state, which can lead to erroneous interpretation of the observed excited-state dynamics. In this study, the possibility of using DAPI to unambiguously report the environmental dynamics in restricted environments including DNA is explored. The dynamics of the probe is studied in bulk solvents, biomimetics like micelles and reverse micelles, and genomic DNA using steady-state and picosecond-resolved fluorescence spectroscopies.

  7. Photoconversion of DAPI following UV or violet excitation can cause DAPI to fluoresce with blue or cyan excitation.

    PubMed

    Piterburg, M; Panet, H; Weiss, A

    2012-04-01

    4'-6-Diamidino-2-phenylindole is a fluorescent dye commonly used to visualize deoxyribonucleic acid or cell nuclei in fixed cell preparations, and is often used together with fluorescein or green fluorescent protein, which can be excited without exciting 4'-6-Diamidino-2-phenylindole. It is assumed that when using typical fluorescein or green fluorescent protein filter cubes, 4'-6-Diamidino-2-phenylindole will not be observed. In this paper, we show that following observation of 4'-6-Diamidino-2-phenylindole using UV or violet excitation, it may become sensitive to the blue/cyan excitation used in fluorescein/green fluorescent protein filter cubes. This has serious implications for the use of 4'-6-Diamidino-2-phenylindole together with widely used green fluorophores in double labelling experiments.

  8. Quantification of DNA synthesis in multicellular organisms by a combined DAPI and BrdU technique.

    PubMed

    Knobloch, Jürgen; Kunz, Werner; Grevelding, Christoph G

    2002-12-01

    The development of a novel method to detect and quantify mitotic activity in multicellular organisms is reported. The method is based on the combinatorial use of 4',6-diamidino-2-phenylindole (DAPI) as a dye for the specific staining of DNA and the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) as a marker for DNA synthesis. It is shown that on nitrocellulose filters, the amount of DNA can be determined by DAPI as a prerequisite for the subsequent quantification of mitotic activity by BrdU. As a model system to prove the applicability of this technique, the blood fluke Schistosoma mansoni has been used. It is demonstrated that the DNA synthesis rate is higher in adult female schistosomes than in adult males. Furthermore, dimethyl sulfoxide, a widely used solvent for many mitogens and inhibitors of mitosis, has no influence on mitotic activity in adult schistosomes.

  9. The meaning of DAPI bands observed after C-banding and FISH procedures.

    PubMed

    Barros e Silva, A E; Guerra, M

    2010-04-01

    Under specific technical conditions chromosome staining with 4',6-diamidino-2-phenylindole (DAPI) permits characterization of heterochromatic regions as AT-rich (DAPI(+)) or AT-poor (DAPI(-)), especially when the chromosomes are counterstained with chromomycin A(3) (CMA), which preferentially binds to GC-rich DNA. DAPI(+) bands also often have been observed after C-banding or FISH. In these cases, however, it is not clear whether only AT-rich regions stain positively with DAPI or other heterochromatins with different base compositions also are stained. We evaluated the meaning of DAPI bands observed after C-banding and FISH using three plant species bearing different types of heterochromatin: DAPI(+)/CMA(-), DAP(-)/CMA(+) and DAPI(0)/CMA(0) (neutral bands). Additional tests were performed using propidium iodide, a fluorochrome without preferential affinity for AT or GC. Our results indicate that AT-rich heterochromatin stains as DAPI(+) bands after C-banding or FISH, but other kinds of heterochromatin also may be stained by DAPI.

  10. DAPI diffusion after intravitreal injection of mesenchymal stem cells in the injured retina of rats.

    PubMed

    Castanheira, Paula; Torquetti, Leonardo Torquetti; Magalhãs, Débora Rodrigues Soares; Nehemy, Marcio B; Goes, Alfredo M

    2009-01-01

    To evaluate DAPI (4',6-diamidino-2-phenylindole) as a nuclear tracer of stem cell migration and incorporation it was observed the pattern of retinal integration and differentiation of mesenchymal stem cells (MSCs) injected into the vitreous cavity of rat eyes with retinal injury. For this purpose adult rat retinas were submitted to laser damage followed by transplantation of DAPI-labeled BM-MSCs grafts and double-labeled DAPI and quantum dot-labeled BM-MSCs. To assess a possible DAPI diffusion as well as the integration and differentiation of DAPI-labeled BM-MSCs in laser-injured retina, host retinas were evaluated 8 weeks after injury/transplantation. It was demonstrated that, 8 weeks after the transplant, most of the retinal cells in all neural retinal presented nuclear DAPI labeling, specifically in the outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL). Meanwhile, at this point, most of the double-labeled BM-MSCs (DAPI and quantum dot) remained in the vitreous cavity and no retinal cells presented the quantum dot marker. Based on these evidences we concluded that DAPI diffused to adjacent retinal cells while the nanocrystals remained labeling only the transplanted BM-MSCs. Therefore, DAPI is not a useful marker for stem cells in vivo tracing experiments because the DAPI released from dying cells in moment of the transplant are taken up by host cells in the tissue.

  11. High sensitivity, quantitative measurements of polyphosphate using a new DAPI-based approach.

    PubMed

    Aschar-Sobbi, Roozbeh; Abramov, Andrey Y; Diao, Catherine; Kargacin, Margaret E; Kargacin, Gary J; French, Robert J; Pavlov, Evgeny

    2008-09-01

    Polyphosphate (poly-P) is an important metabolite and signaling molecule in prokaryotes and eukaryotes. DAPI (4',6-diamidino-2-phenylindole), a widely used fluorescent label for DNA, also interacts with polyphosphate. Binding of poly-P to DAPI, shifts its peak emission wavelength from 475 to 525 nm (excitation at 360 nm), allowing use of DAPI for detection of poly-P in vitro, and in live poly-P accumulating organisms. This approach, which relies on detection of a shift in fluorescence emission, allows use of DAPI only for qualitative detection of relatively high concentrations of poly-P, in the microg/ml range. Here, we report that long-wavelength excitation (> or = 400 nm) of the DAPI-poly-P complex provides a dramatic increase in the sensitivity of poly-P detection. Using excitation at 415 nm, fluorescence of the DAPI-poly-P complex can be detected at a higher wavelength (550 nm) for as little as 25 ng/ml of poly-P. Fluorescence emission from free DAPI and DAPI-DNA are minimal at this wavelength, making the DAPI-poly-P signal highly specific and essentially independent of the presence of DNA. In addition, we demonstrate the use of this protocol to measure the activity of poly-P hydrolyzing enzyme, polyphosphatase and demonstrate a similar signal from the mitochondrial region of cultured neurons.

  12. Karyotype of Zea luxurians and Z. mays subsp. mays using FISH/DAPI, and analysis of meiotic behavior of hybrids.

    PubMed

    González, Graciela E; Poggio, Lidia

    2011-01-01

    The karyotypes of Zea luxurians and a race of maize from northwestern Argentina are described and compared using 4′,6-diamidino-2-phenylindole (DAPI) banding and fluorescent in situ hybridization (FISH) to localize the 180 bp knobs. The meiotic behavior of the F₁ artificial hybrids Z. luxurians × maize is also analyzed to determine the genomic relationships between both species. Neocentromere activity at knobs in the meiosis of the hybrids is particularly discussed. The meiotic behavior and the high pollen sterility of the hybrid revealed genetical and (or) chromosomal divergences, leading to postzygotic reproductive isolation among their parents. Here, we propose that maize shows lower genomic affinity to Z. luxurians than to other species of the genus with 2n = 20.

  13. Characterization of human OCT1-mediated transport of DAPI as a fluorescent probe substrate.

    PubMed

    Yasujima, Tomoya; Ohta, Kinya; Inoue, Katsuhisa; Yuasa, Hiroaki

    2011-09-01

    The present study was conducted to assess the functional characteristics of human organic cation transporter 1 (hOCT1) for the transport of 4',6-diamidino-2-phenylindol (DAPI), a fluorescent compound that may be used as a probe substrate for rapid assays of its functionality. The specific uptake of DAPI by hOCT1 heterologously introduced into Madin-Darby canine kidney II cells by stable transfection was found to be, when assessed by DAPI-derived fluorescence intensity, rapid and saturable with a Michaelis constant of 8.94 µM, indicating that DAPI is a good substrate of hOCT1. The specific uptake of DAPI was insensitive to the membrane potential and extracellular pH, indicating a mode of operation different from that for typical cationic substrates such as tetraethylammonium (TEA), for which hOCT1 has been suggested to be driven by an inside-negative membrane potential and favor higher pH for optimal operation. However, many organic cations were found to inhibit the specific DAPI uptake with extents well correlated with those of inhibition of the specific uptake of [(14) C]TEA, indicating comparable performances of both substrates as probes in identifying inhibitors. Thus, DAPI can be an alternative probe substrate that enables fluorometric rapid assays of the functionality of hOCT1.

  14. Effects of the fluorescence dye DAPI on microtubule structure in vitro: formation of novel types of tubulin assembly products.

    PubMed

    Vater, W; Böhm, K J; Unger, E

    1993-02-01

    It has been found that the DNA fluorescence dye 4',6-diamidino-2-phenylindole (DAPI) is able to stain also microtubules. However, electron microscopy revealed that DAPI changed microtubule structure and induced the formation of a broad spectrum of polymorphic tubulin assembly products. Upon addition of DAPI to microtubules assembled from 10 to 15 mumol tubulin (molar DAPI/tubulin ratios of 10 to 40) in the presence of microtubule-associated proteins, most of the microtubules were decorated with additional protofilaments usually running parallel to the protofilaments of the microtubule wall (microtubule-protofilament complexes). When DAPI was already present during assembly, curved C- and S-shaped protofilament ribbons and microtubule-ribbon complexes with 6-shaped profiles were the most prominent products, beside microtubules. Additionally, protofilament bundles, some flat sheets, and hoops occurred. Electrophoresis revealed that DAPI lowered the amount of associated proteins, especially of tau-proteins, bound to the assembly products. Nevertheless, DAPI stimulated the assembly, enabled pure tubulin to assemble even at concentrations as low as 10 mumol, and stabilized the assembly products against cold. The microtubule-protofilament complexes, observed for the first time, are interpreted as the result of DAPI-induced protofilament linking as well as of activation of an additional tubulin-tubulin binding site which is possibly identical to that involved in the formation of microtubule doublets.

  15. Time-Resolved Fluorescence Spectroscopy and Imaging of DNA Labeled with DAPI and Hoechst 33342 Using Three-Photon Excitation

    PubMed Central

    Lakowicz, Joseph R.; Gryczynski, Ignacy; Malak, Henryk; Schrader, Martin; Engelhardt, Peter; Kano, Hiroski; Hell, Stefan W.

    1997-01-01

    We examined the fluorescence spectral properties of the DNA stains DAPI (4′,6-diamidino-2-phenylindole, hydrochloride) and Hoechst 33342 (bis-benzimide, or 2,5′-bi-1H-benzimidazole2′-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)) with two-photon (2hν) and three-photon (3hν) excitation using femtosecond pulses from a Ti:sapphire laser from 830 to 885 nm. The mode of excitation of DAPI bound to DNA changed from two-photon at 830 nm to three-photon at 885 nm. In contrast, Hoechst 33342 displayed only two-photon excitation from 830 to 885 nm. DAPI-DNA displayed the same emission spectra and decay times for 2hν and 3hν excitation. Hoechst 33342-DNA displayed the same intensity decay for excitation at 830 and 885 nm. Both probes displayed higher anisotropies for multiphoton excitation as compared to one-photon excitation with ultraviolet wavelengths, and DAPI-DNA displays a higher anisotropy for 3hν at 885 nm than for 2hν at 830 nm. We used 970-nm excitation of DAPI-stained chromosomes to obtain the first three-dimensional images with three-photon excitation. Three-photon excitation of DAPI-stained chromosomes at 970 nm was demonstrated by the power dependence in the fluorescence microscope. ImagesFIGURE 10FIGURE 11FIGURE 12 PMID:9017187

  16. Analysis of heterochromatin by combination of C-banding and CMA3 and DAPI staining in two fish species (Pimelodidae, Siluriformes).

    PubMed

    Swarça, Ana C; Fenocchio, Alberto S; Cestari, Marta M; Dias, Ana L

    2003-09-01

    The chromosomes of Steindachneridion sp. (2n = 56) and Rhamdia quelen (2n = 58) were analyzed by C-banding (CB) and Chromomycin A3 (CMA3) and 4,6-diamidino-2-phenylindole (DAPI) staining, separately and consecutively, in order to understand the role of base-specific fluorochrome treatment after CB. Both species' chromosomes shared common staining profiles as follows. CB with Giemsa (CBG) revealed weak heterochromatic blocks in the telomeric regions of some chromosomes and conspicuous bands on the short arms of one chromosome pair, where nucleolar organizer regions (NORs) were evidenced by silver-staining. Without CB pretreatment, the NORs were stained conspicuously with CMA3, but not with DAPI. The latter uniformly stained all chromosomes, but leaving the NORs pale. Combination of CMA3 or DAPI staining with CB showed distinctive fluorescent blocks in the NOR-bearing short arms of the single chromosome pair along with several bright fluorescent signals on other chromosomes, which were not evidenced by single CMA3 or DAPI staining. These results suggest a modification of chromatin structure by CB treatment, which may increase the stainability of CMA3 and DAPI.

  17. A new chromosome fluorescence banding technique combining DAPI staining with image analysis in plants.

    PubMed

    Liu, Jing Yu; She, Chao Wen; Hu, Zhong Li; Xiong, Zhi Yong; Liu, Li Hua; Song, Yun Chun

    2004-08-01

    In this study, a new chromosome fluorescence banding technique was developed in plants. The technique combined 4',6-diamidino-2-phenylindole (DAPI) staining with software analysis including three-dimensional imaging after deconvolution. Clear multiple and adjacent DAPI bands like G-bands were obtained by this technique in the tested species including Hordeum vulgare L., Oryza officinalis, Wall & Watt, Triticum aestivum L., Lilium brownii, Brown, and Vicia faba L. During mitotic metaphase, the numbers of bands for the haploid genomes of these species were about 185, 141, 309, 456 and 194, respectively. Reproducibility analysis demonstrated that banding patterns within a species were stable at the same mitotic stage and they could be used for identifying specific chromosomes and chromosome regions. The band number fluctuated: the earlier the mitotic stage, the greater the number of bands. The technique enables genes to be mapped onto specific band regions of the chromosomes by only one fluorescence in situ hybridisation (FISH) step with no chemical banding treatments. In this study, the 45S and 5S rDNAs of some tested species were located on specific band regions of specific chromosomes and they were all positioned at the interbands with the new technique. Because no chemical banding treatment was used, the banding patterns displayed by the technique should reflect the natural conformational features of chromatin. Thus it could be expected that this technique should be suitable for all eukaryotes and would have widespread utility in chromosomal structure analysis and physical mapping of genes.

  18. Evidence for DAPI intercalation in CG sites of DNA oligomer [d(CGACGTCG)]2: a 1H NMR study.

    PubMed Central

    Trotta, E; D'Ambrosio, E; Ravagnan, G; Paci, M

    1995-01-01

    The interaction between 4',6-diamidino-2-phenylindole (DAPI) and the DNA oligomer [d(CGACGTCG)]2 has been investigated by proton one- and two-dimensional NMR spectroscopy in solution. Compared with the minor groove binding of the drug to [d(GCGATCGC)]2, previously studied by NMR spectroscopy, the interaction of DAPI with [d(CGACGTCG)]2 appears markedly different and gives results typical of a binding mechanism by intercalation. C:G imino proton signals of the [d(CGACGTCG)]2 oligomer as well as DAPI resonances appear strongly upfield shifted and sequential dipolar connectivities between cytosine and guanine residues show a clear decrease upon binding. Moreover, protons lying in both the minor and major grooves of the DNA double helix appear involved in the interaction, as evidenced principally by intermolecular drug-DNA NOEs. In particular, the results indicate the existence of two stereochemically non-equivalent intercalation binding sites located in the central and terminal adjacent C:G base pairs of the palindromic DNA sequence. Different lifetimes of the complexes were also observed for the two sites of binding. Moreover, due to the fast exchange on the NMR timescale between free and bound species, different interactions in dynamic equilibrium with the observed intercalative bindings were not excluded. PMID:7753623

  19. Solution structure of DAPI selectively bound in the minor groove of a DNA T.T mismatch-containing site: NMR and molecular dynamics studies.

    PubMed Central

    Trotta, E; Paci, M

    1998-01-01

    The solution structure of the complex between 4', 6-diamidino-2-phenylindole (DAPI) and DNA oligomer [d(GCGATTCGC)]2, containing a central T.T mismatch, has been characterized by combined use of proton one- and two-dimensional NMR spectroscopy, molecular mechanics and molecular dynamics computations including relaxation matrix refinement. The results show that the DAPI molecule binds in the minor groove of the central region 5'-ATT-3' of the DNA oligomer, which predominantly adopts a duplex structure with a global right-handed B-like conformation. In the final models of the complex, the DAPI molecule is located nearly isohelical with its NH indole proton oriented towards the DNA helix axis and forming a bifurcated hydrogen bond with the carbonyl O2 groups of a mismatched T5 and the T6 residue of the opposite strand. Mismatched thymines adopt a wobble base pair conformation and are found stacked between the flanking base pairs, inducing only minor local conformational changes in global duplex structure. In addition, no other binding mechanisms were observed, showing that minor groove binding of DAPI to the mismatch-containing site is favoured in comparison with any other previously reported interaction with G.C sequences. PMID:9753740

  20. Fluorescence anisotropy of DNA/DAPI complex: torsional dynamics and geometry of the complex.

    PubMed Central

    Barcellona, M L; Gratton, E

    1996-01-01

    Fluorescence depolarization of synthetic polydeoxynucleotide/4'-6-diamidino-2-phenylindole dihydrochloride complexes has been investigated as a function of dye/polymer coverage. At low coverage, fluorescence depolarization is due to local torsional motions of the DNA segment where the dye resides. At relatively high coverage, fluorescence depolarization is dominated by energy transfer to other dye molecules along the DNA. The extent of the observed depolarization due to torsional motion depends on the angle the dye molecule forms with the DNA helical axis. A large torsional motion and a small angle produce the same depolarization as a small torsional motion and a large projection angle. Furthermore, the extent of transfer critically depends on the relative orientation of dye molecules along the DNA. The effect of multiple transfer is examined using a Monte Carlo approach. The measurement of depolarization with transfer, at high coverage, allows determination of the dye orientation about the DNA helical axis. The value of the torsional spring constant is then determined, at very low coverage, for few selected polydeoxynucleotides. Images FIGURE 3 PMID:9172758

  1. An investigation of the photophysical properties of minor groove bound and intercalated DAPI through quantum-mechanical and spectroscopic tools.

    PubMed

    Biancardi, Alessandro; Biver, Tarita; Secco, Fernando; Mennucci, Benedetta

    2013-04-07

    The fluorescent probe 4',6-diamidino-2-phenylindole (DAPI) is a dye known to interact with polynucleotides in a non-univocal manner, both intercalation and minor groove binding modes being possible, and to specifically change its photophysical properties according to the different environments. To investigate this behavior, quantum-mechanical calculations using time-dependent density functional theory (TDDFT), coupled with polarizable continuum and/or atomistic models, were performed in combination with spectroscopic measurements of the probe in the different environments, ranging from a homogeneous solution to the minor groove or intercalation pockets of double stranded nucleic acids. According to our simulation, the electronic transition involves a displacement of the electron charge towards the external amidine groups and this feature makes the absorption energies very environment-sensitive while a much smaller sensitivity is seen in the fluorescence energies. Moreover, the calculations show that the DAPI molecule, when minor groove bound to the nucleic acid, presents both a reduced geometrical flexibility because of the rigid DNA pocket and a reduced polarization due to the very "apolar" microenvironment. All these effects can be used to better understand the observed enhancement of the fluorescence, which makes it an excellent marker for DNA.

  2. Quantification of sPLA2-induced early and late apoptosis changes in neuronal cell cultures using combined TUNEL and DAPI staining.

    PubMed

    Daniel, Bron; DeCoster, Mark A

    2004-08-01

    The terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) stain is in wide use for measuring apoptosis in neurons, as well as in other cell types. TUNEL may give false positive results due to variations in labeling technique as well as staining of cells that have undergone non-apoptotic DNA strand breaks. Therefore, in isolation, TUNEL is not a certain indicator of apoptosis. Recently, we have demonstrated the potent apoptotic effect of secreted phospholipase A2 from group III (sPLA2-III) on primary cortical neurons from rat. Here we describe a computer-assisted method for quantifying TUNEL-positive neurons after sPLA2-III induced apoptosis. Extent of TUNEL is normalized to total nuclear content using 4',6-diamidino-2-phenylindole (DAPI) staining. Furthermore, DAPI counterstaining allows for determination of a nuclear morphology indicator, based on nuclear size and roundness, which we call the nuclear area factor. We found that the nuclear area factor is an early indicator of cell death (significant after 4 h post treatment), while TUNEL staining is significant at later times (26 h). Thus, the independent staining techniques using TUNEL and DAPI complement each other, and with commercially available image analysis software, may be used to indicate early as well as delayed cell injury processes.

  3. Dynamics in the DNA recognition by DAPI: exploration of the various binding modes.

    PubMed

    Banerjee, Debapriya; Pal, Samir Kumar

    2008-01-24

    Two distinct modes of interaction of the fluorescent probe 4',6-diamidino-2-phenylindole (DAPI), depending on the sequence of DNA, have been reported in the literature. In the present study, the dynamics of solvation has been utilized to explore the binding interaction of DAPI to DNA oligomers of different sequences. Picosecond-resolved fluorescence and polarization-gated anisotropy have been used to characterize the binding of DAPI to the different oligomers. In the double-stranded dodecamer of sequence CGCGAATTCGCG (oligo1), the solvation relaxation dynamics of the probe reveals time constants of 0.130 ns (75%) and 2.35 ns (25%). Independent exploration of the minor-groove environment of oligo1 using another well-known minor-groove binder Hoechst 33258 (H258) shows similar timescales, further confirming minor-groove binding of DAPI to oligo1. In the double-stranded dodecamer (oligo2) having the sequence GCGCGCGCGCGC, where intercalation has been reported in the literature, no solvation is observed in our experimental window. DAPI bound to oligo2 shows quenching of fluorescence compared to that of DAPI in a buffer. The quenching of fluorescence of DAPI intercalated in DNA is also borne out by the appearance of a fast component of 30 ps in the fluorescence lifetime, revealing electron transfer to DAPI from GC base pairs, between which it intercalates. In addition to this, the excited-state lifetime of the probe in the DAPI-DNA complex also shows a time constant similar to that of the dye in a buffer, indicating that the excited-state photoprocesses associated with the free dye is also operative in this binding mode, consistent with the binding geometry of the DAPI in the DNA. The dynamics of DAPI in calf thymus DNA having a random sequence of base pairs is similar to that associated with the DNA minor groove. Our studies clearly explore the structure-dynamics correlation of the DAPI-DNA complex in the two distinct modes of interaction of DAPI with DNA.

  4. Effect of compartmentalization of donor and acceptor on the ultrafast resonance energy transfer from DAPI to silver nanoclusters

    NASA Astrophysics Data System (ADS)

    Prajapati, Roopali; Chatterjee, Surajit; Kannaujiya, Krishna K.; Mukherjee, Tushar Kanti

    2016-06-01

    The mechanism and dynamics of excitation energy transfer (EET) from photo-excited 4',6-diamidino-2-phenylindole (DAPI) to silver nanoclusters (Ag NCs) and its subsequent modulation in the presence of cationic polymer poly(diallyldimethylammonium chloride) (PDADMAC) and Calf Thymus DNA (CT-DNA) have been demonstrated using steady-state fluorescence and femtosecond fluorescence upconversion techniques. The synthesized Ag NCs were characterized using FTIR, mass spectrometry, XPS, HRTEM, DLS, UV-Vis and PL spectroscopy. Mass spectrometric analysis reveals the formation of ultrasmall Ag4 NCs with a small amount of Ag5 NCs. UV-Vis and PL spectra reveal distinct molecular-like optoelectronic behaviour of these ultrasmall Ag NCs. The dihydrolipoic acid-capped Ag NCs strongly quench the fluorescence of DAPI with concomitant increase in its photoluminescence (PL) intensity at 675 nm. This steady-state fluorescence quenching proceeds with a significant shortening of the fluorescence lifetime of DAPI in the presence of Ag NCs, signifying the nonradiative Förster resonance energy transfer (FRET) from DAPI to Ag NCs. Various energy transfer parameters have been estimated from FRET theory. The present FRET pair shows a characteristic Förster distance of 2.45 nm and can be utilized as a reporter of short-range distances in various FRET based applications. Moreover, this nonradiative FRET is completely suppressed in the presence of both 0.2 wt% PDADMAC and CT-DNA. Our results reveal selective compartmentalization of Ag NCs and DAPI in the presence of 0.2 wt% PDADMAC and CT-DNA, respectively. This selective compartmentalization of donor and acceptor and the subsequent modification of the FRET process may find application in various sensing, photovoltaic, and light harvesting applications.The mechanism and dynamics of excitation energy transfer (EET) from photo-excited 4',6-diamidino-2-phenylindole (DAPI) to silver nanoclusters (Ag NCs) and its subsequent modulation in the presence

  5. Cytogenetic analyses using C-banding and DAPI/CMA3 staining of four populations of the maize weevil Sitophilus zeamais Motschulsky, 1855 (Coleoptera, Curculionidae)

    PubMed Central

    da Silva, Alexandra A.; Braga, Lucas S.; Guedes, Raul Narciso C.; Tavares, Mara G.

    2015-01-01

    Abstract Cytogenetic data avalaible for the maize weevil Sitophilus zeamais Motschulsky, 1855 (Coleoptera: Curculionidae), one of the most destructive pests of stored cereal grains, are controversial. Earlier studies focused on single populations and emphasized chromosome number and sex determination system. In this paper, the karyotypes of four populations of Sitophilus zeamais were characterized by conventional staining, C-banding and sequential staining with the fluorochromes chromomycin-A3/4-6-diamidino-2-phenylindole (CMA3/DAPI). The analyses of metaphases obtained from the cerebral ganglia of last instar larvae and the testes of adults showed that the species had 2n = 22 chromosomes, with 10 autosomal pairs and a sex chromosome pair (XX in females and Xyp in males). Chromosome number, however, ranged from 2n = 22 to 26 due to the presence of 0–4 supernumerary chromosomes in individuals from the populations of Viçosa, Unai and Porto Alegre. With the exception of the Y chromosome, which was dot-like, all other chromosomes of this species were metacentric, including the supernumeraries. The heterochromatin was present in the centromeric regions of all autosomes and in the centromere of the X chromosome. The B chromosomes were partially or totally heterochromatic, and the Y chromosome was euchromatic. The heterochromatic regions were labeled with C-banding and DAPI, which showed that they were rich in AT base pairs. PMID:25893077

  6. Cytogenetic analyses using C-banding and DAPI/CMA3 staining of four populations of the maize weevil Sitophiluszeamais Motschulsky, 1855 (Coleoptera, Curculionidae).

    PubMed

    da Silva, Alexandra A; Braga, Lucas S; Guedes, Raul Narciso C; Tavares, Mara G

    2015-01-01

    Cytogenetic data avalaible for the maize weevil Sitophiluszeamais Motschulsky, 1855 (Coleoptera: Curculionidae), one of the most destructive pests of stored cereal grains, are controversial. Earlier studies focused on single populations and emphasized chromosome number and sex determination system. In this paper, the karyotypes of four populations of Sitophiluszeamais were characterized by conventional staining, C-banding and sequential staining with the fluorochromes chromomycin-A3/4-6-diamidino-2-phenylindole (CMA3/DAPI). The analyses of metaphases obtained from the cerebral ganglia of last instar larvae and the testes of adults showed that the species had 2n = 22 chromosomes, with 10 autosomal pairs and a sex chromosome pair (XX in females and Xyp in males). Chromosome number, however, ranged from 2n = 22 to 26 due to the presence of 0-4 supernumerary chromosomes in individuals from the populations of Viçosa, Unai and Porto Alegre. With the exception of the Y chromosome, which was dot-like, all other chromosomes of this species were metacentric, including the supernumeraries. The heterochromatin was present in the centromeric regions of all autosomes and in the centromere of the X chromosome. The B chromosomes were partially or totally heterochromatic, and the Y chromosome was euchromatic. The heterochromatic regions were labeled with C-banding and DAPI, which showed that they were rich in AT base pairs.

  7. Effect of compartmentalization of donor and acceptor on the ultrafast resonance energy transfer from DAPI to silver nanoclusters.

    PubMed

    Prajapati, Roopali; Chatterjee, Surajit; Kannaujiya, Krishna K; Mukherjee, Tushar Kanti

    2016-07-14

    The mechanism and dynamics of excitation energy transfer (EET) from photo-excited 4',6-diamidino-2-phenylindole (DAPI) to silver nanoclusters (Ag NCs) and its subsequent modulation in the presence of cationic polymer poly(diallyldimethylammonium chloride) (PDADMAC) and Calf Thymus DNA (CT-DNA) have been demonstrated using steady-state fluorescence and femtosecond fluorescence upconversion techniques. The synthesized Ag NCs were characterized using FTIR, mass spectrometry, XPS, HRTEM, DLS, UV-Vis and PL spectroscopy. Mass spectrometric analysis reveals the formation of ultrasmall Ag4 NCs with a small amount of Ag5 NCs. UV-Vis and PL spectra reveal distinct molecular-like optoelectronic behaviour of these ultrasmall Ag NCs. The dihydrolipoic acid-capped Ag NCs strongly quench the fluorescence of DAPI with concomitant increase in its photoluminescence (PL) intensity at 675 nm. This steady-state fluorescence quenching proceeds with a significant shortening of the fluorescence lifetime of DAPI in the presence of Ag NCs, signifying the nonradiative Förster resonance energy transfer (FRET) from DAPI to Ag NCs. Various energy transfer parameters have been estimated from FRET theory. The present FRET pair shows a characteristic Förster distance of 2.45 nm and can be utilized as a reporter of short-range distances in various FRET based applications. Moreover, this nonradiative FRET is completely suppressed in the presence of both 0.2 wt% PDADMAC and CT-DNA. Our results reveal selective compartmentalization of Ag NCs and DAPI in the presence of 0.2 wt% PDADMAC and CT-DNA, respectively. This selective compartmentalization of donor and acceptor and the subsequent modification of the FRET process may find application in various sensing, photovoltaic, and light harvesting applications.

  8. A simple and high-throughput method to assess maturation status of bovine oocytes: comparison of anti-lamin A/C-DAPI with an aceto-orcein staining technique.

    PubMed

    Prentice-Biensch, J R; Singh, J; Alfoteisy, B; Anzar, M

    2012-10-15

    A precise, accurate, nonambiguous and high-throughput method is required to assess nuclear maturation of mammalian oocytes. The objectives of this study were to compare the efficiency and ease of use of a simplified fluorescence imaging (anti-lamin A/C and 4',6-diamidino-2-phenylindole [DAPI]) technique to the existing technique (aceto-orcein staining) for the evaluation of nuclear maturation of bovine oocytes, and to determine the kinetics of bovine oocyte maturation using an anti-lamin A/C-DAPI technique. In Experiment 1, oocytes were matured in vitro and stained with aceto-orcein and anti-lamin A/C-DAPI staining techniques. The proportions of oocytes lost during procedures and those that could not be classified (because of ambiguous morphology) during evaluation were lower (P < 0.0001) in oocytes stained with anti-lamin A/C-DAPI (9% and 2%) than those stained with aceto-orcein (31% and 13%), respectively. Anti-lamin A/C-DAPI was a quick procedure which could be completed within 7 h after completion of the maturation (compared with > 24 h for the aceto-orcein method). Furthermore, > 200 oocytes could be stained in one batch with anti-lamin A/C-DAPI technique. In Experiment 2, nuclear maturation kinetics of bovine oocytes at various time intervals (0, 6, 12, and 22 h) during in vitro maturation (IVM) was evaluated using the anti-lamin A/C-DAPI technique. Germinal vesicle, germinal vesicle breakdown, metaphase I, and metaphase II oocytes were predominant at 0, 6, 12, and 22 h of IVM, respectively. We concluded that the anti-lamin A/C-DAPI was an efficient and simple technique for nonambiguous evaluation of nuclear maturation status of large numbers of oocytes in a short interval.

  9. Detection of immunomagnetically captured 4',6-diamidino-2-phenyl-indole (DAPI)-labeled Escherichia coli 0157:H7 by fluorescent microscopic imaging

    NASA Astrophysics Data System (ADS)

    Tu, Shu-I.; Uknalis, Joseph; Patterson, Deidre; Gehring, Andrew G.

    1999-01-01

    Live cells of E. coliO157:H7 were captured by goat anti-E. coliO157 serum coated on the surface of polystyrene based immunomagnetic beads (IMB). The captured bacteria were labeled by 4',6-diamidino-2-phenylindole (DAPI), a nucleic acid stain, for observation by epifluorescent microscopy. The beads with captured bacteria were then concentrated by magnetic separators. The efficiency of this magnetic concentration step was less than that of using high speed centrifugation. The antibody-captured and IMB-immobilized bacteria were then applied on HF-treated, bovine serum albumin (BSA)-coated microscope slides mounted on an automated stage, and magnetically aligned before fluorescence distribution was measured by a cooled CCD attached to an inverted microscope. Since the beads were concentrated and linearly aligned along the edge of the magnetic field, image capture along the edge for a few field widths was sufficient to account for most of captured bacteria. We applied this approach to determine the bacterial counts in spiked beef hamburger patties. The results showed that after a 6-hour enrichment, sufficient number of the bacteria could be detected from the samples spiked with 1 CFU of E. coliO157:H7 per gram of the hamburger.

  10. Use of lycorine and DAPI staining in Saccharomyces cerevisiae to differentiate between rho0 and rho- cells in a cce1/delta cce1 nuclear background.

    PubMed

    Massardo, D R; Zweifel, S G; Gunge, N; Miyakawa, I; Sando, N; Del Giudice, A; Wolf, K; Del Giudice, L

    2000-11-01

    In the yeast Saccharomyces cerevisiae, mutants are viable with large deletions (rho-), or even complete loss of the mitochondrial genome (rho0). One class of rho- mutants, which is called hypersuppressive, is characterised by a high transmission of the mutated mitochondrial genome to the diploid progeny when mated to a wild-type (rho+) haploid. The nuclear gene CCE1 encodes a cruciform cutting endonuclease, which is located in the mitochondrion and is responsible for the highly biased transmission of the hypersuppressive rho- genome. CCE1 is a Holliday junction specific endonuclease that resolves recombination intermediates in mitochondrial DNA. The cleavage activity shows a strong preference for cutting after a 5'-CT dinucleotide. In the absence of the CCE1 gene product, the mitochondrial genomes remain interconnected and have difficulty segregating to the daughter cells. As a consequence, there is an increase in the fraction of daughter cells that are rho0. In this paper we demonstrate the usefulness of lycorine, together with staining by 4',6-diamidino-2-phenylindole (DAPI), to assay for the mitotic stability of a variety of mitochondrial genomes. We have found that rho+ and rho- strains that contain CT sequences produce a large fraction of rho0 progeny in the absence of CCE1 activity. Only those rho- mitochondrial genomes lacking the CT recognition sequence are unaffected by the cce1 allele.

  11. RNA targeting by DNA binding drugs: structural, conformational and energetic aspects of the binding of quinacrine and DAPI to A-form and H(L)-form of poly(rC).poly(rG).

    PubMed

    Sinha, Rangana; Hossain, Maidul; Kumar, Gopinatha Suresh

    2007-12-01

    A key step in the rational design of new RNA binding small molecules necessitates a complete elucidation of the molecular aspects of the binding of existing molecules to RNA structures. This work focuses towards the understanding of the interaction of a DNA intercalator, quinacrine and a minor groove binder 4',6-diamidino-2-phenylindole (DAPI) with the right handed Watson-Crick base paired A-form and the left-handed Hoogsteen base paired H(L)-form of poly(rC).poly(rG) evaluated by multifaceted spectroscopic and viscometric techniques. The energetics of their interaction has also been elucidated by isothermal titration calorimetry. Results of this study converge to suggest that (i) quinacrine intercalates to both A-form and H(L)-form of poly(rC).poly(rG); (ii) DAPI shows both intercalative and groove-binding modes to the A-form of the RNA but binds by intercalative mode to the H(L)-form. Isothermal calorimetric patterns of quinacrine binding to both the forms of RNA and of DAPI binding to the H(L)-form are indicative of single binding while the binding of DAPI to the A-form reveals two kinds of binding. The binding of both the drugs to both conformations of RNA is exothermic; while the binding of quinacrine to both conformations and DAPI to the A-form (first site) is entropy driven, the binding of DAPI to the second site of A-form and H(L)-conformation is enthalpy driven. Temperature dependence of the binding enthalpy revealed that the RNA-ligand interaction reactions are accompanied by small heat capacity changes that are nonetheless significant. We conclude that the binding affinity characteristics and energetics of interaction of these DNA binding molecules to the RNA conformations are significantly different and may serve as data for the development of effective structure selective RNA-based antiviral drugs.

  12. Molecular analyses of dinosaur osteocytes support the presence of endogenous molecules.

    PubMed

    Schweitzer, Mary Higby; Zheng, Wenxia; Cleland, Timothy P; Bern, Marshall

    2013-01-01

    The discovery of soft, transparent microstructures in dinosaur bone consistent in morphology with osteocytes was controversial. We hypothesize that, if original, these microstructures will have molecular features in common with extant osteocytes. We present immunological and mass spectrometry evidence for preservation of proteins comprising extant osteocytes (Actin, Tubulin, PHEX, Histone H4) in osteocytes recovered from two non-avian dinosaurs. Furthermore, antibodies to DNA show localized binding to these microstructures, which also react positively with DNA intercalating stains propidium iodide (PI) and 4',6'-diamidino-2-phenylindole dihydrochloride (DAPI). Each antibody binds dinosaur cells in patterns similar to extant cells. These data are the first to support preservation of multiple proteins and to present multiple lines of evidence for material consistent with DNA in dinosaurs, supporting the hypothesis that these structures were part of the once living animals. We propose mechanisms for preservation of cells and component molecules, and discuss implications for dinosaurian cellular biology.

  13. Human colon cell culture models of different transformation stages to assess conjugated linoleic acid and conjugated linolenic acid metabolism: Challenges and chances.

    PubMed

    Degen, Christian; Habermann, Nina; Piegholdt, Stefanie; Glei, Michael; Jahreis, Gerhard

    2012-09-01

    Both cellular transformation status and cell culture conditions affect fatty acid metabolism. Hence, the incorporation and metabolism of c9,t11-CLA (conjugated linoleic acid) and other CFAs (conjugated fatty acids) were compared in colon cells (LT-97, adenoma; HT-29, adenocarcinoma). Growth inhibition by CFA in LT-97 cells was assessed via the DAPI (4',6-diamidino-2-phenylindole dihydrochloride) assay. Basal gene expression of desaturases (Δ5, Δ6 and Δ9) and elongases (1, 2, 5 and 6) was determined in LT-97 using PCR. Analysis of cellular fatty acids revealed a 2-fold higher incorporation of c9,t11-CLA (40 and 80μM) in HT-29 cells compared to LT-97 cells. The β-oxidized and elongated conjugated dienoic (CD) fatty acids differed by 8-fold (CD-C16:2/CD-C20:2; HT-29: 8:1; LT-97: 1:1). Notably, LT-97 cells were shown to convert conjugated linolenic acid (CLnA) to CLA. Moreover, LT-97 cells revealed no basal expression of elongase 2. CLnA caused stronger growth inhibition (≤80μM) compared to CLA (200μM). The results indicate that LT-97 cells represent a superior model to carry out elongation and desaturation studies of unsaturated and conjugated fatty acids compared to HT-29 cells. Nevertheless, further in-depth metabolic and transcriptomic analyses are required to confirm this suggestion.

  14. Hispolon from Phellinus linteus induces G0/G1 cell cycle arrest and apoptosis in NB4 human leukaemia cells.

    PubMed

    Chen, Yi-Chuan; Chang, Heng-Yuan; Deng, Jeng-Shyan; Chen, Jian-Jung; Huang, Shyh-Shyun; Lin, I-Hsin; Kuo, Wan-Lin; Chao, Wei; Huang, Guan-Jhong

    2013-01-01

    Hispolon (a phenolic compound isolated from Phellinus linteus) has been shown to possess strong antioxidant, anti-inflammatory, anticancer, and antidiabetic properties. In this study, we investigated the antiproliferative effect of hispolon on human hepatocellular carcinoma NB4 cells using the MTT assay, DNA fragmentation, DAPI (4, 6-diamidino-2-phenylindole dihydrochloride) staining, and flow cytometric analysis. Hispolon inhibited the cellular growth of NB4 cells in a dose-dependent manner through the induction of cell cycle arrest at G0/G1 phase measured using flow cytometric analysis and apoptotic cell death, as demonstrated by DNA laddering. Exposure of NB4 cells to hispolon-induced apoptosis-related protein expressions, such as the cleavage form of caspase 3, caspase 8, caspase 9, poly (ADP ribose) polymerase, and the proapoptotic Bax protein. Western blot analysis showed that the protein levels of extrinsic apoptotic proteins (Fas and FasL), intrinsic related proteins (cytochrome c), and the ratio of Bax/Bcl-2 were increased in NB4 cells after hispolon treatment. Hispolon-induced G0/G1-phase arrest was associated with a marked decrease in the protein expression of p53, cyclins D1, and cyclins E, and cyclin-dependent kinases (CDKs) 2, and 4, with concomitant induction of p21waf1/Cip1 and p27Kip1. We conclude that hispolon induces both of extrinsic and intrinsic apoptotic pathways in NB4 human leukemia cells in vitro.

  15. Biogenic terbium oxide nanoparticles as the vanguard against osteosarcoma

    NASA Astrophysics Data System (ADS)

    Iram, Sana; Khan, Salman; Ansary, Abu Ayoobul; Arshad, Mohd; Siddiqui, Sahabjada; Ahmad, Ejaz; Khan, Rizwan H.; Khan, Mohd Sajid

    2016-11-01

    The synthesis of inner transition metal nanoparticles via an ecofriendly route is quite difficult. This study, for the first time, reports synthesis of terbium oxide nanoparticles using fungus, Fusarium oxysporum. The biocompatible terbium oxide nanoparticles (Tb2O3 NPs) were synthesized by incubating Tb4O7 with the biomass of fungus F. oxysporum. Multiple physical characterization techniques, such as UV-visible and photoluminescence spectroscopy, TEM, SAED, and zeta-potential were used to confirm the synthesis, purity, optical and surface characteristics, crystallinity, size, shape, distribution, and stability of the nanoemulsion of Tb2O3 NPs. The Tb2O3 NPs were found to inhibit the propagation of MG-63 and Saos-2 cell-lines (IC50 value of 0.102 μg/mL) and remained non-toxic up to a concentration of 0.373 μg/mL toward primary osteoblasts. Cell viability decreased in a concentration-dependent manner upon exposure to 10 nm Tb2O3 NPs in the concentration range 0.023-0.373 μg/mL. Cell toxicity was evaluated by observing changes in cell morphology, cell viability, oxidative stress parameters, and FACS analysis. Morphological examinations of cells revealed cell shrinkage, nuclear condensation, and formation of apoptotic bodies. The level of ROS within the cells-an indicator of oxidative stress was significantly increased. The induction of apoptosis at concentrations ≤ IC50 was corroborated by 4‧,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining (DNA damage and nuclear fragmentation). Flow-cytometric studies indicated that the response was dose dependent with a threshold effect.

  16. Citric acid induces cell-cycle arrest and apoptosis of human immortalized keratinocyte cell line (HaCaT) via caspase- and mitochondrial-dependent signaling pathways.

    PubMed

    Ying, Tsung-Ho; Chen, Chia-Wei; Hsiao, Yu-Ping; Hung, Sung-Jen; Chung, Jing-Gung; Yang, Jen-Hung

    2013-10-01

    Citric acid is an alpha-hydroxyacid (AHA) widely used in cosmetic dermatology and skincare products. However, there is concern regarding its safety for the skin. In this study, we investigated the cytotoxic effects of citric acid on the human keratinocyte cell line HaCaT. HaCaT cells were treated with citric acid at 2.5-12.5 mM for different time periods. Cell-cycle arrest and apoptosis were investigated by 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining, flow cytometry, western blot and confocal microscopy. Citric acid not only inhibited proliferation of HaCaT cells in a dose-dependent manner, but also induced apoptosis and cell cycle-arrest at the G2/M phase (before 24 h) and S phase (after 24 h). Citric acid increased the level of Bcl-2-associated X protein (BAX) and reduced the levels of B-cell lymphoma-2 (BCL-2), B-cell lymphoma-extra large (BCL-XL) and activated caspase-9 and caspase-3, which subsequently induced apoptosis via caspase-dependent and caspase-independent pathways. Citric acid also activated death receptors and increased the levels of caspase-8, activated BH3 interacting-domain death agonist (BID) protein, Apoptosis-inducing factor (AIF), and Endonuclease G (EndoG). Therefore, citric acid induces apoptosis through the mitochondrial pathway in the human keratinocyte cell line HaCaT. The study results suggest that citric acid is cytotoxic to HaCaT cells via induction of apoptosis and cell-cycle arrest in vitro.

  17. Prokaryotes in salt marsh sediments of Ria de Aveiro: Effects of halophyte vegetation on abundance and diversity

    NASA Astrophysics Data System (ADS)

    Oliveira, Vanessa; Santos, Ana L.; Aguiar, Claúdia; Santos, Luisa; Salvador, Ângelo C.; Gomes, Newton C. M.; Silva, Helena; Rocha, Sílvia M.; Almeida, Adelaide; Cunha, Ângela

    2012-09-01

    The aim of this study was to investigate the influence of monospecific colonization of sediment stands by Spartina maritima or Halimione portulacoides on benthic prokaryote assemblages in a salt marsh located in Ria de Aveiro (Portugal). The distribution of Bacteria, Archaea and sulfate-reducing bacteria (SRB) in sediments with monospecific plant stands and in unvegetated sediments was characterized by Fluorescence In Situ Hybridization (FISH). Total prokaryote abundance (0.4 × 109-1.7 × 109 cells gdw-1) was highest in sediments from the surface layer. The domain Bacteria comprised approximately 40% of total prokaryote communities with the highest percentages occurring in the surface layer. Archaeal cells corresponded to an average of 25% of total prokaryote population, with higher abundance in the vegetation banks, and displaying homogeneous vertical distribution. The relative abundance of SRB represented approximately 3% of total 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) stained cells at unvegetated sediment and H. portulacoides stand and 7% at S. maritima stand. Headspace solid-phase microextraction (HS-SPME) combined with Comprehensive Two-Dimensional Gas Chromatography-Time-of-Flight Mass Spectrometry (GC × GC-ToFMS) was used to analyse the volatile and semi-volatile fraction of root exudates. A total of 171 compounds were identified and Principal Component Analysis showed a clear separation between the chemical composition (volatile and semi-volatile organic compounds) of the exudates of the two plants. The patterns of vertical distribution and differences in the proportion of SRB and Archaea in the prokaryote communities developing in sediments colonized by Spartina maritima or Halimione portulacoides suggest the existence of plant-specific interactions between halophyte vegetation and estuarine sediment bacteria in Ria de Aveiro salt marshes, exerted via sediment lithology and root-derived exudates.

  18. Characteristic Thickened Cell Walls of the Bracts of the ‘Eternal Flower’ Helichrysum bracteatum

    PubMed Central

    Nishikawa, Kuniko; Ito, Hiroaki; Awano, Tatsuya; Hosokawa, Munetaka; Yazawa, Susumu

    2008-01-01

    Background and Aims Helichrysum bracteatum is called an ‘eternal flower’ and has large, coloured, scarious bracts. These maintain their aesthetic value without wilting or discoloration for many years. There have been no research studies of cell death or cell morphology of the scarious bract, and hence the aim of this work was to elucidate these characteristics for the bract of H. bracteatum. Methods DAPI (4'6-diamidino-2-phenylindol dihydrochloride) staining and fluorescence microscopy were used for observation of cell nuclei. Light microscopy (LM), transmission electron microscopy (TEM) and polarized light microscopy were used for observation of cells, including cell wall morphology. Key Results Cell death occurred at the bract tip during the early stage of flower development. The cell wall was the most prominent characteristic of H. bracteatum bract cells. Characteristic thickened secondary cell walls on the inside of the primary cell walls were observed in both epidermal and inner cells. In addition, the walls of all cells exhibited birefringence. Characteristic thickened secondary cell walls have orientated cellulose microfibrils as well as general secondary cell walls of the tracheary elements. For comparison, these characters were not observed in the petal and bract tissues of Chrysanthemum morifolium. Conclusions Bracts at anthesis are composed of dead cells. Helichrysum bracteatum bracts have characteristic thickened secondary cell walls that have not been observed in the parenchyma of any other flowers or leaves. The cells of the H. bracteatum bract differ from other tissues with secondary cell walls, suggesting that they may be a new cell type. PMID:18436550

  19. Hemocyanin from Shrimp Litopenaeus vannamei Has Antiproliferative Effect against HeLa Cell In Vitro.

    PubMed

    Zheng, Liyuan; Zhao, Xianliang; Zhang, Pei; Chen, Chuandao; Liu, Shangjie; Huang, Runqing; Zhong, Mingqi; Wei, Chiju; Zhang, Yueling

    2016-01-01

    Hemocyanin (HMC) has been shown to participate in multiple roles of immune defence. In this study, we investigated the antiproliferative effect and underpinning mechanism of HMC from Litopenaeus vannamei in vitro. Sulforhodamine B (SRB) assay indicated that HMC could dramatically inhibit the growth of HeLa cells, but not 293T cells under the same conditions. Moreover, typical morphological features of apoptosis in HeLa cells including the formation of apoptotic body-like vesicles, chromatin condensation and margination were observed by using 4, 6-diamidino-2- phenylindole dihydrochloride (DAPI) staining and fluorescence analysis. An apoptotic DNA ladder from 180 to 300 bp was also detected. Furthermore, 10 variation proteins associated with apoptosis pathway, viz. G3PDH isoforms 1/2 (G3PDH1/2), aldosereductase, ectodemal dysplasia receptor associated death receptor domain isoform CRA_a (EDARADD), heat shock 60kD protein 1 variant 1 (HSP60), heat shock 70kDa protein 5 precursor (HSP70), heat shock protein 90kDa beta member 1 precursor (HSP90), 14-3-3 protein ζ/δ, Ran and ubiquitin activating enzyme E1(UBE1), were identified from HMC-treated HeLa cells by the proteomic and quantitative real-time RT-PCR strategies. Importantly, the reactive oxygen species (ROS), mitochondrial membrane potential (Δψm) and caspase-9/3 activities were changed significantly in HMC-treated HeLa cells. Together, the data suggests that L. vannamei HMC mediates antiproliferative properties through the apoptosis mechanism involving the mitochondria triggered pathway.

  20. Hemocyanin from Shrimp Litopenaeus vannamei Has Antiproliferative Effect against HeLa Cell In Vitro

    PubMed Central

    Chen, Chuandao; Liu, Shangjie; Huang, Runqing; Zhong, Mingqi; Wei, Chiju; Zhang, Yueling

    2016-01-01

    Hemocyanin (HMC) has been shown to participate in multiple roles of immune defence. In this study, we investigated the antiproliferative effect and underpinning mechanism of HMC from Litopenaeus vannamei in vitro. Sulforhodamine B (SRB) assay indicated that HMC could dramatically inhibit the growth of HeLa cells, but not 293T cells under the same conditions. Moreover, typical morphological features of apoptosis in HeLa cells including the formation of apoptotic body-like vesicles, chromatin condensation and margination were observed by using 4, 6-diamidino-2- phenylindole dihydrochloride (DAPI) staining and fluorescence analysis. An apoptotic DNA ladder from 180 to 300 bp was also detected. Furthermore, 10 variation proteins associated with apoptosis pathway, viz. G3PDH isoforms 1/2 (G3PDH1/2), aldosereductase, ectodemal dysplasia receptor associated death receptor domain isoform CRA_a (EDARADD), heat shock 60kD protein 1 variant 1 (HSP60), heat shock 70kDa protein 5 precursor (HSP70), heat shock protein 90kDa beta member 1 precursor (HSP90), 14-3-3 protein ζ/δ, Ran and ubiquitin activating enzyme E1(UBE1), were identified from HMC-treated HeLa cells by the proteomic and quantitative real-time RT-PCR strategies. Importantly, the reactive oxygen species (ROS), mitochondrial membrane potential (Δψm) and caspase-9/3 activities were changed significantly in HMC-treated HeLa cells. Together, the data suggests that L. vannamei HMC mediates antiproliferative properties through the apoptosis mechanism involving the mitochondria triggered pathway. PMID:27007573

  1. Infusion of imaging and therapeutic molecules into the plant virus-based carrier cowpea mosaic virus: cargo-loading and delivery.

    PubMed

    Yildiz, Ibrahim; Lee, Karin L; Chen, Kevin; Shukla, Sourabh; Steinmetz, Nicole F

    2013-12-10

    This work is focused on the development of a plant virus-based carrier system for cargo delivery, specifically 30nm-sized cowpea mosaic virus (CPMV). Whereas previous reports described the engineering of CPMV through genetic or chemical modification, we report a non-covalent infusion technique that facilitates efficient cargo loading. Infusion and retention of 130-155 fluorescent dye molecules per CPMV using DAPI (4',6-diamidino-2-phenylindole dihydrochloride), propidium iodide (3,8-diamino-5-[3-(diethylmethylammonio)propyl]-6-phenylphenanthridinium diiodide), and acridine orange (3,6-bis(dimethylamino)acridinium chloride), as well as 140 copies of therapeutic payload proflavine (PF, acridine-3,6-diamine hydrochloride), is reported. Loading is achieved through interaction of the cargo with the CPMV's encapsidated RNA molecules. The loading mechanism is specific; empty RNA-free eCPMV nanoparticles could not be loaded. Cargo-infused CPMV nanoparticles remain chemically active, and surface lysine residues were covalent modified with dyes leading to the development of dual-functional CPMV carrier systems. We demonstrate cargo-delivery to a panel of cancer cells (cervical, breast, and colon): CPMV nanoparticles enter cells via the surface marker vimentin, the nanoparticles target the endolysosome, where the carrier is degraded and the cargo is released allowing imaging and/or cell killing. In conclusion, we demonstrate cargo-infusion and delivery to cells; the methods discussed provide a useful means for functionalization of CPMV toward its application as drug and/or contrast agent delivery vehicle.

  2. Bacteria and Archaea Physically Associated with Gulf of Mexico Gas Hydrates

    PubMed Central

    Lanoil, Brian D.; Sassen, Roger; La Duc, Myron T.; Sweet, Stephen T.; Nealson, Kenneth H.

    2001-01-01

    Although there is significant interest in the potential interactions of microbes with gas hydrate, no direct physical association between them has been demonstrated. We examined several intact samples of naturally occurring gas hydrate from the Gulf of Mexico for evidence of microbes. All samples were collected from anaerobic hemipelagic mud within the gas hydrate stability zone, at water depths in the ca. 540- to 2,000-m range. The δ13C of hydrate-bound methane varied from −45.1‰ Peedee belemnite (PDB) to −74.7‰ PDB, reflecting different gas origins. Stable isotope composition data indicated microbial consumption of methane or propane in some of the samples. Evidence of the presence of microbes was initially determined by 4,6-diamidino 2-phenylindole dihydrochloride (DAPI) total direct counts of hydrate-associated sediments (mean = 1.5 × 109 cells g−1) and gas hydrate (mean = 1.0 × 106 cells ml−1). Small-subunit rRNA phylogenetic characterization was performed to assess the composition of the microbial community in one gas hydrate sample (AT425) that had no detectable associated sediment and showed evidence of microbial methane consumption. Bacteria were moderately diverse within AT425 and were dominated by gene sequences related to several groups of Proteobacteria, as well as Actinobacteria and low-G + C Firmicutes. In contrast, there was low diversity of Archaea, nearly all of which were related to methanogenic Archaea, with the majority specifically related to Methanosaeta spp. The results of this study suggest that there is a direct association between microbes and gas hydrate, a finding that may have significance for hydrocarbon flux into the Gulf of Mexico and for life in extreme environments. PMID:11679338

  3. Consequences of stoichiometric error on nuclear DNA content evaluation in Coffea liberica var. dewevrei using DAPI and propidium iodide.

    PubMed

    Noirot, Michel; Barre, Philippe; Louarn, Jacques; Duperray, Christophe; Hamon, Serge

    2002-04-01

    The genome size of coffee trees (Coffea sp.) was assessed using flow cytometry. Nuclear DNA was stained with two dyes [4',6-diamino-2-phenylindole dihydrochloride hydrate (DAPI) and propidium iodide (PI)]. Fluorescence in coffee tree nuclei (C-PI or C-DAPI) was compared with that of the standard, petunia (P-PI or P-DAPI). If there is no stoichiometric error, then the ratio between fluorescence of the target nuclei and that of the standard nuclei (R-PI or R-DAPI) is expected to be proportional to the genome size. Between-tree differences in target : standard fluorescence ratios were noted in Coffea liberica var. dewevrei using propidium iodide and DAPI. For both dyes, between-tree differences were due to a lack of proportionality when comparing locations of the coffee peak and the petunia peak. Intraspecific genome size variations clearly cannot explain variations in the target : standard fluorescence ratio. The origin of the lack of proportionality between target and standard fluorescences differed for the two dyes. With propidium iodide, there was a regression line convergence point, and no between-tree differences were noted in this respect, whereas there was no such convergence with DAPI. An accurate estimate of genome size can thus be obtained with PI. Implications with respect to accessibility and binding mode are discussed.

  4. Consequences of Stoichiometric Error on Nuclear DNA Content Evaluation in Coffea liberica var. dewevrei using DAPI and Propidium Iodide

    PubMed Central

    NOIROT, MICHEL; BARRE, PHILIPPE; LOUARN, JACQUES; DUPERRAY, CHRISTOPHE; HAMON, SERGE

    2002-01-01

    The genome size of coffee trees (Coffea sp.) was assessed using flow cytometry. Nuclear DNA was stained with two dyes [4′,6‐diamino‐2‐phenylindole dihydrochloride hydrate (DAPI) and propidium iodide (PI)]. Fluorescence in coffee tree nuclei (C‐PI or C‐DAPI) was compared with that of the standard, petunia (P‐PI or P‐DAPI). If there is no stoichiometric error, then the ratio between fluorescence of the target nuclei and that of the standard nuclei (R‐PI or R‐DAPI) is expected to be proportional to the genome size. Between‐tree differences in target : standard fluorescence ratios were noted in Coffea liberica var. dewevrei using propidium iodide and DAPI. For both dyes, between‐tree differences were due to a lack of proportionality when comparing locations of the coffee peak and the petunia peak. Intraspecific genome size variations clearly cannot explain variations in the target : standard fluorescence ratio. The origin of the lack of proportionality between target and standard fluorescences differed for the two dyes. With propidium iodide, there was a regression line convergence point, and no between‐tree differences were noted in this respect, whereas there was no such convergence with DAPI. An accurate estimate of genome size can thus be obtained with PI. Implications with respect to accessibility and binding mode are discussed. PMID:12096798

  5. A cautionary (spectral) tail: red-shifted fluorescence by DAPI-DAPI interactions.

    PubMed

    Omelon, Sidney; Georgiou, John; Habraken, Wouter

    2016-02-01

    The fluorescent dye DAPI is useful for its association with and consequent amplification of an ∼460 nm emission maximum upon binding to dsDNA. Labelling with higher DAPI concentrations is a technique used to reveal Pi polymers [polyphosphate (polyP)], with a red-shift to ∼520-550 nm fluorescence emission. DAPI-polyP emissions of ∼580 nm are also generated upon 415 nm excitation. Red-shifted DAPI emission has been associated with polyP and RNA and has more recently been reported with polyadenylic acid (polyA), specific inositol phosphates (IPs) and heparin. We find that amorphous calcium phosphate (ACP) also demonstrates red-shifted DAPI emission at high DAPI concentrations. This DAPI spectral shift has been attributed to DAPI-DAPI electrostatic interactions enabled by molecules with high negative charge density that increase the local DAPI concentration and favour DAPI molecular proximity, as observed by increasing the dye/phosphate ratio. Excitation of dry DAPI (∼360 nm) confirmed a red-shifted DAPI emission. Whereas enzymatic approaches to modify substrates can help define the nature of DAPI fluorescence signals, multiple approaches beyond red-shifted DAPI excitation/emission are advised before conclusions are drawn about DAPI substrate identification.

  6. Functional cloning and expression of emeA, and characterization of EmeA, a multidrug efflux pump from Enterococcus faecalis.

    PubMed

    Lee, Eun-Woo; Chen, Jing; Huda, Md Nazmul; Kuroda, Teruo; Mizushima, Tohru; Tsuchiya, Tomofusa

    2003-02-01

    A fragment of chromosomal DNA from Enterococcus faecalis ATCC 29212 was cloned using Escherichia coli KAM32 host cells lacking major multidrug efflux pumps. E. coli KAM32 cells were sensitive to many antimicrobial agents, and the transformed cells harboring a recombinant plasmid became resistant to several structurally unrelated antimicrobial agents such as tetraphenylphosphonium chloride, 4',6-diamidino-2-phenylindole (DAPI), Hoechst 33342, acriflavine, benzalkonium chloride, norfloxacin and ethidium bromide. This suggests that the cloned DNA fragment carries a gene(s) encoding a multidrug efflux pump. Determination of the nucleotide sequence of the cloned DNA revealed a gene designated as emeA. The transformed E. coli cells showed efflux activity of several antimicrobial agents such as DAPI, Hoechst 33342 and acriflavine. Efflux of DAPI via EmeA was strongly inhibited by reserpine.

  7. The antibacterial mechanism of berberine against Actinobacillus pleuropneumoniae.

    PubMed

    Kang, Shuai; Li, Zhengwen; Yin, Zhongqiong; Jia, Renyong; Song, Xu; Li, Li; Chen, Zhenzhen; Peng, Lianci; Qu, Jing; Hu, Zhiqiang; Lai, Xin; Wang, Guangxi; Liang, Xiaoxia; He, Changliang; Yin, Lizi

    2015-01-01

    This study demonstrated berberine to be a potential natural compound against Actinobacillus pleuropneumoniae. Liquid doubling dilution, transmission electron microscopy (TEM), SDS-PAGE and 4',6-diamidino-2-phenylindole (DAPI) staining were employed to elucidate the antibacterial activity and mechanism of berberine. The minimal inhibitory concentration of berberine was 0.3125 mg/mL, and time-kill curves showed concentration and time dependence. The TEM micrographs displayed damaged cell wall, concentrated cytoplasm, cytoplasmic content leakage and cell death. SDS-PAGE and DAPI assays revealed that berberine can restrain DNA and protein syntheses. Berberine inhibited the synthesis of proteins associated with the growth and cleavage of bacteria and then blocked the division and development of bacteria. The compound ultimately induced cytoplasm pyknosis and bacterial death.

  8. Lactobacillus Casei Decreases Organophosphorus Pesticide Diazinon Cytotoxicity in Human HUVEC Cell Line

    PubMed Central

    Bagherpour Shamloo, Hasan; Golkari, Saber; Faghfoori, Zeinab; Movassaghpour, AliAkbar; Lotfi, Hajie; Barzegari, Abolfazl; Yari Khosroushahi, Ahmad

    2016-01-01

    Purpose: Exposure to diazinon can trigger acute and chronic toxicity and significantly induces DNA damage and proapoptotic effects in different human cells. Due to the significance of probiotic bacteria antitoxin effect, this study aimed to investigate the effect of Lactobacillus casei on diazinon (DZN) cytotoxicity in human umbilical vein endothelial cells (HUVEC) in vitro. Methods: The cytotoxicity assessments were performed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test, DAPI (4',6-diamidino-2-phenylindole) staining and flow cytometric methodologies. Results: Cytotoxic assessments through flow cytometry/ DAPI staining demonstrated that apoptosis is the main cytotoxic mechanism of diazinon in HUVEC cells and L. casei could decrease the diazinon cytotoxic effects on toxicants. Conclusion: the screen of total bacterial secreted metabolites can be considered as a wealthy source to find the new active compounds to introduce as reducing agricultural remained pesticide cytotoxicity effects on the human food chain. PMID:27478782

  9. Inositol phosphates induce DAPI fluorescence shift.

    PubMed

    Kolozsvari, Bernadett; Parisi, Federica; Saiardi, Adolfo

    2014-06-15

    The polymer inorganic polyP (polyphosphate) and inositol phosphates, such as IP6 (inositol hexakisphosphate; also known as phytic acid), share many biophysical features. These similarities must be attributed to the phosphate groups present in these molecules. Given the ability of polyP to modify the excitation-emission spectra of DAPI we decided to investigate whether inositol phosphates possess the same property. We discovered that DAPI-IP6 complexes emit at approximately 550 nm when excited with light of wavelength 410-420 nm. IP5 (inositol pentakisphosphate) is also able to induce a similar shift in DAPI fluorescence. Conversely, IP3 (inositol trisphosphate) and IP4 (inositol tetrakisphosphate) are unable to shift DAPI fluorescence. We have employed this newly discovered feature of DAPI to study the enzymatic activity of the inositol polyphosphate multikinase and to monitor phytase phosphatase reactions. Finally, we used DAPI-IP6 fluorescence to determine the amount of IP6 in plant seeds. Using an IP6 standard curve this straight-forward analysis revealed that among the samples tested, borlotti beans possess the highest level of IP6 (9.4 mg/g of dry mass), whereas the Indian urad bean the lowest (3.2 mg/g of dry mass). The newly identified fluorescence properties of the DAPI-IP5 and DAPI-IP6 complexes allow the levels and enzymatic conversion of these two important messengers to be rapidly and reliably monitored.

  10. Biological activities of green silver nanoparticles synthesized with Acorous calamus rhizome extract.

    PubMed

    Nakkala, Jayachandra Reddy; Mata, Rani; Gupta, Arvind Kumar; Sadras, Sudha Rani

    2014-10-06

    Nanomedicine utilize biocompatible nanomaterials for diagnostic and therapeutic purposes. This study reports the synthesis of silver nanoparticles using aqueous rhizome extract of Acorus calamus (ACRE) and evaluation of antioxidant, antibacterial as well as anticancer effects of synthesized A. calamus silver nanoparticles (ACAgNPs). The formation of ACAgNPs was confirmed by UV-visible spectroscopy and their average size was found to be 31.83 nm by DLS particle size analyzer. Scanning electron micrograph (SEM) revealed spherical shape of ACAgNPs and energy dispersive spectroscopy (EDX) data showed the presence of metallic silver. Fourier transform infrared spectroscopy (FTIR) analysis indicated the presence of phenol/alcohol, aromatic amine and carbonyl groups in ACRE that were involved in reduction and capping of nanoparticles. ACRE and ACAgNPs exhibited substantial free radical quenching ability in various in vitro antioxidant assays performed in this study. ACAgNPs also displayed appreciable antibacterial activity against three different pathogenic bacteria and the growth kinetic study with Escherichia coli designated the inhibition of bacterial growth at the log phase. The cytotoxic effect of ACAgNPs was assessed by MTT assay in HeLa and A549 cells. The IC50 value of ACAgNPs respectively after 24 and 48 h was found to be 92.48 and 69.44 μg/ml in HeLa cells and in A549 cells it was 53.2 and 32.1 μg/ml. Apoptotic cell death in ACAgNPs treated cells was indicated by acridine orange/ethidium bromide (AO/EB) and annexinV-Cy3 staining techniques. Staining with propidium iodide (PI) and 4', 6-diamidino-2-phenylindole, dihydrochloride (DAPI) also confirmed nuclear changes such as condensation and fragmentation. Further, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay showed distribution of ACAgNPs treated cells in the late apoptotic stage. These findings emphasize that such biocompatible green nanoparticles with multifaceted biological

  11. Caspofungin kills Candida albicans by causing both cellular apoptosis and necrosis.

    PubMed

    Hao, Binghua; Cheng, Shaoji; Clancy, Cornelius J; Nguyen, M Hong

    2013-01-01

    Caspofungin exerts candidacidal activity by inhibiting cell wall (1,3)-β-d-glucan synthesis. We investigated the physiologic mechanisms of caspofungin-induced Candida albicans cell death. Apoptosis (programmed cell death) and necrosis were studied after C. albicans SC5314 cells were exposed to caspofungin at 0.06, 0.125, and 0.5 μg/ml (0.5×, 1×, and 4× the MIC, respectively) for 3 h. Caspofungin at 0.125 and 0.5 μg/ml reduced cellular viability by >50%, as measured by colony counts and methylene blue exclusion. Apoptosis and necrosis were demonstrated by annexin V and propidium iodide staining for phosphatidylserine externalization and loss of membrane integrity, respectively. At all concentrations of caspofungin, 20 to 25% and 5 to 7% of C. albicans cells exhibited early apoptosis and late apoptosis/necrosis, respectively (P value was not significant [NS]). Necrosis, on the other hand, was significantly greater at 0.125 (43%) and 0.5 (48%) μg/ml than at 0.06 μg/ml (26%) (P values of 0.003 and 0.003, respectively). The induction of apoptosis at concentrations less than or equal to the MIC was corroborated by dihydrorhodamine 123 (DHR-123) and dihydroethidium (DHE) staining (reactive oxygen species production), JC-1 staining (mitochondrial membrane potential dissipation), and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) staining (DNA damage and nuclear fragmentation). Moreover, electron microscopy of cells exposed to 0.125 μg/ml of caspofungin showed hallmark apoptotic features like chromatin margination and condensation and nuclear blebs. Apoptosis was associated with metacaspase 1 activation, as demonstrated by D2R staining. Caspofungin exerts activity against C. albicans by directly killing cells (resulting in necrosis) and causing others to undergo programmed cell death (apoptosis). Apoptosis is initiated at subinhibitory concentrations, suggesting that strategies to target

  12. Anticancer potential of pyrrole (1, 2, a) pyrazine 1, 4, dione, hexahydro 3-(2-methyl propyl) (PPDHMP) extracted from a new marine bacterium, Staphylococcus sp. strain MB30.

    PubMed

    Lalitha, P; Veena, V; Vidhyapriya, P; Lakshmi, Pragna; Krishna, R; Sakthivel, N

    2016-05-01

    Marine bacterium, strain MB30 isolated from the deep sea sediment of Bay of Bengal, India, exhibited antimicrobial activity against human pathogenic bacteria. Based on the 16S rRNA sequence homology and subsequent phylogenetic tree analysis, the strain MB30 was identified as Staphylococcus sp. The bioactive metabolite produced by the strain MB30 was purified through silica gel column chromatography and preparative HPLC. Purified metabolite was further characterized by FT-IR, LC-MS and NMR analyses. On the basis of spectroscopic data, the metabolite was identified as pyrrole (1, 2, a) pyrazine 1, 4, dione, hexahydro 3-(2-methyl propyl) (PPDHMP). The PPDHMP exhibited in vitro anticancer potential against lung (A549) and cervical (HeLa) cancer cells in a dose-dependent manner with the IC50 concentration of 19.94 ± 1.23 and 16.73 ± 1.78 μg ml(-1) respectively. The acridine orange (AO)/ethidium bromide (EB) and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining of the IC50 concentration of PPDHMP-treated cancer cells exhibited an array of morphological changes such as nuclear condensation, cell shrinkage and formation of apoptotic bodies. The PPDHMP-treated cancer cells induced the progressive accumulation of fragmented DNA in a time-dependent manner. Based on the flow cytometric analysis, it has become evident that the compound was also effective in arresting the cell cycle at G1 phase. Further, the Western blotting analysis confirmed the down-regulation of cyclin-D1, cyclin dependent kinase (CDK-2), anti-apoptotic Bcl-2 family proteins (Bcl-2 and Bcl-xL), activation of caspase-9 and 3 with the cleavage of PARP. The PPDHMP-treated cancer cells also showed the inhibition of migration and invasive capacity of cancer cells. In the present investigation, for the first time, we have reported the extraction, purification and characterization of an anticancer metabolite, PPDHMP from a new marine bacterium, Staphylococcus sp. strain MB30.

  13. Hydrothermal synthesis of titanium dioxide nanoparticles: mosquitocidal potential and anticancer activity on human breast cancer cells (MCF-7).

    PubMed

    Murugan, Kadarkarai; Dinesh, Devakumar; Kavithaa, Krishnamoorthy; Paulpandi, Manickam; Ponraj, Thondhi; Alsalhi, Mohamad Saleh; Devanesan, Sandhanasamy; Subramaniam, Jayapal; Rajaganesh, Rajapandian; Wei, Hui; Kumar, Suresh; Nicoletti, Marcello; Benelli, Giovanni

    2016-03-01

    Mosquito vectors (Diptera: Culicidae) are responsible for transmission of serious diseases worldwide. Mosquito control is being enhanced in many areas, but there are significant challenges, including increasing resistance to insecticides and lack of alternative, cost-effective, and eco-friendly products. To deal with these crucial issues, recent emphasis has been placed on plant materials with mosquitocidal properties. Furthermore, cancers figure among the leading causes of morbidity and mortality worldwide, with approximately 14 million new cases and 8.2 million cancer-related deaths in 2012. It is expected that annual cancer cases will rise from 14 million in 2012 to 22 million within the next two decades. Nanotechnology is a promising field of research and is expected to give major innovation impulses in a variety of industrial sectors. In this study, we synthesized titanium dioxide (TiO2) nanoparticles using the hydrothermal method. Nanoparticles were subjected to different analysis including UV-Vis spectrophotometry, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), zeta potential, and energy-dispersive spectrometric (EDX). The synthesized TiO2 nanoparticles exhibited dose-dependent cytotoxicity against human breast cancer cells (MCF-7) and normal breast epithelial cells (HBL-100). After 24-h incubation, the inhibitory concentrations (IC50) were found to be 60 and 80 μg/mL on MCF-7 and normal HBL-100 cells, respectively. Induction of apoptosis was evidenced by Acridine Orange (AO)/ethidium bromide (EtBr) and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) staining. In larvicidal and pupicidal experiments conducted against the primary dengue mosquito Aedes aegypti, LC50 values of nanoparticles were 4.02 ppm (larva I), 4.962 ppm (larva II), 5.671 ppm (larva III), 6.485 ppm (larva IV), and 7.527 ppm (pupa). Overall, our results suggested that TiO2 nanoparticles may be considered as

  14. Karyological features of wild and cultivated forms of myrtle (Myrtus communis, Myrtaceae).

    PubMed

    Serçe, S; Ekbiç, E; Suda, J; Gündüz, K; Kiyga, Y

    2010-03-09

    Myrtle is an evergreen shrub or small tree widespread throughout the Mediterranean region. In Turkey, both cultivated and wild forms, differing in plant and fruit size and fruit composition, can be found. These differences may have resulted from the domestication of the cultivated form over a long period of time. We investigated whether wild and cultivated forms of myrtle differ in karyological features (i.e., number of somatic chromosomes and relative genome size). We sampled two wild forms and six cultivated types of myrtle. All the samples had the same chromosome number (2n = 2x = 22). The results were confirmed by 4',6-diamidino-2-phenylindole (DAPI) flow cytometry. Only negligible variation (approximately 3%) in relative fluorescence intensity was observed among the different myrtle accessions, with wild genotypes having the smallest values. We concluded that despite considerable morphological differentiation, cultivated and wild myrtle genotypes in Turkey have similar karyological features.

  15. Inhibition of Staphylococcus aureus biofilm by a copper-bearing 317L-Cu stainless steel and its corrosion resistance.

    PubMed

    Sun, Da; Xu, Dake; Yang, Chunguang; Chen, Jia; Shahzad, M Babar; Sun, Ziqing; Zhao, Jinlong; Gu, Tingyue; Yang, Ke; Wang, Guixue

    2016-12-01

    The present study investigated the antibacterial performance, corrosion resistance and surface properties of antibacterial austenitic 317L-Cu stainless steel (317L-Cu SS). After 4.5wt% copper was added to 317L stainless steel (317L SS), the new alloy underwent solid solution and aging heat treatment. Fluorescent staining using 4',6-diamidino-2-phenylindole (DAPI) revealed that the 317L-Cu SS showed strong antibacterial efficacy, achieving a 99% inhibition rate of sessile Staphylococcus aureus cells after 5days. The corrosion data obtained by potentiodynamic polarization curves indicated that in comparison with 317L SS, the pitting potential and corrosion current density of 317L-Cu slightly decreased due to the addition of Cu. The 317L-Cu SS exhibited no cytotoxicity against zebrafish (Danio rerio) embryos. The experimental results in this study demonstrated that the new alloy has potential applications in medical and daily uses.

  16. Induction of microtubule damage in Allium cepa meristematic cells by pharmaceutical formulations of thiabendazole and griseofulvin.

    PubMed

    Andrioli, Nancy B; Soloneski, Sonia; Larramendy, Marcelo L; Mudry, Marta D

    2014-09-15

    Microtubules (MT) are formed by the assembly of α- and β-tubulins and MT-associated proteins. We characterized the effects of pharmaceutical formulations containing the microtubule disruptors thiabendazole (TBZ) and griseofulvin (GF) on the mitotic machinery of plant (A. cepa) meristematic cells. GF concentrations between 10 and 250 μg/ml were tested. GF induced mitotic index inhibition and genotoxic effects, including chromosome fragments, bridges, lagged chromosomes, C-metaphases, tripolar cell division, disorganized anaphases and nuclear abnormalities in interphase cells. Efects on the mitotic machinery were studied by direct immunofluorescence with β-tubulin labeling and by DNA counterstaining with 4',6-diamidino-2-phenylindole (DAPI). Exposure of meristematic root cells to TBZ or GF, 100 μg/ml, caused microtubular damage which led to abnormal MT arrays. Our results suggest that GF induces abnormalities in spindle symmetry/polarity, while TBZ causes chromosome missegregation, polyploidy, and lack of cytokinesis.

  17. Induction of growth arrest in colorectal cancer cells by cold plasma and gold nanoparticles

    PubMed Central

    Irani, Shiva; Shahmirani, Zhohreh; Mirpoor, Shahriar

    2015-01-01

    Introduction Guided treatments with nanoparticles and cold atmospheric plasma are a new approach in cancer therapy. Plasma is an ionized gas that has reactive and energetic particles and can be produced in the laboratory by different methods. Material and methods Plasma jet therapy was employed to irradiate HCT-116 cells (human colorectal cancer cells) which were cultured in the presence of gold nanoparticles (GNPs). Cell cytotoxicity was tested with 3-[4, 5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT), and cancerous cell apoptosis was shown by 4’,6-diamidino-2-phenylindole (DAPI) staining. Results The results showed that cell death was increased significantly with p < 0.001 by cold atmospheric plasma in the presence of gold nanoparticles. Conclusions It appears that non-thermal plasma and gold nanoparticles synergism is a promising approach in colon cancer therapy. PMID:26788092

  18. New method for estimating bacterial cell abundances in natural samples by use of sublimation.

    PubMed

    Glavin, Daniel P; Cleaves, H James; Schubert, Michael; Aubrey, Andrew; Bada, Jeffrey L

    2004-10-01

    We have developed a new method based on the sublimation of adenine from Escherichia coli to estimate bacterial cell counts in natural samples. To demonstrate this technique, several types of natural samples, including beach sand, seawater, deep-sea sediment, and two soil samples from the Atacama Desert, were heated to a temperature of 500 degrees C for several seconds under reduced pressure. The sublimate was collected on a cold finger, and the amount of adenine released from the samples was then determined by high-performance liquid chromatography with UV absorbance detection. Based on the total amount of adenine recovered from DNA and RNA in these samples, we estimated bacterial cell counts ranging from approximately 10(5) to 10(9) E. coli cell equivalents per gram. For most of these samples, the sublimation-based cell counts were in agreement with total bacterial counts obtained by traditional DAPI (4,6-diamidino-2-phenylindole) staining.

  19. New method for estimating bacterial cell abundances in natural samples by use of sublimation

    NASA Technical Reports Server (NTRS)

    Glavin, Daniel P.; Cleaves, H. James; Schubert, Michael; Aubrey, Andrew; Bada, Jeffrey L.

    2004-01-01

    We have developed a new method based on the sublimation of adenine from Escherichia coli to estimate bacterial cell counts in natural samples. To demonstrate this technique, several types of natural samples, including beach sand, seawater, deep-sea sediment, and two soil samples from the Atacama Desert, were heated to a temperature of 500 degrees C for several seconds under reduced pressure. The sublimate was collected on a cold finger, and the amount of adenine released from the samples was then determined by high-performance liquid chromatography with UV absorbance detection. Based on the total amount of adenine recovered from DNA and RNA in these samples, we estimated bacterial cell counts ranging from approximately 10(5) to 10(9) E. coli cell equivalents per gram. For most of these samples, the sublimation-based cell counts were in agreement with total bacterial counts obtained by traditional DAPI (4,6-diamidino-2-phenylindole) staining.

  20. Anticancer activity of Moringa oleifera mediated silver nanoparticles on human cervical carcinoma cells by apoptosis induction.

    PubMed

    Vasanth, Karunamoorthy; Ilango, Kaliappan; MohanKumar, Ramasamy; Agrawal, Aruna; Dubey, Govind Prasad

    2014-05-01

    Silver nanomaterial plays a crucial role in the growing field of nanotechnology as there is an increasing commercial demand for silver nanoparticles (AgNPs) owing to their wide biological applications. The present investigation aims at developing anti-cancerous colloidal silver using Moringa olifera stem bark extract. Electron and atomic force microscopic images were taken to analyze the surface morphology of the synthesized AgNPs. The effects of synthesized AgNPs were tested against human cervical carcinoma cells (HeLa) and cell morphology was further evaluated using 4,6-diamidino-2-phenylindole (DAPI) staining. The efficiency of green synthesized AgNPs was studied with the help of fluorescence activated cell sorting (FACS) and was shown to induce apoptosis through reactive oxygen species (ROS) generation in HeLa cells.

  1. Biofilms from a Brazilian water distribution system include filamentous fungi.

    PubMed

    Siqueira, V M; Oliveira, H M B; Santos, C; Paterson, R R M; Gusmão, N B; Lima, N

    2013-03-01

    Filamentous fungi in drinking water can block water pipes, can cause organoleptic biodeterioration, and are a source of pathogens. There are increasing reports of the involvement of the organisms in biofilms. This present study describes a sampling device that can be inserted directly into pipes within water distribution systems, allowing biofilm formation in situ. Calcofluor White M2R staining and fluorescent in situ hybridization with morphological analyses using epifluorescent microscopy were used to analyse biofilms for filamentous fungi, permitting direct observation of the fungi. DAPI (4',6-diamidino-2-phenylindole) was applied to detect bacteria. Filamentous fungi were detected in biofilms after 6 months on coupons exposed to raw water, decanted water and at the entrance of the water distribution system. Algae, yeast, and bacteria were also observed. The role of filamentous fungi requires further investigations.

  2. Simultaneous UV Spectrophotometric Estimation of Ambroxol Hydrochloride and Levocetirizine Dihydrochloride

    PubMed Central

    Prabhu, S. Lakshmana; Shirwaikar, A. A.; Shirwaikar, Annie; Kumar, C. Dinesh; Kumar, G. Aravind

    2008-01-01

    A novel, simple, sensitive and rapid spectrophotometric method has been developed for simultaneous estimation of ambroxol hydrochloride and levocetirizine dihydrochloride. The method involved solving simultaneous equations based on measurement of absorbance at two wavelengths 242 nm and 231 nm, the γ max of ambroxol hydrochloride and levocetirizine dihydrochloride, respectively. Beer's law was obeyed in the concentration range 10–50 μg/ml and 8–24 μg/ml for ambroxol hydrochloride and levocetirizine dihydrochloride respectively. Results of the method were validated statistically and by recovery studies. PMID:20046721

  3. 21 CFR 522.2120 - Spectinomycin dihydrochloride injection.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... dihydrochloride pentahydrate used in manufacturing the drug is the antibiotic substance produced by the growth of Streptomyces flavopersicus (var. Abbott) or the same antibiotic substance produced by any other means....

  4. 21 CFR 522.2120 - Spectinomycin dihydrochloride injection.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... dihydrochloride pentahydrate used in manufacturing the drug is the antibiotic substance produced by the growth of Streptomyces flavopersicus (var. Abbott) or the same antibiotic substance produced by any other means....

  5. 21 CFR 522.2120 - Spectinomycin dihydrochloride injection.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... dihydrochloride pentahydrate used in manufacturing the drug is the antibiotic substance produced by the growth of Streptomyces flavopersicus (var. Abbott) or the same antibiotic substance produced by any other means....

  6. 21 CFR 522.2120 - Spectinomycin dihydrochloride injection.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... dihydrochloride pentahydrate used in manufacturing the drug is the antibiotic substance produced by the growth of Streptomyces flavopersicus (var. Abbott) or the same antibiotic substance produced by any other means....

  7. 21 CFR 522.2120 - Spectinomycin dihydrochloride injection.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... dihydrochloride pentahydrate used in manufacturing the drug is the antibiotic substance produced by the growth of Streptomyces flavopersicus (var. Abbott) or the same antibiotic substance produced by any other means....

  8. Characterization of a DAPI-RIT-DAPI system for gas-phase ion/molecule and ion/ion reactions.

    PubMed

    Lin, Ziqing; Tan, Lei; Garimella, Sandilya; Li, Linfan; Chen, Tsung-Chi; Xu, Wei; Xia, Yu; Ouyang, Zheng

    2014-01-01

    The discontinuous atmospheric pressure interface (DAPI) has been developed as a facile means for efficiently introducing ions generated at atmospheric pressure to an ion trap in vacuum [e.g., a rectilinear ion trap (RIT)] for mass analysis. Introduction of multiple beams of ions or neutral species through two DAPIs into a single RIT has been previously demonstrated. In this study, a home-built instrument with a DAPI-RIT-DAPI configuration has been characterized for the study of gas-phase ion/molecule and ion/ion reactions. The reaction species, including ions or neutrals, can be introduced from both ends of the RIT through the two DAPIs without complicated ion optics or differential pumping stages. The primary reactant ions were isolated prior to reaction and the product ions were mass analyzed after controlled reaction time period. Ion/molecule reactions involving peptide radical ions and proton-transfer ion/ion reactions have been carried out using this instrument. The gas dynamic effect due to the DAPI operation on internal energy deposition and the reactivity of peptide radical ions has been characterized. The DAPI-RIT-DAPI system also has a unique feature for allowing the ion reactions to be carried out at significantly elevated pressures (in 10(-1) Torr range), which has been found to be helpful to speed up the reactions. The viability and flexibility of the DAPI-RIT-DAPI system for the study of gas-phase ion reactions have been demonstrated.

  9. Characterization of a DAPI-RIT-DAPI System for Gas-Phase Ion/Molecule and Ion/Ion Reactions

    NASA Astrophysics Data System (ADS)

    Lin, Ziqing; Tan, Lei; Garimella, Sandilya; Li, Linfan; Chen, Tsung-Chi; Xu, Wei; Xia, Yu; Ouyang, Zheng

    2014-01-01

    The discontinuous atmospheric pressure interface (DAPI) has been developed as a facile means for efficiently introducing ions generated at atmospheric pressure to an ion trap in vacuum [e.g., a rectilinear ion trap (RIT)] for mass analysis. Introduction of multiple beams of ions or neutral species through two DAPIs into a single RIT has been previously demonstrated. In this study, a home-built instrument with a DAPI-RIT-DAPI configuration has been characterized for the study of gas-phase ion/molecule and ion/ion reactions. The reaction species, including ions or neutrals, can be introduced from both ends of the RIT through the two DAPIs without complicated ion optics or differential pumping stages. The primary reactant ions were isolated prior to reaction and the product ions were mass analyzed after controlled reaction time period. Ion/molecule reactions involving peptide radical ions and proton-transfer ion/ion reactions have been carried out using this instrument. The gas dynamic effect due to the DAPI operation on internal energy deposition and the reactivity of peptide radical ions has been characterized. The DAPI-RIT-DAPI system also has a unique feature for allowing the ion reactions to be carried out at significantly elevated pressures (in 10-1 Torr range), which has been found to be helpful to speed up the reactions. The viability and flexibility of the DAPI-RIT-DAPI system for the study of gas-phase ion reactions have been demonstrated.

  10. Interaction of DAPI with double-stranded ribonucleic acids.

    PubMed Central

    Manzini, G; Xodo, L; Barcellona, M L; Quadrifoglio, F

    1985-01-01

    The interaction of DAPI with natural and synthetic double-stranded polyribonucleotides was studied with different optical and calorimetric methods. The results were similar to those obtained previously with double-stranded polydeoxynucleotides, i.e. two interaction modes, the first of which shows high affinity for AU clusters and consequent strong fluorescence enhancement. The results suggest caution in the use of DAPI as selective fluorescent staining agent for DNA in the presence of RNA. A narrow groove binding model with hydrogen bonds between DAPI and AU pairs is proposed. An intercalation mechanism can be excluded because of the non planarity of DAPI molecule. PMID:4080554

  11. Comparative analysis of heterochromatin distribution in wild and cultivated Abelmoschus species based on fluorescent staining methods.

    PubMed

    Merita, Keisham; Kattukunnel, Joseph John; Yadav, Shrirang Ramchandra; Bhat, Kangila Venkataramana; Rao, Satyawada Rama

    2015-03-01

    A comparative analysis of fluorochrome-binding pattern in nine taxa of Abelmoschus had shown that the type, amount and distribution pattern of heterochromatin were characteristic for each taxa. The fluorescent chromosome-binding sites obtained by chromomycin A3 (CMA) and 4',6-diamidino-2-phenylindole (DAPI) staining in all the nine species showed constitutive heterochromatin CMA(+), DAPI(+) and CMA(+)/DAPI(+). Large amount of heterozygosity was observed with regard to heterochromatin distribution pattern in all the taxa studied. The CMA(+)-binding sites are comparatively less than DAPI(+)-binding sites which is clearly evident as AT-rich regions are more than GC-rich regions in all the nine taxa analysed in Abelmoschus. These CMA(+) and DAPI(+)-binding sites apparently rise with the increased in chromosome numbers of the different species. This pattern of heterochromatin heterogeneity seems to be a general characteristic feature. Therefore, the differential pattern of distribution of GC- and AT-rich sequences might have played an important role in diversification of the genus Abelmoschus. Polyploidy is an important factor in the evolution of Abelmoschus and the sole reason for range in chromosome numbers in this genus. It may be noted that, though often, but not always, the increase of DNA is caused by an increase in the amount of heterochromatin, i.e. increase of non-coding sections indicating restructuring of the heterochromatin. Thus, cumulative small and direct numerical changes might have played a role in the speciation of Abelmoschus.

  12. Ultrasound assisted enzyme catalyzed degradation of Cetirizine dihydrochloride.

    PubMed

    Sutar, Rahul S; Rathod, Virendra K

    2015-05-01

    Cetirizine dihydrochloride, a pharmaceutical drug of the class antihistamines is frequently detected in wastewater samples. In the present work, the degradation of Cetirizine dihydrochloride is carried out using a novel technique of laccase enzyme as a catalyst under the influence of ultrasound irradiation. Effect of various process parameters such as enzyme loading, temperature, power, duty cycle, frequency and speed of agitation has been studied along with identification of the degradation intermediates. The maximum degradation of 91% is achieved at optimized experimental parameters such as 0.02% enzyme loading (w/v), 50°C temperature, power input of 100 W, 25 kHz frequency and 50% duty cycle with agitation speed of 200 rpm. It is observed that enzymatic degradation of Cetirizine dihydrochloride under the influence of ultrasound irradiation not only enhances the degradation but also reduces the time of degradation as compared to conventional enzymatic degradation technique.

  13. Sapropterin Dihydrochloride Mixed With Common Foods and Beverages

    PubMed Central

    Jurecki, Elaina R.; Cunningham, Amy; Mahoney, John J.; Tingley, Douglas; Chung, Stanley; James, Neil; Cohen-Pfeffer, Jessica L.

    2014-01-01

    Sapropterin dihydrochloride is used to lower blood phenylalanine levels in tetrahydrobiopterin-responsive phenylketonuria in conjunction with a phenylalanine-restricted diet. This study investigated the solubility and stability of sapropterin tablets and a sapropterin powder formulation when mixed in selected beverages and foods. Solubility was partial for the tablets and complete for the powder. The stability testing showed that 93% or more of active sapropterin dihydrochloride is present at 1 hour after either tablets or powders are mixed with certain foods and beverages. Mixing sapropterin powder with foods and beverages might facilitate its administration in patients who have difficulty swallowing the drug according to prescribing information. PMID:25382934

  14. Monitoring intracellular polyphosphate accumulation in enhanced biological phosphorus removal systems by quantitative image analysis.

    PubMed

    Mesquita, Daniela P; Amaral, A Luís; Leal, Cristiano; Carvalheira, Mónica; Cunha, Jorge R; Oehmen, Adrian; Reis, Maria A M; Ferreira, Eugénio C

    2014-01-01

    A rapid methodology for intracellular storage polyphosphate (poly-P) identification and monitoring in enhanced biological phosphorus removal (EBPR) systems is proposed based on quantitative image analysis (QIA). In EBPR systems, 4',6-diamidino-2-phenylindole (DAPI) is usually combined with fluorescence in situ hybridization to evaluate the microbial community. The proposed monitoring technique is based on a QIA procedure specifically developed for determining poly-P inclusions within a biomass suspension using solely DAPI by epifluorescence microscopy. Due to contradictory literature regarding DAPI concentrations used for poly-P detection, the present work assessed the optimal DAPI concentration for samples acquired at the end of the EBPR aerobic stage when the accumulation occurred. Digital images were then acquired and processed by means of image processing and analysis. A correlation was found between average poly-P intensity values and the analytical determination. The proposed methodology can be seen as a promising alternative procedure for quantifying intracellular poly-P accumulation in a faster and less labour-intensive way.

  15. Cytogenetic characterization of the invasive mussel species Xenostrobus securis Lmk. (Bivalvia: Mytilidae).

    PubMed

    Pérez-García, Concepción; Morán, Paloma; Pasantes, Juan J

    2011-09-01

    The chromosomes of the invasive black-pigmy mussel (Xenostrobus securis (Lmk. 1819)) were analyzed by means of 4',6-diamidino-2-phenylindole (DAPI) / propidium iodide (PI) and chromomycin A3 (CMA) / DAPI fluorescence staining and fluorescent in situ hybridization using major rDNA, 5S rDNA, core histone genes, linker histone genes, and telomeric sequences as probes. The diploid chromosome number in this species is 2n = 30. The karyotype is composed of seven metacentric, one meta/submetacentric, and seven submetacentric chromosome pairs. Telomeric sequences appear at both ends of every single chromosome. Major rDNA clusters appear near the centromeres on chromosome pairs 1 and 3 and are associated with bright CMA fluorescence and dull DAPI fluorescence. This species shows five 5S rDNA clusters close to the centromeres on four chromosome pairs (2, 5, 6, and 8). Three of the four core histone gene clusters map to centromeric positions on chromosome pairs 7, 10, and 13. The fourth core histone gene cluster occupies a terminal position on chromosome pair 8, also bearing a 5S rDNA cluster. The two linker histone gene clusters are close to the centromeres on chromosome pairs 12 and 14. Therefore, the use of these probes allows the unequivocal identification of 11 of the 15 chromosome pairs that compose the karyotype of X. securis.

  16. Power-Law Solvation Dynamics in G-Quadruplex DNA: Role of Hydration Dynamics on Ligand Solvation inside DNA.

    PubMed

    Pal, Nibedita; Shweta, Him; Singh, Moirangthem Kiran; Verma, Sachin Dev; Sen, Sobhan

    2015-05-07

    G-quadruplex DNA (GqDNA) structures act as promising anticancer targets for small-molecules (ligands). Solvation dynamics of a ligand (DAPI: 4',6-diamidino-2-phenylindole) inside antiparallel-GqDNA is studied through direct comparison of time-resolved experiments to molecular dynamics (MD) simulation. Dynamic Stokes shifts of DAPI in GqDNA prepared in H2O buffer and D2O are compared to find the effect of water on ligand solvation. Experimental dynamics (in H2O) is then directly compared with the dynamics computed from 65 ns simulation on the same DAPI-GqDNA complex. Ligand solvation follows power-law relaxation (summed with fast exponential relaxation) from ~100 fs to 10 ns. Simulation results show relaxation below ~5 ps is dominated by water motion, while both water and DNA contribute comparably to dictate long-time power-law dynamics. Ion contribution is, however, found to be negligible. Simulation results also suggest that anomalous solvation dynamics may have origin in subdiffusive motion of perturbed water near GqDNA.

  17. Dermal fibroblasts participate in the formation of new muscle fibres when implanted into regenerating normal mouse muscle.

    PubMed

    Pye, D; Watt, D J

    2001-02-01

    Both in vitro and in vivo studies have described the conversion of fibroblasts to myogenesis when in the presence of dysfunctional myogenic cells. Myogenic conversion of fibroblasts subjected to a normal, as opposed to a diseased muscle environment has only been reported in vitro. The primary aim of this work was to determine if fibroblasts can convert to a myogenic lineage and contribute to new fibre formation when implanted into the regenerating muscle of a normal mouse. Dermal fibroblasts were prepared from neonatal mouse skin and labelled prior to implantation with the fluorescent nuclear marker 4',6-diamidino-2-phenylindole (DAPI). Cells were implanted into muscles of host mice that had been subjected to either cold/crush or minced muscle injury. Some host muscles were x-irradiated to deplete the muscle of endogenous muscle precursor cells. Muscles were removed at 3 wk postimplantation and analysed both histologically and for the presence of DAPI labelled nuclei. Fibres containing DAPI labelled central nuclei indicated that the implanted cells had participated in the regenerative process. Mouse dermal fibroblasts therefore do contribute to muscle fibre formation in regenerating normal mouse muscle but the extent of their contribution is dependent on the nature of the trauma induced in the host muscle. The study also showed that regeneration was more successful in muscles which had not been irradiated, which is contrary to the previous studies where dermal fibroblasts were introduced into myopathic mouse muscle.

  18. Preliminary Research of Relationship between Acute Peritonitis and Celiac Primo Vessels

    PubMed Central

    Wang, Xiaoyu; Shi, Hong; Cui, Jingjing; Bai, Wanzhu; He, Wei; Shang, Hongyan; Su, Yangshuai; Xin, Juanjuan; Jing, Xianghong; Zhu, Bing

    2013-01-01

    Previous studies demonstrated that primo vessels (PVs) were distributed in different parts of the body in mammals, and PVs were also involved in some processes of pathology such as cancer. Whether PVs are intrinsic structures in mammals or not is still ignored. In this study, a peritonitis model rat was induced by i.p. administration of E. coli in rats. PVs were observed in all infected rats, but it appeared less in untreated rats, taking 10.53% (4/38). In addition, we examined cell types in celiac PVs by fluorescent staining with 4′,6-diamidino-2-phenylindole (DAPI) and Alexa Fluor 488 phalloidin, as well as immunofluorescent staining with CD11b and intercellular adhesion molecule-1(ICAM-1), and found the following. (1) The rod-shaped nuclei aligned longitudinally along PVs. (2) DAPI-, phalloidin-, CD11b-, and ICAM-1-positive labeling coexisted in PVs, suggesting that fibroblasts and leucocytes might be two kinds of cell types in PVs for both infected and control rats. (3) The difference was that numerous cells in PVs of the infected rats contained DAPI-labeled multilobal nucleus and were expressed with CD11b- and ICAM-1-positive labeling on the cytoplasm and membrane, showing the typical characteristics of neutrophil. (4) The cells in PVs from the untreated rats are those of loose connective tissue. Therefore, it is reasonably considered that PVs from infected rats might be the pathological products which might be involved in inflammation. PMID:24069050

  19. Dermal fibroblasts participate in the formation of new muscle fibres when implanted into regenerating normal mouse muscle

    PubMed Central

    PYE, DEBORAH; WATT, DIANA J.

    2001-01-01

    Both in vitro and in vivo studies have described the conversion of fibroblasts to myogenesis when in the presence of dysfunctional myogenic cells. Myogenic conversion of fibroblasts subjected to a normal, as opposed to a diseased muscle environment has only been reported in vitro. The primary aim of this work was to determine if fibroblasts can convert to a myogenic lineage and contribute to new fibre formation when implanted into the regenerating muscle of a normal mouse. Dermal fibroblasts were prepared from neonatal mouse skin and labelled prior to implantation with the fluorescent nuclear marker 4′,6-diamidino-2-phenylindole (DAPI). Cells were implanted into muscles of host mice that had been subjected to either cold/crush or minced muscle injury. Some host muscles were x-irradiated to deplete the muscle of endogenous muscle precursor cells. Muscles were removed at 3 wk postimplantation and analysed both histologically and for the presence of DAPI labelled nuclei. Fibres containing DAPI labelled central nuclei indicated that the implanted cells had participated in the regenerative process. Mouse dermal fibroblasts therefore do contribute to muscle fibre formation in regenerating normal mouse muscle but the extent of their contribution is dependent on the nature of the trauma induced in the host muscle. The study also showed that regeneration was more successful in muscles which had not been irradiated, which is contrary to the previous studies where dermal fibroblasts were introduced into myopathic mouse muscle. PMID:11273041

  20. Visualizing light-triggered release of molecules inside living cells.

    PubMed

    Huschka, Ryan; Neumann, Oara; Barhoumi, Aoune; Halas, Naomi J

    2010-10-13

    The light-triggered release of deoxyribonucleic acid (DNA) from gold nanoparticle-based, plasmon resonant vectors, such as nanoshells, shows great promise for gene delivery in living cells. Here we show that intracellular light-triggered release can be performed on molecules that associate with the DNA in a DNA host-guest complex bound to nanoshells. DAPI (4',6-diamidino-2-phenylindole), a bright blue fluorescent molecule that binds reversibly to double-stranded DNA, was chosen to visualize this intracellular light-induced release process. Illumination of nanoshell-dsDNA-DAPI complexes at their plasmon resonance wavelength dehybridizes the DNA, releasing the DAPI molecules within living cells, where they diffuse to the nucleus and associate with the cell's endogenous DNA. The low laser power and irradiation times required for molecular release do not compromise cell viability. This highly controlled co-release of nonbiological molecules accompanying the oligonucleotides could have broad applications in the study of cellular processes and in the development of intracellular targeted therapies.

  1. In vitro effect of octenidine dihydrochloride against Trichomonas vaginalis.

    PubMed

    Küng, Erik; Pietrzak, Jacek; Klaus, Christoph; Walochnik, Julia

    2016-03-01

    Trichomoniasis is the most common non-viral sexually transmitted disease. It is associated with a wide spectrum of complications, including infertility and increased susceptibility to human immunodeficiency virus (HIV). A rising number of reports of Trichomonas vaginalis strains resistant to metronidazole has driven the search for new compounds. In the present study, the in vitro effects of the common antiseptic octenidine dihydrochloride against T. vaginalis were tested on metronidazole-resistant and -susceptible strains. Assays were performed under microaerophilic conditions in three different media containing varying concentrations of protein. It was shown that octenidine dihydrochloride is highly effective against T. vaginalis, with no difference between metronidazole-resistant and -susceptible strains. The 50% effective concentration (EC50) values ranged from 5.7 to 21.37μg/mL after 5min, from 6.48 to 10.82μg/mL after 15min and from 0.68 to 2.11μg/mL after 30min of treatment depending on the protein concentration of the test medium. Octenidine dihydrochloride, already approved in some countries for the treatment of bacterial and fungal vaginal infections, appears to be a promising alternative treatment for trichomoniasis, particularly in mixed vaginal infections or in cases caused by metronidazole-resistant strains.

  2. Karyotype evolution in the genus Jacaranda Juss. (Jacarandeae, Bignoniaceae): chromosome numbers and heterochromatin.

    PubMed

    Cordeiro, J M P; Lima, S A A; Paz, S N; Santos, A M S; Felix, L P

    2016-10-17

    Most taxa in the Bignoniaceae have 2n = 40, but the basal clade Jacarandeae has 2n = 36, suggesting that x = 18 is the ancestral basic number for the family. Variations in heterochromatin band patterns in genera that are numerically stable, such as Jacaranda, could facilitate our understanding of the chromosomal and karyotypic evolution of the family. We characterized heterochromatin distributions in six Jacaranda species using chromomycin A3 (CMA) and 4'6-diamidino-2-phenylindole (DAPI). All of them had 2n = 36, including first counts for Jacaranda bracteata Bureau & K. Schum., Jacaranda irwinii A.H. Gentry, Jacaranda jasminoides (Thunb.) Sandwith, and Jacaranda rugosa A.H. Gentry. Their karyotypes had four to eight terminal CMA(+)/DAPI(-) bands per monoploid set. In the section Monolobos, Jacaranda brasiliana (Lam.) Pers. had eight terminal bands and Jacaranda mimosifolia D. Don had four; in the section Dilobos, J. bracteata had six bands per monoploid set, with the other species having five. While three species in the section Dilobos had the same number of terminal bands, J. irwinii had two additional pericentromeric bands and a proximal heterozygotic band, and J. bracteata had two distended CMA bands. The consistent records of 2n = 36 in Jacaranda may represent a plesiomorphic condition for the Bignoniaceae; therefore, the family originated from an ancestor with x = 18. However, 2n = 36 may represent a derived condition, and the family could have had an ancestral basic number of x = 20 that is still conserved in most representatives of the family.

  3. Applicability of LIVE/DEAD BacLight stain with glutaraldehyde fixation for the measurement of bacterial abundance and viability in rainwater.

    PubMed

    Hu, Wei; Murata, Kotaro; Zhang, Daizhou

    2017-01-01

    Rainwater contains substantial bacteria and rain is an efficient pathway for the dissemination of bacteria from the atmosphere to land and water surfaces. However, quantitative information on rainwater bacteria is very limited due to the lack of a reliable method. In this study, the epifluorescence microscopy enumeration with the LIVE/DEAD BacLight Bacterial Viability Kit stain was verified to quantify the abundance of viable and non-viable bacterial cells in rainwater, with the 4',6-diamidino-2-phenylindole (DAPI) stain for the reference of total cell counts. Results showed that the total counts of bacterial cells by LIVE/DEAD BacLight staining were consistent with those by DAPI staining, and the average detection efficiency was (109±29)%. The ratio of cell count with glutaraldehyde fixation to that without fixation was (106±5)% on average. The bacterial concentration in negative control was usually an order of magnitude lower than that in rainwater samples. However, in case of small precipitation, the abundance in negative control could be more than that in rainwater samples. These results indicate that the enumeration with LIVE/DEAD BacLight bacterial viability assay coupled with glutaraldehyde fixation and careful negative control investigation is an approach applicable to the measurement of the concentration and viability of bacterial cells in rainwater.

  4. Spinal cord injury in rats treated using bone marrow mesenchymal stem-cell transplantation.

    PubMed

    Chen, Yu-Bing; Jia, Quan-Zhang; Li, Dong-Jun; Sun, Jing-Hai; Xi, Shuang; Liu, Li-Ping; Gao, De-Xuan; Jiang, Da-Wei

    2015-01-01

    The aim of this study was to observe the effects of bone marrow mesenchymal stem-cell transplantation (BMSCs) in repairing acute spinal cord damage in rats and to examine the potential beneficial effects. 192 Wistar rats were randomized into 8 groups. Spinal cord injury was created. Behavior and limb functions were scored. Repairing effects of BMSCs transplantation was evaluated and compared. In vitro 4',6-diamidino-2-phenylindole (DAPI)-tagged BMSCs were observed, and whether they migrated to the area of spinal cord injury after intravenous tail injection was investigated. The expression of neuron-specific protein (NSE) on BMSCs was examined. Fifteen days after transplantation, the BMSCs-treated groups scored significantly higher in limb function tests than the untreated group. Pathological sections of the bone marrow after operation showed significant recovery in treated groups in comparison to the control group. After transplantation, small amounts of fluorescent-tagged BMSCs can be found in the blood vessels in the area of spinal cord injury, and fluorescent-tagged BMSCs were diffused in extravascular tissues, whereas the DAPI-tagged BMSCs could not be detected,and BrdU/NSE double-labeled cells were found in the injured marrow. BMSCs improve behavioral responses and can repair spinal cord injuries by migrating to the injured area, where they can differentiate into neurons.

  5. Impacts of viability and purification on the specific gravity of Cryptosporidium oocysts.

    PubMed

    Young, Pamela L; Komisar, Simeon J

    2005-09-01

    The specific gravity of unpurified and purified Cryptosporidium oocysts was measured using an isopycnic gradient centrifugation technique. Specific gravity varied depending on the viability of the oocysts, as defined by permeabilty to 4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI), the presence or absence of internal structures, and whether or not oocysts were purified. The modal range of densities for a population of 1.4-week-old unpurified oocysts, was 1070-1073 kg/m3. This range was higher than that determined for 14-week-old purified oocysts, 1067-1070 kg/m3. Eleven- and 12-week-old unpurified populations exhibited a bimodal distribution of densities, with densities most frequently in the 1005-1041 and in the 1077-1108 kg/m3 range. In these populations, a high percentage of the oocysts having densities greater than 1077 kg/m3 were viable, while oocysts in the 1005-1024 kg/m3 range were predominately nonintact, and oocysts in the 1024-1041 kg/m3 range were intact, but permeable to DAPI and PI (nonviable). This work demonstrates the importance of controlling factors that may impact the viability of oocysts when conducting studies that examine the transport of these microorganisms in the environment and through water treatment processes.

  6. Novel Threadlike Structures May Be Present on the Large Animal Organ Surface: Evidence in Swine Model

    PubMed Central

    Bae, Kyoung-Hee; Park, Sang Hyun; Lee, Byung-Cheon; Nam, Min-Ho; Yoon, Ji Woong; Kwon, Hee-Min; Yoon, Seung Zhoo

    2013-01-01

    Background. The types of embryonic development probably provoke different paths of novel threadlike structure (NTS) development. The authors hypothesized that NTS may be easily observed on the surface of swine intestines by using trypan blue staining method and visualization under an optical microscope. Methods. General anesthesia was administered to 2 Yorkshire pigs. The abdominal walls of the pigs were carefully dissected along the medial alba. NTSs were identified on organ surfaces under a stereoscopic microscope after trypan blue staining. Isolated NTS specimens obtained from the large intestine were subjected to 4′,6-diamidino-2-phenylindole (DAPI) staining and observed using the polarized light microscopy to confirm whether the obtained structure fits the definition of NTS. Results. We found elastic, semitransparent threadlike structures (forming a network structure) that had a milky-white color in situ and in vivo in swine large intestines. The samples showed distinct extinction of polarized light at every 90 degrees, and nucleus was shown to be rod shaped by DAPI staining, indicating that they meet the criteria of NTS. Conclusion. We used a swine model to demonstrate that NTS may be present on large animal organ surfaces. Our results may permit similar studies by using human specimens. PMID:23762159

  7. In vitro Antiviral Activity of Rubia cordifolia Aerial Part Extract against Rotavirus

    PubMed Central

    Sun, Yuanyuan; Gong, Xuepeng; Tan, Jia Y.; Kang, Lifeng; Li, Dongyan; Vikash; Yang, Jihong; Du, Guang

    2016-01-01

    The root of Rubia cordifolia has been used traditionally as a hemostatic agent, while the aerial part of the plant consisting of leaf and stem is known to exhibit anti-diarrheal properties and has been widely used as a remedy in many parts of China. As rotavirus is one of the most commonly associated diarrhea-causing pathogen, this study aims to investigate the anti-rotaviral effect of R. cordifolia aerial part (RCAP). The cytotoxicity of RCAP toward MA-104 cells was evaluated using the WST-8 assay. Colloidal gold method and real time polymerase chain reaction (qPCR) assay were used to confirm the findings of the antiviral assay. Then, 4′,6-diamidino-2-phenylindole (DAPI) staining method was subsequently used to investigate the mode of death among the cells. And the representative components of aqueous extract were isolated and identified. It was shown that both the viability of MA-104 cells and the viral load were reduced with increasing concentration of the extract. DAPI staining showed that virus-induced apoptosis was the cause of the low cell viability and viral load, an effect which was accelerated with incubation in the aqueous herbal extract. The major compounds postulated to exhibit this activity were isolated from the aqueous herbal extract and identified to be compounds Xanthopurpurin and Vanillic Acid. This study showed that RCAP extract effectively inhibited rotavirus multiplication by promoting virus-induced apoptosis in MA-104 cells. PMID:27679574

  8. Fluorometric Quantification of Polyphosphate in Environmental Plankton Samples: Extraction Protocols, Matrix Effects, and Nucleic Acid Interference

    PubMed Central

    Martin, Patrick

    2013-01-01

    Polyphosphate (polyP) is a ubiquitous biochemical with many cellular functions and comprises an important environmental phosphorus pool. However, methodological challenges have hampered routine quantification of polyP in environmental samples. We tested 15 protocols to extract inorganic polyphosphate from natural marine samples and cultured cyanobacteria for fluorometric quantification with 4′,6-diamidino-2-phenylindole (DAPI) without prior purification. A combination of brief boiling and digestion with proteinase K was superior to all other protocols, including other enzymatic digestions and neutral or alkaline leaches. However, three successive extractions were required to extract all polyP. Standard addition revealed matrix effects that differed between sample types, causing polyP to be over- or underestimated by up to 50% in the samples tested here. Although previous studies judged that the presence of DNA would not complicate fluorometric quantification of polyP with DAPI, we show that RNA can cause significant interference at the wavelengths used to measure polyP. Importantly, treating samples with DNase and RNase before proteinase K digestion reduced fluorescence by up to 57%. We measured particulate polyP along a North Pacific coastal-to-open ocean transect and show that particulate polyP concentrations increased toward the open ocean. While our final method is optimized for marine particulate matter, different environmental sample types may need to be assessed for matrix effects, extraction efficiency, and nucleic acid interference. PMID:23104409

  9. A hotspot for cold crenarchaeota in the neuston of high mountain lakes.

    PubMed

    Auguet, Jean-Christophe; Casamayor, Emilio O

    2008-04-01

    We have surveyed the first 1 m of 10 oligotrophic high mountain lakes in the Central Pyrenees (Spain) for both abundance and predominant phylotypes richness of the archaeaplankton assemblage, using CARD-FISH and 16S rRNA gene sequencing respectively. Archaea inhabiting the air-water surface microlayer (neuston) ranged between 3% and 37% of total 4,6-diamidino-2-phenylindole (DAPI) counts and were mainly Crenarchaeota of a new freshwater cluster distantly related to the Marine Group 1.1a. Conversely, most of the Archaea from the underlying waters (the remaining first 1 m integrated) were mainly Euryarchaeota of three distantly related branches ranging between 0.4% and 27% of total DAPI counts. Therefore, a consistent qualitative and quantitative spatial segregation was observed for the two main archaeal phyla between neuston and underlying waters at a regional scale. We also observed a consistent pattern along the lakes surveyed between lake area, lake depth and water residence time, and the archaeal enrichment in the neuston: the larger the lake the higher the proportion of archaea in the neuston as compared with abundances from the underlying waters (n = 10 lakes; R(2) > 0.80; P < 0.001, in all three cases). This is the first report identifying a widespread non-thermophilic habitat where freshwater planktonic Crenarchaeota can be found naturally enriched. High mountain lakes offer great research opportunities to explore the ecology of one of the most enigmatic and far from being understood group of prokaryotes.

  10. Novel threadlike structures may be present on the large animal organ surface: evidence in Swine model.

    PubMed

    Bae, Kyoung-Hee; Park, Sang Hyun; Lee, Byung-Cheon; Nam, Min-Ho; Yoon, Ji Woong; Kwon, Hee-Min; Yoon, Seung Zhoo

    2013-01-01

    Background. The types of embryonic development probably provoke different paths of novel threadlike structure (NTS) development. The authors hypothesized that NTS may be easily observed on the surface of swine intestines by using trypan blue staining method and visualization under an optical microscope. Methods. General anesthesia was administered to 2 Yorkshire pigs. The abdominal walls of the pigs were carefully dissected along the medial alba. NTSs were identified on organ surfaces under a stereoscopic microscope after trypan blue staining. Isolated NTS specimens obtained from the large intestine were subjected to 4',6-diamidino-2-phenylindole (DAPI) staining and observed using the polarized light microscopy to confirm whether the obtained structure fits the definition of NTS. Results. We found elastic, semitransparent threadlike structures (forming a network structure) that had a milky-white color in situ and in vivo in swine large intestines. The samples showed distinct extinction of polarized light at every 90 degrees, and nucleus was shown to be rod shaped by DAPI staining, indicating that they meet the criteria of NTS. Conclusion. We used a swine model to demonstrate that NTS may be present on large animal organ surfaces. Our results may permit similar studies by using human specimens.

  11. A comparative cytogenetic study of Drosophila parasitoids (Hymenoptera, Figitidae) using DNA-binding fluorochromes and FISH with 45S rDNA probe.

    PubMed

    Gokhman, Vladimir E; Bolsheva, Nadezhda L; Govind, Shubha; Muravenko, Olga V

    2016-06-01

    Karyotypes of Leptopilina boulardi (Barbotin, Carton et Keiner-Pillault, 1979) (n = 9), L. heterotoma (Thomson, 1862) (n = 10), L. victoriae Nordlander, 1980 (n = 10) and Ganaspis xanthopoda (Ashmead, 1896) (n = 9) (Hymenoptera, Figitidae) were studied using DNA-binding ligands with different base specificity [propidium iodide (PI), chromomycin A3 (CMA3) and 4',6-diamidino-2-phenylindole (DAPI)], and fluorescence in situ hybridization (FISH) with a 45S rDNA probe. Fluorochrome staining was similar between the different fluorochromes, except for a single CMA3- and PI-positive and DAPI-negative band per haploid karyotype of each species. FISH with 45S rDNA probe detected a single rDNA site in place of the bright CMA3-positive band, thus identifying the nucleolus organizing region (NOR). Chromosomal locations of NORs were similar for both L. heterotoma and L. victoriae, but strongly differed in L. boulardi as well as in G. xanthopoda. Phylogenetic aspects of NOR localization in all studied species are briefly discussed.

  12. Peanut witches' broom (PnWB) phytoplasma-mediated leafy flower symptoms and abnormal vascular bundles development.

    PubMed

    Liu, Chi-Te; Huang, Hsin-Mei; Hong, Syuan-Fei; Kuo-Huang, Ling-Long; Yang, Chiao-Yin; Lin, Yen-Yu; Lin, Chan-Pin; Lin, Shih-Shun

    2015-01-01

    The peanut witches' broom (PnWB) phytoplasma causes virescence symptoms such as phyllody (leafy flower) in infected peanuts. However, the obligate nature of phytoplasma limits the study of host-pathogen interactions, and the detailed anatomy of PnWB-infected plants has yet to be reported. Here, we demonstrate that 4',6'-diamidino-2-phenylindole (DAPI) staining can be used to track PnWB infection. The DAPI-stained phytoplasma cells were observed in phloem/internal phloem tissues, and changes in vascular bundle morphology, including increasing pith rays and thinner cell walls in the xylem, were found. We also discerned the cell types comprising PnWB in infected sieve tube members. These results suggest that the presence of PnWB in phloem tissue facilitates the transmission of phytoplasma via sap-feeding insect vectors. In addition, PnWB in sieve tube members and changes in vascular bundle morphology might strongly promote the ability of phytoplasmas to assimilate nutrients. These data will help further an understanding of the obligate life cycle and host-pathogen interactions of phytoplasma.

  13. Nonuniform spatial patterns of respiratory activity within biofilms during disinfection.

    PubMed Central

    Huang, C T; Yu, F P; McFeters, G A; Stewart, P S

    1995-01-01

    Fluorescent stains in conjunction with cryoembedding and image analysis were applied to demonstrate spatial gradients in respiratory activity within bacterial biofilms during disinfection with monochloramine. Biofilms of Klebsiella pneumoniae and Pseudomonas aeruginosa grown together on stainless steel surfaces in continuous-flow annular reactors were treated with 2 mg of monochloramine per liter (influent concentration) for 2 h. Relatively little biofilm removal occurred as evidenced by total cell direct counts. Plate counts (of both species summed) indicated an average 1.3-log decrease after exposure to 2 mg of monochloramine per liter. The fluorogenic redox indicator 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and the DNA stain 4',6-diamidino-2-phenylindole (DAPI) were used to differentiate respiring and nonrespiring cells in biofilms. Epifluorescence micrographs of frozen biofilm cross sections clearly revealed gradients of respiratory activity within biofilms in response to monochloramine treatment. These gradients in specific respiratory activity were quantified by calculating the ratio of CTC and DAPI intensities measured by image analysis. Cells near the biofilm-bulk fluid interface lost respiratory activity first. After 2 h of biocide treatment, greater respiratory activity persisted deep in the biofilm than near the biofilm-bulk fluid interface. PMID:7793945

  14. A Red to Near-IR Fluorogen: Aggregation-Induced Emission, Large Stokes Shift, High Solid Efficiency and Application in Cell-Imaging.

    PubMed

    Wang, Yi Jia; Shi, Yang; Wang, Zhaoyang; Zhu, Zhenfeng; Zhao, Xinyuan; Nie, Han; Qian, Jun; Qin, Anjun; Sun, Jing Zhi; Tang, Ben Zhong

    2016-07-04

    A tetraphenylethene (TPE) derivative modified with the strong electron acceptor 2-dicyano-methylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (TCF) was obtained in high yield by a simple two-step reaction. The resultant TPE-TCF showed evident aggregation-induced emission (AIE) features and pronounced solvatochromic behavior. Changing the solvent from apolar cyclohexane to highly polar acetonitrile, the emission peak shifted from 560 to 680 nm (120 nm redshift). In an acetonitrile solution and in the solid powder, the Stokes shifts are as large as 230 and 190 nm, respectively. The solid film emits red to near-IR (red-NIR) fluorescence with an emission peak at 670 nm and a quantum efficiency of 24.8 %. Taking the advantages of red-NIR emission and high efficiency, nanoparticles (NPs) of TPE-TCF were fabricated by using tat-modified 1,2-distearoylsn-glycero-3-phosphor-ethanol-amine-N-[methoxy-(polyethyl-eneglycol)-2000] as the encapsulation matrix. The obtained NPs showed perfect membrane penetrability and high fluorescent imaging quality of cell cytoplasm. Upon co-incubation with 4,6-diamidino-2-phenylindole (DAPI) in the presence of tritons, the capsulated TPE-TCF nanoparticles could enter into the nucleus and displayed similar staining properties to those of DAPI.

  15. Determination of the drug-DNA binding modes using fluorescence-based assays.

    PubMed

    Williams, Alicia K; Dasilva, Sofia Cheliout; Bhatta, Ankit; Rawal, Baibhav; Liu, Melinda; Korobkova, Ekaterina A

    2012-03-15

    Therapeutic drugs and environmental pollutants may exhibit high reactivity toward DNA bases and backbone. Understanding the mechanisms of drug-DNA binding is crucial for predicting their potential genotoxicity. We developed a fluorescence analytical method for the determination of the preferential binding mode for drug-DNA interactions. Two nucleic acid dyes were employed in the method: TO-PRO-3 iodide (TP3) and 4',6-diamidino-2-phenylindole (DAPI). TP3 binds DNA by intercalation, whereas DAPI exhibits minor groove binding. Both dyes exhibit significant fluorescence magnification on binding to DNA. We evaluated the DNA binding constant, K(b), for each dye. We also performed fluorescence quenching experiments with 11 molecules of various structures and measured a C(50) value for each compound. We determined preferential binding modes for the aforementioned molecules and found that they bound to DNA consistently, as indicated by other studies. The values of the likelihood of DNA intercalation were correlated with the partition coefficients of the molecules. In addition, we performed nuclear magnetic resonance (NMR) studies of the interactions with calf thymus DNA for the three molecules. The results were consistent with the fluorescence method described above. Thus, we conclude that the fluorescence method we developed provides a reliable determination of the likelihoods of the two different DNA binding modes.

  16. Cytotoxic effects of MgO nanoparticles on human umbilical vein endothelial cells in vitro.

    PubMed

    Ge, S; Wang, G; Shen, Y; Zhang, Q; Jia, D; Wang, H; Dong, Q; Yin, T

    2011-06-01

    The MgO nanoparticles are widely used in many fields. However, the toxicity of these nanoparticles to cells and organs remains fairly undiscovered. In this study, the cytotoxicity of MgO nanoparticles on human umbilical vein endothelial cells (HUVECs) in vitro was examined. The morphology and size of MgO nanoparticles were analysed by the transmission electron microscope (TEM) and nanoparticle size analyser. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2 h-tetrazolium bromide) assay, 4',6-diamidino-2-phenylindole (DAPI) staining analysis, NO release and total antioxidation competence (T-AOC) assay were used to evaluate the cytotoxicity of MgO nanoparticles. The results showed that most MgO nanoparticles were spherical with agglomerated state and the diameter of single particle was about 100 nm. Meanwhile, low concentration (below 200 [micro sign]g/ml) of MgO nanoparticles suspension showed no cytotoxicity by MTT assay. However, once the concentration of MgO nanoparticles was higher than 500 [micro sign]g/ml, the relative growth rate was lower than the control. The DAPI staining analysis results showed no significant difference of the cells morphology between the groups with or without MgO nanoparticles. In addition, the MgO nanoparticles significantly enhanced the NO release and T-AOC content of the HUVECs. The testing results indicated that low concentration of MgO nanoparticles exhibited non-cytotoxicity.

  17. Visualization of Neutrophil Extracellular Traps and Fibrin Meshwork in Human Fibrinopurulent Inflammatory Lesions: III. Correlative Light and Electron Microscopic Study

    PubMed Central

    Onouchi, Takanori; Shiogama, Kazuya; Mizutani, Yasuyoshi; Takaki, Takashi; Tsutsumi, Yutaka

    2016-01-01

    Neutrophil extracellular traps (NETs) released from dead neutrophils at the site of inflammation represent webs of neutrophilic DNA stretches dotted with granule-derived antimicrobial proteins, including lactoferrin, and play important roles in innate immunity against microbial infection. We have shown the coexistence of NETs and fibrin meshwork in varied fibrinopurulent inflammatory lesions at both light and electron microscopic levels. In the present study, correlative light and electron microscopy (CLEM) employing confocal laser scanning microscopy and scanning electron microscopy was performed to bridge light and electron microscopic images of NETs and fibrin fibrils in formalin-fixed, paraffin-embedded, autopsied lung sections of legionnaire’s pneumonia. Lactoferrin immunoreactivity and 4'-6-diamidino-2-phenylindole (DAPI) reactivity were used as markers of NETs, and fibrin was probed by fibrinogen gamma chain. Of note is that NETs light microscopically represented as lactoferrin and DAPI-colocalized dots, 2.5 μm in diameter. CLEM gave super-resolution images of NETs and fibrin fibrils: “Dotted” NETs were ultrastructurally composed of fine filaments and masses of 58 nm-sized globular materials. A fibrin fibril consisted of clusters of smooth-surfaced filaments. NETs filaments (26 nm in diameter) were significantly thinner than fibrin filaments (295 nm in diameter). Of note is that CLEM was applicable to formalin-fixed, paraffin-embedded sections of autopsy material. PMID:27917008

  18. 21 CFR 520.1242c - Levamisole hydrochloride and piperazine dihydrochloride.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... piperazine dihydrochloride equivalent to 3.98 grams of piperazine base. (2) The drug is a soluble powder... and piperazine dihydrochloride equivalent to 5.0 grams of piperazine base. (b) Sponsor. See No. 053501... 100 pounds of body weight. Reconstituted soluble powder: administer by stomach tube 1 fluid ounce...

  19. Characterization of an autotrophic sulfide-oxidizing marine Arcobacter sp. that produces filamentous sulfur.

    PubMed

    Wirsen, C O; Sievert, S M; Cavanaugh, C M; Molyneaux, S J; Ahmad, A; Taylor, L T; DeLong, E F; Taylor, C D

    2002-01-01

    A coastal marine sulfide-oxidizing autotrophic bacterium produces hydrophilic filamentous sulfur as a novel metabolic end product. Phylogenetic analysis placed the organism in the genus Arcobacter in the epsilon subdivision of the Proteobacteria. This motile vibrioid organism can be considered difficult to grow, preferring to grow under microaerophilic conditions in flowing systems in which a sulfide-oxygen gradient has been established. Purified cell cultures were maintained by using this approach. Essentially all 4',6-diamidino-2-phenylindole dihydrochloride-stained cells in a flowing reactor system hybridized with Arcobacter-specific probes as well as with a probe specific for the sequence obtained from reactor-grown cells. The proposed provisional name for the coastal isolate is "Candidatus Arcobacter sulfidicus." For cells cultured in a flowing reactor system, the sulfide optimum was higher than and the CO(2) fixation activity was as high as or higher than those reported for other sulfur oxidizers, such as Thiomicrospira spp. Cells associated with filamentous sulfur material demonstrated nitrogen fixation capability. No ribulose 1,5-bisphosphate carboxylase/oxygenase could be detected on the basis of radioisotopic activity or by Western blotting techniques, suggesting an alternative pathway of CO(2) fixation. The process of microbial filamentous sulfur formation has been documented in a number of marine environments where both sulfide and oxygen are available. Filamentous sulfur formation by "Candidatus Arcobacter sulfidicus" or similar strains may be an ecologically important process, contributing significantly to primary production in such environments.

  20. Alpha-phellandrene-induced DNA damage and affect DNA repair protein expression in WEHI-3 murine leukemia cells in vitro.

    PubMed

    Lin, Jen-Jyh; Wu, Chih-Chung; Hsu, Shu-Chun; Weng, Shu-Wen; Ma, Yi-Shih; Huang, Yi-Ping; Lin, Jaung-Geng; Chung, Jing-Gung

    2015-11-01

    Although there are few reports regarding α-phellandrene (α-PA), a natural compound from Schinus molle L. essential oil, there is no report to show that α-PA induced DNA damage and affected DNA repair associated protein expression. Herein, we investigated the effects of α-PA on DNA damage and repair associated protein expression in murine leukemia cells. Flow cytometric assay was used to measure the effects of α-PA on total cell viability and the results indicated that α-PA induced cell death. Comet assay and 4,6-diamidino-2-phenylindole dihydrochloride staining were used for measuring DNA damage and condensation, respectively, and the results indicated that α-PA induced DNA damage and condensation in a concentration-dependent manner. DNA gel electrophoresis was used to examine the DNA damage and the results showed that α-PA induced DNA damage in WEHI-3 cells. Western blotting assay was used to measure the changes of DNA damage and repair associated protein expression and the results indicated that α-PA increased p-p53, p-H2A.X, 14-3-3-σ, and MDC1 protein expression but inhibited the protein of p53, MGMT, DNA-PK, and BRCA-1.

  1. Peroxiredoxin I and II in human eyes: cellular distribution and association with pterygium and DNA damage.

    PubMed

    Klebe, Sonja; Callahan, Thomas; Power, John H T

    2014-01-01

    Peroxiredoxin I and II are both 2-Cys members of the peroxiredoxin family of antioxidant enzymes and inactivate hydrogen peroxide. On western blotting, both enzymes appeared as 22-kD proteins and were present in the sclera, retina and iris. Immunohistochemistry showed strong cytoplasmic labeling in the basal cells of the corneal epithelial layer and the corneoscleral limbus. The melanocytes within the stroma of the iris and the anterior epithelial cells of the lens also showed strong cytoplasmic labeling. The fibrous structure of the stroma and the posterior surface of the ciliary body were also labeled. There was also strong labeling for both enzymes in the photoreceptors and the inner and outer plexiform layers of the retina. There was increased labeling of peroxiredoxin I and II in pterygium. In normal conjunctiva and cornea, only the basal cell layer showed labeling for peroxiredoxin I and II, whereas, in pterygia, there was strong cytoplasmic labeling in most cells involving the full thickness of the epithelium. Co-localization of the DNA oxidation product 8-hydroxy-2'-deoxyguanosine antibody with the nuclear dye 4',6'-diamidino-2-phenylindole dihydrochloride indicated that the majority of the oxidative damage was cytoplasmic; this suggested that the mitochondrial DNA was most affected by the UV radiation in this condition.

  2. Inhibition of proteasome activity is involved in cobalt-induced apoptosis of human alveolar macrophages.

    PubMed

    Araya, Jun; Maruyama, Muneharu; Inoue, Akira; Fujita, Tadashi; Kawahara, Junko; Sassa, Kazuhiko; Hayashi, Ryuji; Kawagishi, Yukio; Yamashita, Naohiro; Sugiyama, Eiji; Kobayashi, Masashi

    2002-10-01

    Inhalation of particulate cobalt has been known to induce interstitial lung disease. There is growing evidence that apoptosis plays a crucial role in physiological and pathological settings and that the ubiquitin-proteasome system is involved in the regulation of apoptosis. Cadmium, the same transitional heavy metal as cobalt, has been reported to accumulate ubiquitinated proteins in neuronal cells. On the basis of these findings, we hypothesized that cobalt would induce apoptosis in the lung by disturbance of the ubiquitin-proteasome pathway. To evaluate this, we exposed U-937 cells and human alveolar macrophages (AMs) to cobalt chloride (CoCl(2)) and examined their apoptosis by DNA fragmentation assay, 4',6-diamidino-2'-phenylindol dihydrochloride staining, and Western blot analysis. CoCl(2) induced apoptosis and accumulated ubiquitinated proteins. Exposure to CoCl(2) inhibited proteasome activity in U-937 cells. Cobalt-induced apoptosis was mediated via mitochondrial pathway because CoCl(2) released cytochrome c from mitochondria. These results suggest that cobalt-induced apoptosis of AMs may be one of the mechanisms for cobalt-induced lung injury and that the accumulation of ubiquitinated proteins might be involved in this apoptotic process.

  3. Three-dimensional reconstruction of painted human interphase chromosomes: active and inactive X chromosome territories have similar volumes but differ in shape and surface structure

    PubMed Central

    1996-01-01

    This study provides a three-dimensional (3D) analysis of differences between the 3D morphology of active and inactive human X interphase chromosomes (Xa and Xi territories). Chromosome territories were painted in formaldehyde-fixed, three-dimensionally intact human diploid female amniotic fluid cell nuclei (46, XX) with X-specific whole chromosome compositive probes. The colocalization of a 4,6-diamidino-2- phenylindole dihydrochloride-stained Barr body with one of the two painted X territories allowed the unequivocal discrimination of the inactive X from its active counterpart. Light optical serial sections were obtained with a confocal laser scanning microscope. 3D- reconstructed Xa territories revealed a flatter shape and exhibited a larger and more irregular surface when compared to the apparently smoother surface and rounder shape of Xi territories. The relationship between territory surface and volume was quantified by the determination of a dimensionless roundness factor (RF). RF and surface area measurements showed a highly significant difference between Xa and Xi territories (P < 0.001) in contrast to volume differences (P > 0.1). For comparison with an autosome of similar DNA content, chromosome 7 territories were additionally painted. The 3D morphology of the chromosome 7 territories was similar to the Xa territory but differed strongly from the Xi territory with respect to RF and surface area (P < 0.001). PMID:8978813

  4. Mitotic catastrophe is a putative mechanism underlying the weak correlation between sensitivity to carbon ions and cisplatin

    PubMed Central

    Kobayashi, Daijiro; Oike, Takahiro; Shibata, Atsushi; Niimi, Atsuko; Kubota, Yoshiki; Sakai, Makoto; Amornwhichet, Napapat; Yoshimoto, Yuya; Hagiwara, Yoshihiko; Kimura, Yuka; Hirota, Yuka; Sato, Hiro; Isono, Mayu; Yoshida, Yukari; Kohno, Takashi; Ohno, Tatsuya; Nakano, Takashi

    2017-01-01

    In cancer therapy today, carbon ion radiotherapy is used mainly as monotherapy, whereas cisplatin is used concomitantly with X-ray radiotherapy. The effectiveness of concomitant carbon ions and cisplatin is unclear. To obtain the information on the mechanisms potentially shared between carbon ions or X-rays and cisplatin, we assessed the correlation of sensitivity to the single treatments. In 20 human cancer cell lines, sensitivity to X-rays strongly correlated with sensitivity to cisplatin, indicating the presence of potentially shared target mechanisms. Interestingly, the correlation of sensitivity to carbon ions and cisplatin was much weaker than that of sensitivity to X-rays and cisplatin, indicating the presence of potentially different target mechanisms between carbon ions and cisplatin. Assessment of clonogenic cell death by 4′,6-diamidino-2-phenylindole dihydrochloride staining showed that mitotic catastrophe was more efficiently induced by carbon ions than by the same physical dose of X-rays, while apoptosis and senescence were not. These data indicate that the correlation of sensitivity to carbon ions and cisplatin is weaker than that of sensitivity to X-rays and cisplatin, which are helpful as biological basis to understand the potentially shared mechanism among these treatments. Further investigation is mandatory to elucidate the clinical efficacy of carbon ions and cisplatin combination. PMID:28091564

  5. Betahistine dihydrochloride in the treatment of peripheral vestibular vertigo.

    PubMed

    Mira, Eugenio; Guidetti, G; Ghilardi, L; Fattori, B; Malannino, N; Maiolino, L; Mora, R; Ottoboni, S; Pagnini, P; Leprini, M; Pallestrini, E; Passali, D; Nuti, D; Russolo, M; Tirelli, G; Simoncelli, C; Brizi, S; Vicini, C; Frasconi, P

    2003-02-01

    The present study compares the efficacy and safety of betahistine dihydrochloride to that of a placebo in recurrent vertigo resulting from Meniere's disease (MD) or in paroxysmal positional vertigo (PPV) of probable vascular origin. The design was double-blind, multicentre and parallel-group randomised. Eleven Italian centres enrolled 144 patients: 75 of the patients were treated with betahistine (41 MD/34 PPV) and 69 with placebos (40 MD/29 PPV). The betahistine dosage was 16 mg twice per day for 3 months. Compared to the placebo, betahistine had a significant effect on the frequency, intensity and duration of vertigo attacks. Associated symptoms and the quality of life also were significantly improved by betahistine. Both the physician's judgement and the patient's opinion on the efficacy and acceptability of the treatment were in agreement as to the superiority of betahistine. The effective and safe profile of betahistine in the treatment of vertigo due to peripheral vestibular disorders was confirmed.

  6. Quantum dots-based double imaging combined with organic dye imaging to establish an automatic computerized method for cancer Ki67 measurement

    NASA Astrophysics Data System (ADS)

    Wang, Lin-Wei; Qu, Ai-Ping; Liu, Wen-Lou; Chen, Jia-Mei; Yuan, Jing-Ping; Wu, Han; Li, Yan; Liu, Juan

    2016-02-01

    As a widely used proliferative marker, Ki67 has important impacts on cancer prognosis, especially for breast cancer (BC). However, variations in analytical practice make it difficult for pathologists to manually measure Ki67 index. This study is to establish quantum dots (QDs)-based double imaging of nuclear Ki67 as red signal by QDs-655, cytoplasmic cytokeratin (CK) as yellow signal by QDs-585, and organic dye imaging of cell nucleus as blue signal by 4‧,6-diamidino-2-phenylindole (DAPI), and to develop a computer-aided automatic method for Ki67 index measurement. The newly developed automatic computerized Ki67 measurement could efficiently recognize and count Ki67-positive cancer cell nuclei with red signals and cancer cell nuclei with blue signals within cancer cell cytoplasmic with yellow signals. Comparisons of computerized Ki67 index, visual Ki67 index, and marked Ki67 index for 30 patients of 90 images with Ki67 ≤ 10% (low grade), 10% < Ki67 < 50% (moderate grade), and Ki67 ≥ 50% (high grade) showed computerized Ki67 counting is better than visual Ki67 counting, especially for Ki67 low and moderate grades. Based on QDs-based double imaging and organic dye imaging on BC tissues, this study successfully developed an automatic computerized Ki67 counting method to measure Ki67 index.

  7. Cell-death assessment by fluorescent and nonfluorescent cytosolic and nuclear staining techniques.

    PubMed

    Atale, N; Gupta, S; Yadav, U C S; Rani, V

    2014-07-01

    Apoptosis, a genetically programmed cellular event leads to biochemical and morphological changes in cells. Alterations in DNA caused by several factors affect nucleus and ultimately the entire cell leading to compromised function of the organ and organism. DNA, a master regulator of the cellular events, is an important biomolecule with regards to cell growth, cell death, cell migration and cell differentiation. It is therefore imperative to develop the staining techniques that may lead to visualize the changes in nucleus where DNA is housed, to comprehend the cellular pathophysiology. Over the years a number of nuclear staining techniques such as propidium iodide, Hoechst-33342, 4', 6-diamidino-2-phenylindole (DAPI), Acridine orange-Ethidium bromide staining, among others have been developed to assess the changes in DNA. Some nonnuclear staining techniques such as Annexin-V staining, which although does not stain DNA, but helps to identify the events that result from DNA alteration and leads to initiation of apoptotic cell death. In this review, we have briefly discussed some of the most commonly used fluorescent and nonfluorescent staining techniques that identify apoptotic changes in cell, DNA and the nucleus. These techniques help in differentiating several cellular and nuclear phenotypes that result from DNA damage and have been identified as specific to necrosis or early and late apoptosis as well as scores of other nuclear deformities occurring inside the cells.

  8. Three-dimensional direct measurement of cardiomyocyte volume, nuclearity, and ploidy in thick histological sections

    NASA Astrophysics Data System (ADS)

    Bensley, Jonathan Guy; de Matteo, Robert; Harding, Richard; Black, Mary Jane

    2016-04-01

    Quantitative assessment of myocardial development and disease requires accurate measurement of cardiomyocyte volume, nuclearity (nuclei per cell), and ploidy (genome copies per cell). Current methods require enzymatically isolating cells, which excludes the use of archived tissue, or serial sectioning. We describe a method of analysis that permits the direct simultaneous measurement of cardiomyocyte volume, nuclearity, and ploidy in thick histological sections. To demonstrate the utility of our technique, heart tissue was obtained from four species (rat, mouse, rabbit, sheep) at up to three life stages: prenatal, weaning and adulthood. Thick (40 μm) paraffin sections were stained with Wheat Germ Agglutinin-Alexa Fluor 488 to visualise cell membranes, and DAPI (4‧,6-diamidino-2-phenylindole) to visualise nuclei and measure ploidy. Previous methods have been restricted to thin sections (2–10 μm) and offer an incomplete picture of cardiomyocytes. Using confocal microscopy and three-dimensional image analysis software (Imaris Version 8.2, Bitplane AG, Switzerland), cardiomyocyte volume, nuclearity, and ploidy were measured. This method of staining and analysis of cardiomyocytes enables accurate morphometric measurements in thick histological sections, thus unlocking the potential of archived tissue. Our novel time-efficient method permits the entire cardiomyocyte to be visualised directly in 3D, eliminating the need for precise alignment of serial sections.

  9. Comparison of vertical distributions of prokaryotic assemblages in the anoxic Cariaco Basin and Black Sea by use of fluorescence in situ hybridization.

    PubMed

    Lin, Xueju; Wakeham, Stuart G; Putnam, Isabell F; Astor, Yrene M; Scranton, Mary I; Chistoserdov, Andrei Y; Taylor, Gordon T

    2006-04-01

    Individual prokaryotic cells from two major anoxic basins, the Cariaco Basin and the Black Sea, were enumerated throughout their water columns using fluorescence in situ hybridization (FISH) with the fluorochrome Cy3 or horseradish peroxidase-modified oligonucleotide probes. For both basins, significant differences in total prokaryotic abundance and phylogenetic composition were observed among oxic, anoxic, and transitional (redoxcline) waters. Epsilon-proteobacteria, Crenarchaeota, and Euryarchaeota were more prevalent in the redoxclines, where previous studies reported high rates of chemoautotrophic production relative to those in waters above and below the redoxclines. Relative abundances of Archaea in both systems varied between 1% and 28% of total prokaryotes, depending on depth. The prokaryotic community composition varied between the two anoxic basins, consistent with distinct geochemical and physical conditions. In the Black Sea, the relative contributions of group I Crenarchaeota (median, 5.5%) to prokaryotic communities were significantly higher (P < 0.001; n = 20) than those of group II Euryarchaeota (median, 2.9%). In contrast, their proportions were nearly equivalent in the Cariaco Basin. Beta-proteobacteria were unexpectedly common throughout the Cariaco Basin's water column, accounting for an average of 47% of 4',6'-diamidino-2-phenylindole (DAPI)-stained cells. This group was below the detection limit (<1%) in the Black Sea samples. Compositional differences between basins may reflect temporal variability in microbial populations and/or systematic differences in environmental conditions and the populations for which they select.

  10. Morphology of mitochondrial nucleoids, mitochondria, and nuclei during meiosis and sporulation of the yeast Saccharomycodes ludwigii.

    PubMed

    Miyakawa, Isamu; Nakahara, Ayumi; Ito, Kohei

    2012-01-01

    The morphology of mitochondrial nucleoids (mt-nucleoids), mitochondria, and nuclei was investigated during meiosis and sporulation of the diploid cells of the ascosporogenic yeast Saccharomycodes ludwigii. The mt-nucleoids appeared as discrete dots uniformly distributed in stationary-phase cells as revealed by 4',6-diamidino-2-phenylindole (DAPI) staining. Throughout first and second meiotic divisions, the mt-nucleoids moved to be located close to the dividing nuclei with the appearance of dots. On the other hand, mitochondria, which had tubular or fragmented forms in stationary-phase cells, increasingly fused with each other to form elongated mitochondria during meiotic prophase as revealed by 3,3' -dihexyloxacarbocyanine iodide [DiOC(6)(3)] staining. Mitochondria assembled to be located close to dividing nuclei during first and second meiotic divisions, and were finally incorporated into spores. During the first meiotic division, nuclear division occurred in any direction parallel, diagonally, or perpendicular to the longitudinal axis of the cell. In contrast, the second meiotic division was exclusively parallel to the longitudinal axis of the cell. The behavior of dividing nuclei explains the formation of a pair of spores with opposite mating types at both ends of cells. In the course of this study, it was also found that ledges between two spores were specifically stained with DiOC(6)(3).

  11. Microbial life in Champagne Pool, a geothermal spring in Waiotapu, New Zealand.

    PubMed

    Hetzer, Adrian; Morgan, Hugh W; McDonald, Ian R; Daughney, Christopher J

    2007-07-01

    Surveys of Champagne Pool, one of New Zealand's largest terrestrial hot springs and rich in arsenic ions and compounds, have been restricted to geological and geochemical descriptions, and a few microbiological studies applying culture-independent methods. In the current investigation, a combination of culture and culture-independent approaches were chosen to determine microbial density and diversity in Champagne Pool. Recovered total DNA and adenosine 5'-triphosphate (ATP) content of spring water revealed relatively low values compared to other geothermal springs within New Zealand and are in good agreement with low cell numbers of 5.6 +/- 0.5 x 10(6) cells/ml obtained for Champagne Pool water samples by 4',6-diamidino-2-phenylindole (DAPI) staining. Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rRNA (small-subunit ribosomal nucleic acid) gene clone library analyses of environmental DNA indicated the abundance of Sulfurihydrogenibium, Sulfolobus, and Thermofilum-like populations in Champagne Pool. From these results, media were selected to target the enrichment of hydrogen-oxidizing and sulfur-dependent microorganisms. Three isolates were successfully obtained having 16S rRNA gene sequences with similarities of approximately 98% to Thermoanaerobacter tengcongensis, 94% to Sulfurihydrogenibium azorense, and 99% to Thermococcus waiotapuensis, respectively.

  12. Evaluation of USEPA method 1622 for detection of Cryptosporidium oocysts in stream waters

    USGS Publications Warehouse

    Simmons, O. D.; Sobsey, M.D.; Schaefer, F. W.; Francy, D.S.; Nally, R.A.; Heaney, C.D.

    2001-01-01

    To improve surveillance for Cryptosporidium oocysts in water, the US Environmental Protection Agency developed method 1622, which consists of filtration, concentration, immunomagnetic separation, fluorescent antibody and 4, 6-diamidino-2-phenylindole (DAPI) counter-staining, and microscopic evaluation. Two filters were compared for analysis of 11 stream water samples collected throughout the United States. Replicate 10-L stream water samples (unspiked and spiked with 100-250 oocysts) were tested to evaluate matrix effects. Oocyst recoveries from the stream water samples averaged 22% (standard deviation [SD] = ??17%) with a membrane disk and 12% (SD = ??6%) with a capsule filter. Oocyst recoveries from reagent water precision and recovery samples averaged 39% (SD = ??13%) with a membrane disk and 47% (SD = ??19%) with a capsule filter. These results demonstrate that Cryptosporidium oocysts can be recovered from stream waters using method 1622, but recoveries are lower than those from reagent-grade water. This research also evaluated concentrations of indicator bacteria in the stream water samples. Because few samples were oocyst-positive, relationships between detections of oocysts and concentrations of indicator organisms could not be determined.

  13. Nuclear DNA content variation in life history phases of the Bonnemasoniaceae (Rhodophyta).

    PubMed

    Salvador Soler, Noemi; Gómez Garreta, Amelia; Ribera Siguan, Ma Antonia; Kapraun, Donald F

    2014-01-01

    Nuclear DNA content in gametophytes and sporophytes or the prostrate phases of the following species of Bonnemaisoniaceae (Asparagopsis armata, Asparagopsis taxiformis, Bonnemaisonia asparagoides, Bonnemaisonia clavata and Bonnemaisonia hamifera) were estimated by image analysis and static microspectrophotometry using the DNA-localizing fluorochrome DAPI (4', 6-diamidino-2-phenylindole, dilactate) and the chicken erythrocytes standard. These estimates expand on the Kew database of DNA nuclear content. DNA content values for 1C nuclei in the gametophytes (spermatia and vegetative cells) range from 0.5 pg to 0.8 pg, and for 2C nuclei in the sporophytes or the prostrate phases range from 1.15-1.7 pg. Although only the 2C and 4C values were observed in the sporophyte or the prostrate phase, in the vegetative cells of the gametophyte the values oscillated from 1C to 4C, showing the possible start of endopolyploidy. The results confirm the alternation of nuclear phases in these Bonnemaisoniaceae species, in those that have tetrasporogenesis, as well as those that have somatic meiosis. The availability of a consensus phylogenetic tree for Bonnemaisoniaceae has opened the way to determine evolutionary trends in DNA contents. Both the estimated genome sizes and the published chromosome numbers for Bonnemaisoniaceae suggest a narrow range of values consistent with the conservation of an ancestral genome.

  14. Use of fluorochromes for direct enumeration of total bacteria in environmental samples: past and present.

    PubMed Central

    Kepner, R L; Pratt, J R

    1994-01-01

    Understanding the role of bacteria in microbial food webs is intimately connected to the methods applied in the direct enumeration of bacteria. We have examined over 220 papers describing studies in which fluorochrome staining followed by epifluorescent microscopic direct counts was used to estimate total bacterial abundances. In this review, we summarize patterns in the use of 3,6-bis[dimethylamino]acridinium chloride (acridine orange) and 4',6-diamidino-2-phenylindole (DAPI), the two stains most frequently used in bacterial enumeration. The staining of samples with these fluorochromes, followed by filtration and direct counting of bacterial cells on filter surfaces, has become routine over the past 10 years. We examine trends in features of the standard direct count methods, such as sample preservation and preparation techniques, membrane filter types used, applied stain concentrations, duration of staining, and counting strategies, in relation to the types of samples being examined. The high variability in bacterial counts observed within similar sample types may be partially accounted for by differences in methods. Synthesizing review findings, we include a recommended method for the direct enumeration of bacteria in environmental samples. PMID:7854248

  15. Primo Vascular System in the Subarachnoid Space of a Mouse Brain

    PubMed Central

    Moon, Sang-Ho; Cha, Richard; Lim, Jae-Kwan; Soh, Kwang-Sup

    2013-01-01

    Objective. Recently, a novel circulatory system, the primo vascular system (PVS), was found in the brain ventricles and in the central canal of the spinal cord of a rat. The aim of the current work is to detect the PVS along the transverse sinuses between the cerebrum and the cerebellum of a mouse brain. Materials and Methods. The PVS in the subarachnoid space was analyzed after staining with 4′,6-diamidino-2-phenylindole (DAPI) and phalloidin in order to identify the PVS. With confocal microscopy and polarization microscopy, the primo vessel underneath the sagittal sinus was examined. The primo nodes under the transversal sinuses were observed after peeling off the dura and pia maters of the brain. Results. The primo vessel underneath the superior sagittal sinus was observed and showed linear optical polarization, similarly to the rabbit and the rat cases. The primo nodes were observed under the left and the right transverse sinuses at distances of 3,763 μm and 5,967 μm. The average size was 155 μm × 248 μm. Conclusion. The observation of primo vessels was consistent with previous observations in rabbits and rats, and primo nodes under the transverse sinuses were observed for the first time in this work. PMID:23781258

  16. Evidence for the Primo Vascular System above the Epicardia of Rat Hearts

    PubMed Central

    Lee, Ho-Sung; Lee, Jeong Yim; Kang, Dae-In; Kim, Se Hoon; Lee, Inhyung; Park, Sang Hyun; Yoon, Seung Zhoo; Ryu, Yeon Hee; Lee, Byung-Cheon

    2013-01-01

    We for the first time reported evidence for the existence of a novel network, a PVS, abovethe epicardium of the rat heart. (1) We were consecutively able to visualize the PVs and the PNs above the epicardial spaces of five rats' hearts by using Cr-Hx spraying or injection. (2) Hematoxylin and eosin (H&E) and toluidine blue staining of the PVs and the PNs showed that they consisted of a basophilic matrix; specifically the PNs contained several mast cells, some of which were degranulating into pericardial space. Also, 4′, 6-diamidino-2 phenylindole (DAPI) images of the PVs and the PNs showed that they contained various kinds of cells. (3) Transmission electron microscopic (TEM) longitudinal image of the PVs showed that the sinuses contained many granules with high-electron-density cores in parallel with putative endothelial cells. (4) TEM images of the PNs demonstrated that they consisted of lumen-containing cells surrounded by fibers and that they had mast cells that were degranulating toward the epicardium of the rat heart. The above data suggest that mast-cells-containing novel network exists above the epicardium of the rat heart. PMID:24023576

  17. Prevalence and distribution of Cryptosporidium and Giardia in wastewater and the surface, drinking and ground waters in the Lower Rhine, Germany.

    PubMed

    Gallas-Lindemann, C; Sotiriadou, I; Plutzer, J; Karanis, P

    2013-01-01

    Samples from different water sources (n = 396) were collected during 2009 and 2011. Wastewater (2-5 l) was purified by aluminium sulphate flocculation. Surface, ground and drinking waters (400-6400 l) were collected by filtration. Cryptosporidium oocysts and Giardia cysts were further concentrated by sucrose centrifugation. (Oo)cysts were identified by IFT (immunofluorescence test), DAPI (4',6-diamidino-2-phenylindole) staining and DICM (difference interference contrast microscopy). Out of 206 wastewater samples, 134 (65·0%) were found to be positive for Giardia cysts and 64 (31·1%) for Cryptosporidium oocysts. Parasite numbers ranged from 0 to 2436 cysts/l and 0 to 1745 oocysts/l. Eight (4·2%) surface and drinking water samples (n = 190) were found to be positive for Giardia cysts (0-56000/100 l), and 18 (9·5%) for Cryptosporidium oocysts (2400/100 l). The purpose of this study was to establish the prevalence and concentrations of Giardia lamblia and Cryptosporidium spp. by detecting (oo)cysts from water samples. This study provides substantial evidence that G. lamblia cysts and Cryptosporidium spp. oocysts are able to enter and circulate in the aquatic environment with negative implications for public health.

  18. Transport of Cryptosporidium parvum oocysts in soil columns following applications of raw and separated liquid slurries.

    PubMed

    Petersen, Heidi H; Enemark, Heidi L; Olsen, Annette; Amin, M G Mostofa; Dalsgaard, Anders

    2012-09-01

    The potential for the transport of viable Cryptosporidium parvum oocysts through soil to land drains and groundwater was studied using simulated rainfall and intact soil columns which were applied raw slurry or separated liquid slurry. Following irrigation and weekly samplings over a 4-week period, C. parvum oocysts were detected from all soil columns regardless of slurry type and application method, although recovery rates were low (<1%). Soil columns with injected liquid slurry leached 73 and 90% more oocysts compared to columns with injected and surface-applied raw slurries, respectively. Among leachate samples containing oocysts, 44/72 samples yielded viable oocysts as determined by a dye permeability assay (DAPI [4',6'-diamidino-2-phenylindole]/propidium iodide) with the majority (41%) of viable oocysts found in leachate from soil columns with added liquid slurry. The number of viable oocysts was positively correlated (r = 0.63) with the total number of oocysts found. Destructively sampling of the soil columns showed that type of slurry and irrigation played a role in the vertical distribution of oocysts, with more oocysts recovered from soil columns added liquid slurry irrespective of the irrigation status. Further studies are needed to determine the effectiveness of different slurry separation technologies to remove oocysts and other pathogens, as well as whether the application of separated liquid slurry to agricultural land may represent higher risks for groundwater contamination compared to application of raw slurry.

  19. Cell viability after osteotomy and bone harvesting: comparison of piezoelectric surgery and conventional bur.

    PubMed

    Mouraret, S; Houschyar, K S; Hunter, D J; Smith, A A; Jew, O S; Girod, S; Helms, J A

    2014-08-01

    The aim of this study was to evaluate and compare the influence of a piezoelectric device versus a conventional bur on osteocyte viability and osteoblast and osteoclast activity using an in vivo mouse model. Osteotomies were created and bone grafts were harvested using either a conventional bur or a piezoelectric device; the resulting injuries and bone grafts were evaluated over an extended time-course using molecular and cellular assays for cell death (TUNEL assay), cell viability (4',6-diamidino-2-phenylindole (DAPI) staining), the onset of mineralization (alkaline phosphatase activity), and bone remodelling (tartrate-resistant acid phosphatase activity). Osteotomies created with a piezoelectric device showed greater osteocyte viability and reduced cell death. Bone grafts harvested with a piezoelectric device exhibited greater short-term cell viability than those harvested with a bur, and exhibited slightly more new bone deposition and bone remodelling. The difference in response of osteocytes, osteoblasts, and osteoclasts to bone cutting via a bur and via a piezoelectric device is negligible in vivo. Given the improved visibility and the margin of safety afforded by a piezoelectric device, they are the instrument of choice when cutting or harvesting bone to preserve soft tissue.

  20. Scleral fibroblast response to experimental glaucoma in mice

    PubMed Central

    Tezel, Gülgün; Cone-Kimball, Elizabeth; Steinhart, Matthew R.; Jefferys, Joan; Pease, Mary E.; Quigley, Harry A.

    2016-01-01

    Purpose To study the detailed cellular and molecular changes in the mouse sclera subjected to experimental glaucoma. Methods Three strains of mice underwent experimental bead-injection glaucoma and were euthanized at 3 days and 1, 3, and 6 weeks. Scleral protein expression was analyzed with liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) using 16O/18O labeling for quantification in 1- and 6-week tissues. Sclera protein samples were also analyzed with immunoblotting with specific antibodies to selected proteins. The proportion of proliferating scleral fibroblasts was quantified with Ki67 and 4’,6-diamidino-2-phenylindole (DAPI) labeling, and selected proteins were studied with immunohistochemistry. Results Proteomic analysis showed increases in molecules involved in integrin-linked kinase signaling and actin cytoskeleton signaling pathways at 1 and 6 weeks after experimental glaucoma. The peripapillary scleral region had more fibroblasts than equatorial sclera (p=0.001, n=217, multivariable regression models). There was a sixfold increase in proliferating fibroblasts in the experimental glaucoma sclera at 1 week and a threefold rise at 3 and 6 weeks (p=0.0005, univariate regression). Immunoblots confirmed increases for myosin, spectrin, and actinin at 1 week after glaucoma. Thrombospondin-1 (TSP-1), HINT1, vimentin, actinin, and α-smooth muscle actin were increased according to immunohistochemistry. Conclusions Scleral fibroblasts in experimental mouse glaucoma show increases in actin cytoskeleton and integrin-related signaling, increases in cell division, and features compatible with myofibroblast transition. PMID:26900327

  1. The use of bottle caps as submerged aerated filter medium.

    PubMed

    Damasceno de Oliveira, Laurence; Motlagh, Amir Mohaghegh; Goel, Ramesh; de Souza Missagia, Beatriz; Alves de Abreu Filho, Benício; Lautenschlager, Sandro Rogério

    2014-01-01

    In this study, a submerged aerated filter (SAF) using bottle caps as a support medium was evaluated. The system was fed with effluent from an upflow anaerobic sludge blanket system at ETE 2-South wastewater treatment plant, under different volumetric organic load rates (VOLRs). The population of a particular nitrifying microbial community was assessed by fluorescent in situ hybridization with specific oligonucleotide probes. The system showed an average removal of chemical oxygen demand (COD) equal to 76% for VOLRs between 2.6 and 13.6 kg COD m(-3)_media.day(-1). The process of nitrification in conjunction with the removal of organic matter was observed from applying VOLRs lower than 5.5 kg COD m(-3)_media.day(-1) resulting in 78% conversion of NH4(+)-N. As the applied organic load was reduced, an increase in the nitrifying bacteria population was observed compared with total 4'-6-diamidino-2-phenylindole (DAPI) stained cells. Generally, SAF using bottle caps as a biological aerated filter medium treating wastewater from an anaerobic system showed promising removal of chemical oxygen demand (COD) and conversion of NH4(+)-N.

  2. Protective effects of poly(lactic-co-glycolic acid) nanoparticles loaded with erythropoietin stabilized by sodium cholate against glutamate-induced neurotoxicity.

    PubMed

    Jeong, Ji Heun; Kang, Seung Hee; Kim, Jeong Hwan; Yu, Kwang Sik; Lee, In Ho; Lee, Ye Ji; Lee, Je Hun; Lee, Nam Seob; Jeong, Young Gil; Kim, Do Kyung; Kim, Gyu Hyun; Lee, Shin Hye; Hong, Seul Ki; Han, Seung-Yun; Kang, Bo Sun

    2014-11-01

    The final aim of this study was to confirm the neuroprotective effects of recombinant human erythropoietin (rhEPO)-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles stabilized by sodium cholate (rhEPO-Ch-NP) and compare their effects with those of rhEPO using an in vitro model of cerebral ischemia. Glutamate-induced excitotoxic damage on SH-SY5Y cells, a human neuroblastoma cell line, with or without rhEPO-Ch-NPs was quantitatively evaluated. The rhEPO-Ch-NPs were carefully prepared using a water-in-oil-in-water (w/o/w) emulsion solvent evaporation technique with PLGA, sodium cholate hydrate, and ethyl acetate. The rhEPO-Ch-NPs were fully characterized by both transmission electron microscopy (TEM) and differential scanning calorimetry (DSC). In addition, significant intracellular uptake of these particles was monitored by confocal microscopy. Notably, the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and nuclear changes observed by 4',6-diamidino-2-phenylindole (DAPI) staining in SH-SY5Y cells demonstrated that rhEPO-Ch-NPs were safer at any concentration investigated and rescued more neuronal cells, while preserving normocytic features against glutamate-induced excitotoxic damages compared to rhEPO.

  3. Genome size of termites (Insecta, Dictyoptera, Isoptera) and wood roaches (Insecta, Dictyoptera, Cryptocercidae)

    NASA Astrophysics Data System (ADS)

    Koshikawa, Shigeyuki; Miyazaki, Satoshi; Cornette, Richard; Matsumoto, Tadao; Miura, Toru

    2008-09-01

    The evolution of genome size has been discussed in relation to the evolution of various biological traits. In the present study, the genome sizes of 22 dictyopteran species were estimated by Feulgen image analysis densitometry and 6-diamidino-2-phenylindole (DAPI)-based flow cytometry. The haploid genome sizes ( C-values) of termites (Isoptera) ranged from 0.58 to 1.90 pg, and those of Cryptocercus wood roaches (Cryptocercidae) were 1.16 to 1.32 pg. Compared to known values of other cockroaches (Blattaria) and mantids (Mantodea), these values are low. A relatively small genome size appears to be a (syn)apomorphy of Isoptera + Cryptocercus, together with their sociality. In some phylogenetic groups, genome size evolution is thought to be influenced by selective pressure on a particular trait, such as cell size or rate of development. The present results raise the possibility that genome size is influenced by selective pressures on traits associated with the evolution of sociality.

  4. Genome size of termites (Insecta, Dictyoptera, Isoptera) and wood roaches (Insecta, Dictyoptera, Cryptocercidae).

    PubMed

    Koshikawa, Shigeyuki; Miyazaki, Satoshi; Cornette, Richard; Matsumoto, Tadao; Miura, Toru

    2008-09-01

    The evolution of genome size has been discussed in relation to the evolution of various biological traits. In the present study, the genome sizes of 22 dictyopteran species were estimated by Feulgen image analysis densitometry and 6-diamidino-2-phenylindole (DAPI)-based flow cytometry. The haploid genome sizes (C-values) of termites (Isoptera) ranged from 0.58 to 1.90 pg, and those of Cryptocercus wood roaches (Cryptocercidae) were 1.16 to 1.32 pg. Compared to known values of other cockroaches (Blattaria) and mantids (Mantodea), these values are low. A relatively small genome size appears to be a (syn)apomorphy of Isoptera + Cryptocercus, together with their sociality. In some phylogenetic groups, genome size evolution is thought to be influenced by selective pressure on a particular trait, such as cell size or rate of development. The present results raise the possibility that genome size is influenced by selective pressures on traits associated with the evolution of sociality.

  5. A new technique for studying directional cell migration in a hydrogel-based three-dimensional matrix for tissue engineering model systems.

    PubMed

    Topman, Gil; Shoham, Naama; Sharabani-Yosef, Orna; Lin, Feng-Huei; Gefen, Amit

    2013-08-01

    Cell migration has a key role in biological processes, e.g. malignancy, wound healing, immune response and morphogenesis. Studying migration and factors that influence it is beneficial, e.g. for developing drugs to suppress metastasis, heal wounds faster or enhance the response to infection. Though the majority of the literature describes two-dimensional (2D) migration studies in culture dishes, a more realistic approach is to study migration in three-dimensional (3D) constructs. However, simple-to-implement, straight-forward standardized quantitative techniques for analysis of migration rates of cell colonies in 3D are still required in the field. Here, we describe a new model system for quantifying directional migration of colonies in a hyaluronic acid (oxi-HA) and adipic acid dihydrazide (ADH) hydrogel-based 3D matrix. We further demonstrate that our previously reported image processing technique for measuring migration in 2D (Topman et al., 2011, 2012) is extendable for analyzing the rates of migration of cells that directionally migrate in the hydrogel and are fluorescently stained with a 4',6-diamidino-2-phenylindole (DAPI) nuclear stain. Together, the present experimental setup and image processing algorithm provide a standard test bench for measuring migration rates in a fully automated, robust assay which is useful for high-throughput screening in large-scale drug evaluations, where effects on migration in a 3D matrix are sought.

  6. Lycopene induces apoptosis in Candida albicans through reactive oxygen species production and mitochondrial dysfunction.

    PubMed

    Choi, Hyemin; Lee, Dong Gun

    2015-08-01

    Lycopene, a well-known carotenoid pigment found in tomatoes, has shown various biological functions. In our previous report, we showed that lycopene induces two apoptotic hallmarks, plasma membrane depolarization and G2/M cell cycle arrest, in Candida albicans. In this study, we investigated the ability of lycopene to induce apoptosis, and the mechanism by which it regulates apoptosis. FITC-Annexin V staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis, and 4',6-diamidino-2-phenylindole (DAPI) assay showed that lycopene exerted its antifungal activity during the early and late stages of apoptosis in C. albicans. During apoptosis, intracellular reactive oxygen species (ROS) were increased, and specifically the hydroxyl radicals contributed to the fungal cell death. Furthermore, lycopene treatment caused intracellular Ca(2+) overload and mitochondrial dysfunction, such as mitochondrial depolarization and cytochrome c release from the mitochondria to the cytoplasm. At last caspase activation was triggered. In summary, lycopene exerted its antifungal effects against C. albicans by inducing apoptosis via ROS production and mitochondrial dysfunction.

  7. Anticancer effects of oligomeric proanthocyanidins on human colorectal cancer cell line, SNU-C4

    PubMed Central

    Kim, Youn-Jung; Park, Hae-Jeong; Yoon, Seo-Hyun; Kim, Mi-Ja; Leem, Kang-Hyun; Chung, Joo-Ho; Kim, Hye-Kyung

    2005-01-01

    AIM: Oligomeric proanthocyanidins (OPC), natural polyphenolic compounds found in plants, are known to have antioxidant and anti-cancer effects. We investigated whether the anti-cancer effects of the OPC are induced by apoptosis on human colorectal cancer cell line, SNU-C4. METHODS: Colorectal cancer cell line, SNU-C4 was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. The cytotoxic effect of OPC was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenylt-etrazolium bromide (MTT) assay. To find out the apoptotic cell death, 4, 6-diamidino-2-phenylindole (DAPI) staining, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, reverse transcription-polymerase chain reaction (RT-PCR), and caspase-3 enzyme assay were performed. RESULTS: In this study, cytotoxic effect of OPC on SNU-C4 cells appeared in a dose-dependent manner. OPC treatment (100 µg/mL) revealed typical morphological apoptotic features. Additionally OPC treatment (100 µg/mL) increased level of BAX and CASPASE-3, and decreased level of BCL-2 mRNA expression. Caspase-3 enzyme activity was also significantly increased by treatment of OPC (100 µg/mL) compared with control. CONCLUSION: These data indicate that OPC caused cell death by apoptosis through caspase pathways on human colorectal cancer cell line, SNU-C4. PMID:16094708

  8. A Burkholderia pseudomallei Colony Variant Necessary for Gastric Colonization

    PubMed Central

    Austin, C. R.; Goodyear, A. W.; Bartek, I. L.; Stewart, A.; Sutherland, M. D.; Silva, E. B.; Zweifel, A.; Vitko, N. P.; Tuanyok, A.; Highnam, G.; Mittelman, D.; Keim, P.; Schweizer, H. P.; Vázquez-Torres, A.; Dow, S. W. C.

    2015-01-01

    ABSTRACT  Diverse colony morphologies are a hallmark of Burkholderia pseudomallei recovered from infected patients. We observed that stresses that inhibit aerobic respiration shifted populations of B. pseudomallei from the canonical white colony morphotype toward two distinct, reversible, yet relatively stable yellow colony variants (YA and YB). As accumulating evidence supports the importance of B. pseudomallei enteric infection and gastric colonization, we tested the response of yellow variants to hypoxia, acidity, and stomach colonization. Yellow variants exhibited a competitive advantage under hypoxic and acidic conditions and alkalized culture media. The YB variant, although highly attenuated in acute virulence, was the only form capable of colonization and persistence in the murine stomach. The accumulation of extracellular DNA (eDNA) was a characteristic of YB as observed by 4′,6-diamidino-2-phenylindole (DAPI) staining of gastric tissues, as well as in an in vitro stomach model where large amounts of eDNA were produced without cell lysis. Transposon mutagenesis identified a transcriptional regulator (BPSL1887, designated YelR) that when overexpressed produced the yellow phenotype. Deletion of yelR blocked a shift from white to the yellow forms. These data demonstrate that YB is a unique B. pseudomallei pathovariant controlled by YelR that is specifically adapted to the harsh gastric environment and necessary for persistent stomach colonization. PMID:25650400

  9. Novel Parachlamydia acanthamoebae quantification method based on coculture with amoebae.

    PubMed

    Matsuo, Junji; Hayashi, Yasuhiro; Nakamura, Shinji; Sato, Marie; Mizutani, Yoshihiko; Asaka, Masahiro; Yamaguchi, Hiroyuki

    2008-10-01

    Parachlamydia acanthamoebae, belonging to the order Chlamydiales, is an obligately intracellular bacterium that infects free-living amoebae and is a potential human pathogen. However, no method exists to accurately quantify viable bacterial numbers. We present a novel quantification method for P. acanthamoebae based on coculture with amoebae. P. acanthamoebae was cultured either with Acanthamoeba spp. or with mammalian epithelial HEp-2 or Vero cells. The infection rate of P. acanthamoebae (amoeba-infectious dose [AID]) was determined by DAPI (4',6-diamidino-2-phenylindole) staining and was confirmed by fluorescent in situ hybridization. AIDs were plotted as logistic sigmoid dilution curves, and P. acanthamoebae numbers, defined as amoeba-infectious units (AIU), were calculated. During culture, amoeba numbers and viabilities did not change, and amoebae did not change from trophozoites to cysts. Eight amoeba strains showed similar levels of P. acanthamoebae growth, and bacterial numbers reached ca. 1,000-fold (10(9) AIU preculture) after 4 days. In contrast, no increase was observed for P. acanthamoebae in either mammalian cell line. However, aberrant structures in epithelial cells, implying possible persistent infection, were seen by transmission electron microscopy. Thus, our method could monitor numbers of P. acanthamoebae bacteria in host cells and may be useful for understanding chlamydiae present in the natural environment as human pathogens.

  10. Survival of Mycobacterium avium in drinking water biofilms as affected by water flow velocity, availability of phosphorus, and temperature.

    PubMed

    Torvinen, Eila; Lehtola, Markku J; Martikainen, Pertti J; Miettinen, Ilkka T

    2007-10-01

    Mycobacterium avium is a potential pathogen occurring in drinking water systems. It is a slowly growing bacterium producing a thick cell wall containing mycolic acids, and it is known to resist chlorine better than many other microbes. Several studies have shown that pathogenic bacteria survive better in biofilms than in water. By using Propella biofilm reactors, we studied how factors generally influencing the growth of biofilms (flow rate, phosphorus concentration, and temperature) influence the survival of M. avium in drinking water biofilms. The growth of biofilms was followed by culture and DAPI (4',6'-diamidino-2-phenylindole) staining, and concentrations of M. avium were determined by culture and fluorescence in situ hybridization methods. The spiked M. avium survived in biofilms for the 4-week study period without a dramatic decline in concentration. The addition of phosphorus (10 microg/liter) increased the number of heterotrophic bacteria in biofilms but decreased the culturability of M. avium. The reason for this result is probably that phosphorus increased competition with other microbes. An increase in flow velocity had no effect on the survival of M. avium, although it increased the growth of biofilms. A higher temperature (20 degrees C versus 7 degrees C) increased both the number of heterotrophic bacteria and the survival of M. avium in biofilms. In conclusion, the results show that in terms of affecting the survival of slowly growing M. avium in biofilms, temperature is a more important factor than the availability of nutrients like phosphorus.

  11. Minor groove binding of the food colorant carmoisine to DNA: spectroscopic and calorimetric characterization studies.

    PubMed

    Basu, Anirban; Kumar, Gopinatha Suresh

    2014-01-08

    The interaction of the food additive carmoisine with herring testes DNA was studied by multifaceted biophysical techniques. Carmoisine exhibited hypochromic effects in absorbance, whereas in fluorescence the intensity enhanced upon complexation with DNA. Energy transfer from the DNA base pairs to carmoisine molecules occurred upon complexation. A groove binding model of interaction was envisaged for carmoisine-DNA complexation from 4',6-diamidino-2-phenylindole (DAPI) and Hoechst displacement studies. The binding of carmoisine stabilized the DNA structure against thermal denaturation. The binding induced moderate conformational perturbations in the B-form structure of DNA. The binding affinity (10(4) M(-1)) values, calculated from absorbance and fluorescence data, and calorimetry titrations were in close agreement with each other. The binding was characterized to be exothermic and favored by small negative enthalpic and large positive entropic contributions. Salt-dependent calorimetric studies revealed that the binding reaction was dominated by nonpolyelectrolytic forces. The negative heat capacity value suggested the role of hydrophobic effect in the interaction.

  12. Pro-apoptotic properties of morphine in neuroblastoma × glioma NG108-15 hybrid cells: modulation by yohimbine.

    PubMed

    Polanco, María José; Alguacil, Luis Fernando; González-Martín, Carmen

    2014-01-01

    Short-term incubation with pharmacologically relevant concentrations of morphine has been shown to transiently affect the metabolism and redox status of NG108-15 cells through δ-opioid receptor stimulation, but apparently did not provoke cell death. The present work tries to determine if incubation with morphine at longer time intervals (24 h) provokes apoptosis and/or necrosis, as it has been described in other cell lines. We have also checked the potential modulatory role of yohimbine on these effects, on the basis of the previously described interactions between this drug and opioid receptor ligands. Incubation with morphine 0.1 and 10 μM provoked the appearance of images compatible with apoptosis (bebbling, pyknotic cells with cytoplasmic and nuclear condensation) and necrosis (cells swollen with vacuolated cytoplasm lacking cell processes) that could be observed directly and/or after staining with methylene blue, crystal violet and propidium iodide/4',6-diamidino-2-phenylindole (IP/DAPI). Quantification of apoptosis by activation of caspases 3 and 7 and DNA fragmentation with the Tunel assay revealed a modest but significant increase after incubation with the two concentrations of morphine used. Co-incubation with 10 μM yohimbine prevented all these effects of the opioid. The results extend previous findings of a yohimbine-sensitive, neurotoxic effect of morphine on NG108-15 cells.

  13. Three-dimensional direct measurement of cardiomyocyte volume, nuclearity, and ploidy in thick histological sections

    PubMed Central

    Bensley, Jonathan Guy; De Matteo, Robert; Harding, Richard; Black, Mary Jane

    2016-01-01

    Quantitative assessment of myocardial development and disease requires accurate measurement of cardiomyocyte volume, nuclearity (nuclei per cell), and ploidy (genome copies per cell). Current methods require enzymatically isolating cells, which excludes the use of archived tissue, or serial sectioning. We describe a method of analysis that permits the direct simultaneous measurement of cardiomyocyte volume, nuclearity, and ploidy in thick histological sections. To demonstrate the utility of our technique, heart tissue was obtained from four species (rat, mouse, rabbit, sheep) at up to three life stages: prenatal, weaning and adulthood. Thick (40 μm) paraffin sections were stained with Wheat Germ Agglutinin-Alexa Fluor 488 to visualise cell membranes, and DAPI (4′,6-diamidino-2-phenylindole) to visualise nuclei and measure ploidy. Previous methods have been restricted to thin sections (2–10 μm) and offer an incomplete picture of cardiomyocytes. Using confocal microscopy and three-dimensional image analysis software (Imaris Version 8.2, Bitplane AG, Switzerland), cardiomyocyte volume, nuclearity, and ploidy were measured. This method of staining and analysis of cardiomyocytes enables accurate morphometric measurements in thick histological sections, thus unlocking the potential of archived tissue. Our novel time-efficient method permits the entire cardiomyocyte to be visualised directly in 3D, eliminating the need for precise alignment of serial sections. PMID:27048757

  14. The anticancer effects of Saccharina japonica on 267B1/K-ras human prostate cancer cells.

    PubMed

    Jo, Mi Jeong; Kim, Hyung Rak; Kim, Gun Do

    2012-11-01

    Saccharina japonica (S. japonica), a brown macro-alga, has been used as a traditional medicine in Korea for thousands of years. In this study, the potential anticancer effects of S. japonica were evaluated on 267B1/K-ras human prostate cancer cells. The exposure of cells to the extract induced inhibition of cell growth by increasing the number of apoptotic cells with cell shrinkage and inhibition of cell cycle progression. The effects of the extract on the cells were assessed by studying the cleavage of caspases and the target proteins of caspases. The increased expression of various cleaved caspases and changed expression of other proteins related in the apoptosis pathway were observed. 4'-6-Diamidino-2-phenylindole (DAPI) and immunofluorescence staining showed the cells undergoing apoptosis. Apoptosis induced changes in the expression of proteins involved in a variety of signaling pathways such as endocellular reticulum (ER) stress, death receptor and mammalian target of rapamycin (mTOR)-FoxO-mediated pathways. The data suggest that the extract (n-hexane sub-fraction) of S. japonica, induces apoptosis and cell cycle arrest in 267B1/K-ras human prostate cancer cells, and has potential as a complementary agent for cancer prevention.

  15. Detection of DNA in ancient bones using histochemical methods.

    PubMed

    Guarino, F M; Angelini, F; Odierna, G; Bianco, M R; Di Bernardo, G; Forte, A; Cascino, A; Cipollaro, M

    2000-05-01

    We describe histochemical techniques for detecting DNA within the osteocytic lacunae of ancient bones. The bones examined were fragments of femurs from two human individuals found in the Pompeian C. I. Polybius house and fragments of metacarpals from two horses (Equus sp.) found in the Pompeian "Casti Amanti" house. Both buildings were buried by the 79 A. D. Vesuvius eruption. Fragments of femurs from a modern horse, a modern swine and a modern amphibian also were studied as controls. Some bone sections were stained with two different DNA-specific fluorochromes, 4'-'6-diamidino-2-phenylindole (DAPI) and chromomycin A3 (CMA), while others were stained by the Feulgen reaction. All of the techniques gave a positive reaction within the osteocytic lacunae. Histological analysis of the undecalcified, ground and unstained sections agreed well with results of bone sections stained with either the fluorochromes or the Feulgen reaction. Bones showing good histology also were positive by our DNA-specific stain. Histochemical and histological analyses correlated well with the success of DNA extraction and amplification. Using conventional DNA-specific histochemical techniques in conjunction with histological analysis can be useful in the study of DNA extracted from ancient bone remains while reducing both the amount of time and cost.

  16. Anticancer Effect of Fucoidan on DU-145 Prostate Cancer Cells through Inhibition of PI3K/Akt and MAPK Pathway Expression

    PubMed Central

    Choo, Gang-Sik; Lee, Hae-Nim; Shin, Seong-Ah; Kim, Hyeong-Jin; Jung, Ji-Youn

    2016-01-01

    In this study, we showed that PI3K/Akt signaling mediates fucoidan’s anticancer effects on prostate cancer cells, including suppression of proliferation. Fucoidan significantly decreased viability of DU-145 cancer cells in a concentration-dependent manner as shown by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The drug also significantly increased chromatin condensation, which indicates apoptosis, in a concentration-dependent manner as shown by DAPI (4′,6-diamidino-2-phenylindole) staining. Fucoidan increased expression of Bax, cleaved poly-ADP ribose polymerase and cleaved caspase-9, and decreased of the Bcl-2, p-Akt, p-PI3K, p-P38, and p-ERK in a concentration-dependent manner. In vivo, fucoidan (at 5 and 10 mg/kg) significantly decreased tumor volume, and increased apoptosis as assessed by the TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay, confirming the tumor inhibitory effect. The drug also increased expression of p-Akt and p-ERK as shown by immunohistochemistry staining. Therefore, fucoidan may be a promising cancer preventive medicine due to its growth inhibitory effects and induction of apoptosis in human prostate cancer cells. PMID:27399727

  17. Investigating the effect of surface topography on hydrophobicity and bacterial attachment on polystyrene

    NASA Astrophysics Data System (ADS)

    Mohamad, A. J.; Zhu, X.; Tian, Y.; Wellington, E. M. H.; Pfleging, W.; Liu, X.

    2014-04-01

    In this paper, we present an investigation into the effects of surface topography (roughness) and hydrophobicity (contact angle measurement) on bacteria adhesion for polystyrene materials. The surfaces of polystyrene substrates were patterned using UV-laser radiation with a wavelength of 193 nm under different conditions. Various surface topographies were fabricated and were measured by an optical surface profiler and contact angle measurements were recorded. For the bacterial adhesion experiments, an assay of Escherichia coli (E. coli) was developed and used for measurements on both as-received and modified polystyrene surfaces. The method is based on the staining of attached bacterial cells with the nucleic acid-binding, green fluorescent DAPI (4‧, 6-diamidino-2-phenylindole) stain. The preliminary results show that laser-assisted modification induced by laser ablation can make polystyrene substrates either more hydrophilic (with oxygen) or more hydrophobic (with air). The contact angle can be varied from 37° to 108°. The results on bacterial attachment show that the polystyrene substrates as received have no bacteria attached, indicating a good anti-bacterial performance. The treated substrates show some bacterial attachment and, in particular, the surfaces with a high contact angle have much higher numbers of bacterial cells attached. This indicates that such a laser-assisted process with air can make polystyrene surfaces more attractive to E. coli bacteria.

  18. [Tripartite motif-containing protein 34 (TRIM34) colocalized with micronuclei chromosome and hampers its movement to equatorial plate during the metaphase stage of mitosis].

    PubMed

    Sun, Dakang; An, Xinye; Ji, Bing; Cheng, Yanli; Gao, Honglian; Tian, Mingming

    2016-06-01

    Objective To examine whether tripartite motif-containing protein 34 (TRIM34) is colocalized with micronuclei and investigate the influence on the movement of micronuclei chromosome in mitosis. Methods The eukaryotic expression vector TRIM34-pEGFP-N3 was constructed, identified and then transfected into HEK293T cells. With 4', 6-diamidino-2-phenylindole 2HCI (DAPI) staining, the colocalization between TRIM34 and micronuclei was observed under a fluorescence microscope. Moreover, MitoTracker(R)Deep Red was used to identify the colocalization between the complex of TRIM34-micronulei and mitochondria under a confocal microscope. Finally, the effect of TRIM34 on the movement of micronuclei chromosome in mitosis was examined. Results DNA sequencing confirmed that the vector TRIM34-pEGFP-N3 was constructed successfully. A fluorescence microscope revealed that TRIM34 could be colocalized with micronuclei in HEK293T cells transfected with TRIM34-pEGFP-N3. In the same manner, a confocal microscope distinctly showed that TRIM34 was colocalized with micronuclei similarly in appearance. However, there was no distinguished colocalization relationship between the complex of TRIM34-micronulei and mitochondria. Interestingly, the micronuclei chromosome conjugated with TRIM34 was hardly transferred to equatorial plate during the metaphase stage of mitosis. Conclusion TRIM34 is colocalized with micronuclei chromosome and hampers its movement to equatorial plate in mitosis.

  19. G2/M arrest and apoptosis of human colorectal cancer cells induced by water extract from residues of jelly fig achene.

    PubMed

    Chou, Wing-Ming; Chen, Chun-Nan; Hsieh, Hsiao-Ting; Lo, Tsui-Yun; Juan, Pei-Yi; Mai, Fu-Der

    2015-01-01

    Jelly fig (Ficus awkeotsang) achenes have been utilized to prepare a traditional drink in Taiwan. Herein, we evaluated the effect of water extract from jelly fig seed residues (WERJFA) on cancer cells. WERJFA could inhibit the growth of human colorectal cancer cells, COLO205 and HT29 in both dose- and time-dependent manners. The flow cytometric analysis with propidium iodide (PI) showed that WERJFA primarily arrested COLO205 and HT29 cells at the G2/M phase of cell cycle as the concentration reached to at least 0.5 mg/ml. WERJFA induced apoptosis of these two cell lines, as evidenced by annexin V-FITC/PI and 4', 6-diamidino-2-phenylindole (DAPI) staining using flow cytometry and confocal microscopy, respectively. Reactive oxygen species (ROS) production and the loss of mitochondrial membrane potential in WERJFA-treated cells were detected by flow cytometry with H2DCF-DA and 5,5', 6,6'-Tetrachloro-1, 1', 3,3'-tetraethylbenzimidazolocarbocyanine iodide (JC-1). Our results showed that WERJFA exerted anti-proliferative and apoptotic effects on colorectal cancer cells. WERJFA arrested cell cycle, and caused apoptotic death in these cancer cells possibly via mitochondrial pathway involved with exceeding ROS level.

  20. G2/M arrest and apoptosis of human colorectal cancer cells induced by water extract from residues of jelly fig achene.

    PubMed

    Chou, Wing-Ming; Chen, Chun-Nan; Hsieh, Hsiao-Ting; Lo, Tsui-Yun; Juan, Pei-Yi; Mai, Fu-Der

    2015-09-10

    Jelly fig (Ficus awkeotsang) achenes have been utilized to prepare a traditional drink in Taiwan. Herein, we evaluated the effect of water extract from jelly fig seed residues (WERJFA) on cancer cells. WERJFA could inhibit the growth of human colorectal cancer cells, COLO205 and HT29 in both dose- and time-dependent manners. The flow cytometric analysis with propidium iodide (PI) showed that WERJFA primarily arrested COLO205 and HT29 cells at the G2/M phase of cell cycle as the concentration reached to at least 0.5 mg/ml. WERJFA induced apoptosis of these two cell lines, as evidenced by annexin V-FITC/PI and 4', 6-diamidino-2-phenylindole (DAPI) staining using flow cytometry and confocal microscopy, respectively. Reactive oxygen species (ROS) production and the loss of mitochondrial membrane potential in WERJFA-treated cells were detected by flow cytometry with H2DCF-DA and 5,5', 6,6'-Tetrachloro-1, 1', 3,3'-tetraethylbenzimidazolocarbocyanine iodide (JC-1). Our results showed that WERJFA exerted anti-proliferative and apoptotic effects on colorectal cancer cells. WERJFA arrested cell cycle, and caused apoptotic death in these cancer cells possibly via mitochondrial pathway involved with exceeding ROS level.

  1. Concentration and size distribution of total airborne microbes in hazy and foggy weather.

    PubMed

    Dong, Lijie; Qi, Jianhua; Shao, Congcong; Zhong, Xi; Gao, Dongmei; Cao, Wanwan; Gao, Jiawei; Bai, Ran; Long, Gaoyuan; Chu, Congcong

    2016-01-15

    Atmospheric bioaerosol particles were collected using a bioaerosol sampler from Oct. 2013 to Aug. 2014 in the coastal region of Qingdao. The total microbes were measured using an epifluorescence microscope after staining with DAPI (4',6-diamidino-2-phenylindole). The concentration of total airborne microbes showed seasonal variation, with the highest value in winter and the lowest in summer. The mean concentration of total microbes was 6.55 × 10(5)Cells/m(3) on non-hazy days. The total microbe concentration increased to 7.09 × 10(5) and 9.00 × 10(5)Cells/m(3) on hazy and foggy days, respectively. The particle sizes of the total microbes presented a bimodal distribution on sunny days, with one peak at 1.1-2.1 μm and another at 4.7-7.0 μm. The size distribution of total microbes showed an increase in the fine fraction on hazy days and an increase in the coarse fraction on foggy days. However, the size distribution became unimodal during a heating period. Spearman correlation analysis showed that temperature and O3 had a significant negative correlation with the airborne microbe concentration, while PM2.5, SO2, NO2, CO and the air quality index (AQI) had significant positive correlations with the airborne microbe concentration during hazy days. The increased number of airborne microbes will affect the air quality on hazy days.

  2. Primo vascular system in the subarachnoid space of a mouse brain.

    PubMed

    Moon, Sang-Ho; Cha, Richard; Lee, Geo-Lyong; Lim, Jae-Kwan; Soh, Kwang-Sup

    2013-01-01

    Objective. Recently, a novel circulatory system, the primo vascular system (PVS), was found in the brain ventricles and in the central canal of the spinal cord of a rat. The aim of the current work is to detect the PVS along the transverse sinuses between the cerebrum and the cerebellum of a mouse brain. Materials and Methods. The PVS in the subarachnoid space was analyzed after staining with 4',6-diamidino-2-phenylindole (DAPI) and phalloidin in order to identify the PVS. With confocal microscopy and polarization microscopy, the primo vessel underneath the sagittal sinus was examined. The primo nodes under the transversal sinuses were observed after peeling off the dura and pia maters of the brain. Results. The primo vessel underneath the superior sagittal sinus was observed and showed linear optical polarization, similarly to the rabbit and the rat cases. The primo nodes were observed under the left and the right transverse sinuses at distances of 3,763  μ m and 5,967  μ m. The average size was 155  μ m × 248  μ m. Conclusion. The observation of primo vessels was consistent with previous observations in rabbits and rats, and primo nodes under the transverse sinuses were observed for the first time in this work.

  3. Evidence for the Primo Vascular System above the Epicardia of Rat Hearts.

    PubMed

    Lee, Ho-Sung; Lee, Jeong Yim; Kang, Dae-In; Kim, Se Hoon; Lee, Inhyung; Park, Sang Hyun; Yoon, Seung Zhoo; Ryu, Yeon Hee; Lee, Byung-Cheon

    2013-01-01

    We for the first time reported evidence for the existence of a novel network, a PVS, abovethe epicardium of the rat heart. (1) We were consecutively able to visualize the PVs and the PNs above the epicardial spaces of five rats' hearts by using Cr-Hx spraying or injection. (2) Hematoxylin and eosin (H&E) and toluidine blue staining of the PVs and the PNs showed that they consisted of a basophilic matrix; specifically the PNs contained several mast cells, some of which were degranulating into pericardial space. Also, 4', 6-diamidino-2 phenylindole (DAPI) images of the PVs and the PNs showed that they contained various kinds of cells. (3) Transmission electron microscopic (TEM) longitudinal image of the PVs showed that the sinuses contained many granules with high-electron-density cores in parallel with putative endothelial cells. (4) TEM images of the PNs demonstrated that they consisted of lumen-containing cells surrounded by fibers and that they had mast cells that were degranulating toward the epicardium of the rat heart. The above data suggest that mast-cells-containing novel network exists above the epicardium of the rat heart.

  4. Single-event analysis of the packaging of bacteriophage T7 DNA concatemers in vitro.

    PubMed

    Sun, M; Louie, D; Serwer, P

    1999-09-01

    Bacteriophage T7 packages its double-stranded DNA genome in a preformed protein capsid (procapsid). The DNA substrate for packaging is a head-to-tail multimer (concatemer) of the mature 40-kilobase pair genome. Mature genomes are cleaved from the concatemer during packaging. In the present study, fluorescence microscopy is used to observe T7 concatemeric DNA packaging at the level of a single (microscopic) event. Metabolism-dependent cleavage to form several fragments is observed when T7 concatemers are incubated in an extract of T7-infected Escherichia coli (in vitro). The following observations indicate that the fragment-producing metabolic event is DNA packaging: 1) most fragments have the hydrodynamic radius (R(H)) of bacteriophage particles (+/-3%) when R(H) is determined by analysis of Brownian motion; 2) the fragments also have the fluorescence intensity (I) of bacteriophage particles (+/-6%); 3) as a fragment forms, a progressive decrease occurs in both R(H) and I. The decrease in I follows a pattern expected for intracapsid steric restriction of 4',6-diamidino-2-phenylindole (DAPI) binding to packaged DNA. The observed in vitro packaging of a concatemer's genomes always occurs in a synchronized cluster. Therefore, the following hypothesis is proposed: the observed packaging of concatemer-associated T7 genomes is cooperative.

  5. Identification of a novel antimicrobial peptide, scolopendin 1, derived from centipede Scolopendra subspinipes mutilans and its antifungal mechanism.

    PubMed

    Choi, H; Hwang, J-S; Lee, D G

    2014-12-01

    In this study, a novel antimicrobial peptide, scolopendin 1, was identified from adult centipedes, Scolopendra subspinipes mutilans using RNA sequencing. Scolopendin 1 exerted an antimicrobial activity without inducing haemolysis of human erythrocytes. In order to understand the antifungal mechanism, a reactive oxygen species (ROS) assay was performed, which indicated that scolopendin 1 induced ROS accumulation in Candida albicans. Evaluation of fungal viability using N-acetyl cysteine, a ROS scavenger, suggested that ROS are a major factor in scolopendin 1-induced fungal cell death. Co-staining of annexin V-fluorescein isothiocyanate (FITC) and propidium iodide, and TUNEL and 4',6-diamidino-2-phenylindole (DAPI) assays confirmed that ROS-induced fungal cell death is associated with apoptosis. To further investigate the mechanism that facilitates the progression of apoptosis, changes in intracellular Ca(2+) concentration and mitochondrial dysfunction were examined. Ca(2+) , a signalling molecule in the apoptotic pathway, was increased in the cytosol and mitochondria, and ROS accumulation triggered mitochondrial depolarization and the release of cytochrome c, a pro-apoptotic factor, from the mitochondria to the cytosol. Finally, the released cytochrome c activated intracellular caspase. The present study suggests that scolopendin 1 could emerge as a model molecule that targets the apoptotic pathway and provides a novel remedy.

  6. Glycogen Synthase Kinase-3 is involved in glycogen metabolism control and embryogenesis of Rhodnius prolixus.

    PubMed

    Mury, Flávia B; Lugon, Magda D; DA Fonseca, Rodrigo Nunes; Silva, Jose R; Berni, Mateus; Araujo, Helena M; Fontenele, Marcio Ribeiro; Abreu, Leonardo Araujo DE; Dansa, Marílvia; Braz, Glória; Masuda, Hatisaburo; Logullo, Carlos

    2016-10-01

    Rhodnius prolixus is a blood-feeding insect that transmits Trypanosoma cruzi and Trypanosoma rangeli to vertebrate hosts. Rhodnius prolixus is also a classical model in insect physiology, and the recent availability of R. prolixus genome has opened new avenues on triatomine research. Glycogen synthase kinase 3 (GSK-3) is classically described as a key enzyme involved in glycogen metabolism, also acting as a downstream component of the Wnt pathway during embryogenesis. GSK-3 has been shown to be highly conserved among several organisms, mainly in the catalytic domain region. Meanwhile, the role of GSK-3 during R. prolixus embryogenesis or glycogen metabolism has not been investigated. Here we show that chemical inhibition of GSK-3 by alsterpaullone, an ATP-competitive inhibitor of GSK3, does not affect adult survival rate, though it alters oviposition and egg hatching. Specific GSK-3 gene silencing by dsRNA injection in adult females showed a similar phenotype. Furthermore, bright field and 4'-6-diamidino-2-phenylindole (DAPI) staining analysis revealed that ovaries and eggs from dsGSK-3 injected females exhibited specific morphological defects. We also demonstrate that glycogen content was inversely related to activity and transcription levels of GSK-3 during embryogenesis. Lastly, after GSK-3 knockdown, we observed changes in the expression of the Wingless (Wnt) downstream target β-catenin as well as in members of other pathways such as the receptor Notch. Taken together, our results show that GSK-3 regulation is essential for R. prolixus oogenesis and embryogenesis.

  7. Nuclear DNA Content Estimates in Multicellular Green, Red and Brown Algae: Phylogenetic Considerations

    PubMed Central

    KAPRAUN, DONALD F.

    2005-01-01

    • Background and Aims Multicellular eukaryotic algae are phylogenetically disparate. Nuclear DNA content estimates have been published for fewer than 1 % of the described species of Chlorophyta, Phaeophyta and Rhodophyta. The present investigation aims to summarize the state of our knowledge and to add substantially to our database of C-values for theses algae. • Methods The DNA-localizing fluorochrome DAPI (4′, 6-diamidino-2-phenylindole) and RBC (chicken erythrocyte) standard were used to estimate 2C values with static microspectrophotometry. • Key Results 2C DNA contents for 85 species of Chlorophyta range from 0·2–6·1 pg, excluding the highly polyploidy Charales and Desmidiales with DNA contents of up to 39·2 and 20·7 pg, respectively. 2C DNA contents for 111 species of Rhodophyta range from 0·1–2·8 pg, and for 44 species of Phaeophyta range from 0·2–1·8 pg. • Conclusions New availability of consensus higher-level molecular phylogenies provides a framework for viewing C-value data in a phylogenetic context. Both DNA content ranges and mean values are greater in taxa considered to be basal. It is proposed that the basal, ancestral genome in each algal group was quite small. Both mechanistic and ecological processes are discussed that could have produced the observed C-value ranges. PMID:15596456

  8. Effect of dung burial by the dung beetle Bubas bison on numbers and viability of Cryptosporidium oocysts in cattle dung.

    PubMed

    Ryan, Una; Yang, Rongchang; Gordon, Cameron; Doube, Bernard

    2011-09-01

    Cryptosporidium oocysts were inoculated into fresh dung (∼1.2×10(4) oocysts per gram wet weight) and fed to dung beetles to assess the effect of dung burial by the dung beetle Bubas bison on the distribution of the oocysts in small cores of soil in the laboratory. The experiment consisted of five replicates of each of two treatments; controls (dung but no dung beetles) and the experimental treatment (inoculated dung and seven pairs of dung beetles). After 5 days, when approximately 90% of the dung was buried, the surface and buried dung was recovered and subsampled. The oocysts in the subsamples were recovered and enumerated using qPCR. Oocyst viability was evaluated using an assay based on the exclusion or inclusion of two fluorogenic vital dyes, 4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI). Results revealed that overall 13.7% of oocysts remained on the surface and 86.3% of oocysts were buried. The viability of oocysts in buried dung was only 10% compared to oocysts the surface dung (58%). Therefore, widespread dung burial by B. bison during the winter months could substantially reduce the numbers of Cryptosporidium oocysts available to be washed into waterways following winter rains.

  9. Multicolor fluorescence microscopic imaging of cancer cells on the plasmonic chip (Presentation Recording)

    NASA Astrophysics Data System (ADS)

    Tawa, Keiko; Sasakawa, Chisato; Yamamura, Shohei; Shibata, Izumi; Kataoka, Masatoshi

    2015-09-01

    A plasmonic chip which is a metal coated substrate with grating structure can provide the enhanced fluorescence by the grating-coupled surface plasmon field. In our previous studies, bright epi-fluorescence microscopic imaging of neuron cells and sensitive immunosesnsing have been reported. In this study, two kinds of breast cancer cells, MCF-7 and MDA-MB231, were observed with epi-fluorescence microscope on the plasmonic chip with 2D hole-arrays . They were multicolor stained with 4', 6-diamidino-2-phenylindole (DAPI) and allophycocyanin (APC)-labeled anti-epithelial cell adhesion molecule (EpCAM) antibody. Our plasmonic chip provided the brighter fluorescence images of these cells compared with the glass slide. Even in the cells including few EpCAM, the distribution of EpCAM was clearly observed in the cell membrane. It was found that the plasmonic chip can be one of the powerful tools to detect the marker protein existing around the chip surface even at low concentration.

  10. Mixed ligand complexes of Cu(II)/Zn(II) ions containing (m-)/(p-) carboxylato phenyl azo pentane 2,4-dione and 2,2‧-bipyridine/1,10 phenanthroline: Synthesis, characterization, DNA binding, nuclease and topoisomerase I inhibitory activity

    NASA Astrophysics Data System (ADS)

    Hasan, Md. Amin; Kumari, Niraj; Singh, Kanhaiya; Singh, Kiran; Mishra, Lallan

    2016-01-01

    Metal complexes of type [Cu(L1H)2(bpy)] (1), [Zn(L1H)2(bpy)] (2), [Cu(L2H)2(bpy)] (3) and [Cu(L2H)2(Phen)] (4) (L1H2 = 3-[N‧-(1-acetyl-2-oxo-propylidene)-hydrazino]-benzoic acid, L2H2 = 4-[N‧-(1-acetyl-2-oxo-propylidene)-hydrazino]-benzoic acid, bpy = 2,2‧-bipyridine, Phen = 1,10 phenanthroline) are synthesized and characterized using spectroscopic techniques (FT-IR, 1H NMR, 13C NMR, electronic absorption and emission) and elemental analysis data. The assembly of the complexes involving intramolecular H-bonding is displayed using corresponding crystal structure. Binding of the complexes separately with Calf Thymus DNA is monitored using UV-vis spectral titrations. The displacement of ethidium bromide (EB) bound to DNA by the complexes, in phosphate buffer solution (pH ∼ 7.2) is monitored using fluorescence spectral titrations. Nuclease activity of the complexes follow the order 4 > 3 > 1 > 2. The gel electrophoretic mobility assay measurement in presence of minor groove binder 4‧,6-diamidino-2-phenylindole (DAPI), suggests that complexes preferably bind with the minor groove of DNA. Topoisomerase I inhibitory activity of the complexes 3 and 4 inhibit topoisomerase I activity with IC50 values of 112 and 87 μM respectively.

  11. Analysis of a Partial Male-Sterile Mutant of Arabidopsis thaliana Isolated from a Low-Energy Argon Ion Beam Mutagenized Pool

    NASA Astrophysics Data System (ADS)

    Xu, Min; Bian, Po; Wu, Yuejin; Yu, Zengliang

    2008-04-01

    A screen for Arabidopsis fertility mutants, mutagenized by low-energy argon ion beam, yielded two partial male-sterile mutants tc243-1 and tc243-2 which have similar phenotypes. tc243-2 was investigated in detail. The segregation ratio of the mutant phenotypes in the M2 pools suggested that mutation behaved as single Mendelian recessive mutations. tc243 showed a series of mutant phenotypes, among which partial male-sterile was its striking mutant characteristic. Phenotype analysis indicates that there are four factors leading to male sterility. a. Floral organs normally develop inside the closed bud, but the anther filaments do not elongate sufficiently to position the locules above the stigma at anthesis. b. The anther locules do not dehisce at the time of flower opening (although limited dehiscence occurs later). c. Pollens of mutant plants develop into several types of pollens at the trinucleated stage, as determined by staining with DAPI (4',6-diamidino-2-phenylindole), which shows a variable size, shape and number of nucleus. d. The viability of pollens is lower than that of the wild type on the germination test in vivo and vitro.

  12. Nuclear DNA content analysis of plant seeds by flow cytometry.

    PubMed

    Sliwinska, Elwira

    2006-02-01

    Procedures describing the utilization of seeds or their parts for flow cytometric determination of plant ploidy and endopolyploidy, genome size, and cell cycle activity are presented. The methods have been developed for a single-fluorescence-parameter flow cytometer, equipped with light sources for 488-nm and UV-light illumination. The procedures presented in this unit utilize the two most widely used fluorochromes for plant DNA content analysis, propidium iodide (PI) and 4',6-diamidino-2-phenylindole (DAPI). These methods provide an alternative to estimation of DNA content based on the fluorescence of DNA in cell nuclei isolated from plant leaves. In some instances seeds are more suitable for analysis than leaves, e.g., when plant material must be transported for a long distances or stored for prolonged periods before flow cytometric analysis, or when leaves contain fluorochrome-staining inhibitors. In addition, flow cytometric determination of nuclear replication stages in seeds gives information about their physiological status (e.g., maturity, advancement of germination), which is valuable to seed producers and technologists.

  13. Solid state stability and solubility of triethylenetetramine dihydrochloride.

    PubMed

    Henriet, Théo; Gana, Inès; Ghaddar, Carine; Barrio, Maria; Cartigny, Yohann; Yagoubi, Najet; Do, Bernard; Tamarit, Josep-Lluis; Rietveld, Ivo B

    2016-09-10

    The API triethylenetetramine dihydrochloride used as an alternative treatment of Wilson's disease is sensitive to water and it exhibits polymorphism. As this may become an issue for the drug formulation, the physical stability has been studied by differential scanning calorimetry, high-pressure thermal analysis, dynamic vapor sorption, and X-ray diffraction as a function of temperature. In addition, high-pressure liquid chromatography and mass spectrometry have been used to study the purity and chemical stability of the API. A pressure-temperature phase diagram of the pure compound has been constructed and it can be concluded that form II is monotropic in relation to form I, which is the only stable solid. The solubilities of the different solid forms have been determined with the help of a temperature - composition phase diagram. The API is very soluble, at 20° C about 10% of the saturated solution with respect to the dihydrate consists of API and the solubility of the pure form I is twice as high. Moreover, it has been shown that at 20°C, a relative humidity above 40% induces the formation of the dihydrate and at 70% a saturated solution appears. At higher temperatures, the formation of the dihydrate appears at lower relative humidity values. A clear link has been established between the API's chemical stability, its physical stability and the relative humidity in the air. Humidity levels above 40% are detrimental to the quality of the API.

  14. Blood Vessel Tumorigenesis by 1,2-Dimethylhydrazine Dihydrochloride (Symmetrical)

    PubMed Central

    Toth, Bela; Wilson, Richard B.

    1971-01-01

    Administration of 0.001% 1,2-dimethylhydrazine dihydrochloride, symmetrical, in the drinking water of 7-week-old randomly bred Swiss mice for the remainder of their lifetime induced blood vessel tumors and enhanced the incidence of lung neoplasms. Ninety-eight percent of the females and 92% of the males developed vascular lesions, whereas among the controls the incidence was 3% in the females and 1% in the males. In addition, the incidence of lung tumors rose from 12 to 44% in the females and from 10 to 24% in the males, as compared with the controls. The occurrence of the vascular tumors in order of decreasing frequency was as follows: muscle, pararenal, fat, liver, parametrial, paraepididymal tissues, etc. Gross, light and electron microscopic examinations of vascular lesions revealed the characteristic appearance of angiosarcomas. The type and extent of macroscopic and histologic involvements of the various tissues by the tumors are presented. The ultrastructural descriptions of hemorrhagic areas, vascular spaces, neoplastic endothelial cells, their cytoplasms and organelles are illustrated in detail. In conclusion, whereas hydrazine enhanced the development of lung tumors, when the dimethyl group was attached to it at symmetrical positions, it evoked vascular tumors. Thus, the present study provides evidence for the possible relationship between chemical structure and tumor induction at specific organ sites. ImagesFig 10Fig 9Fig 11Fig 12Fig 1Fig 2Fig 3Fig 4Fig 5Fig 6Fig 7Fig 8 PMID:5133519

  15. Development of taste masked fast disintegrating films of levocetirizine dihydrochloride for oral use.

    PubMed

    Mahesh, A; Shastri, Nalini; Sadanandam, M

    2010-01-01

    Fast disintegrating films of levocetirizine dihydrochloride useful for the treatment of acute allergic rhinitis and chronic urticaria have been developed by using the taste masking ability of cyclodextrins. The fast disintegrating films were prepared by solvent casting method. The films contained water-soluble polymers such as Kollicoat IR or pullulan, aspartame and sucralose as sweeteners and pre-gelatinized starch as disintegrant. Levocetirizine dihydrochloride was incorporated into these films by in-situ complex formation with hydroxy propyl beta-cyclodextrin. The optimized films were evaluated for weight variation, film thickness, folding endurance, tackiness, tensile strength, assay, content uniformity, in vitro disintegration and dissolution, in vivo disintegration and taste masking ability by human gustatory sensation test. Results revealed that the organoleptic properties of levocetirizine dihydrochloride were improved by complexation with hydroxy propyl beta-cyclodextrin and the complex could be successfully formulated into a fast disintegrating film.

  16. Green approach towards the determination of hydroxyzine dihydrochloride in pure and pharmaceutical dosage forms.

    PubMed

    Mumtaz, Amina; Hussain, Shahid; Yasir, Muhammad

    2014-09-01

    A simple eco-friendly method has been developed for detection of hydroxyzine dihydrochloride in pure and pharmaceutical dosage forms. Both conventional system and microwave assisted procedures are used for the development of color. The blue coloured complex is measured spectrophotometrically at 750nm. Peak shift in FT-IR spectra also indicated the formation of complex. The reaction obeys Beer's law over the concentration range of 50- 250βg/mL of hydroxyzine dihydrochloride. The precision value (intra-day and inter-day RSD) for the drug is not greater than 0.79% and recoveries were found to be in range of 99.01-99.99%. The designed method is applicable for periodic determination of hydroxyzine dihydrochloride in pure and pharmaceutical dosage forms.

  17. The infarcted cardiac microenvironment cannot selectively promote embryonic stem cell differentiation into cardiomyocytes.

    PubMed

    Chen, You-Ren; Li, Yang; Chen, Li; Yang, Xin-Chun; Su, Pi-Xiong; Cai, Jun

    2011-01-01

    Postinfarct congestive heart failure is one of the leading causes of morbidity and mortality in industrialized countries. It is controversial whether embryonic stem cells are feasible sources for in situ cardiac regeneration in infarcted hearts. In order to investigate whether the infarcted cardiac microenvironment could selectively promote embryonic stem cell differentiation into cardiomyocytes, we assessed the cardiac differentiation potential of mouse embryonic stem cells (mESCs) injected into normal (n=16) or acutely infarcted rat hearts (n=18). We found that the transplanted 4',6-diamidino-2-phenylindole (DAPI)-labeled mESCs were able to survive and form stable intracardiac grafts both in normal and infarcted hearts, along with macrophages found specifically in the engraftment area. Two to four weeks after mESC transplantation, we found that more DAPI-positive mESCs differentiated into cardiomyocytes, marked by cardiac troponin T (cTnT), in normal than those in infarcted hearts (2.67±0.79% vs. 1.06±0.52%, P<.01). However, the discrepancy between the percentage of DAPI-positive cells that express cTnT in normal and that in infarcted hearts was diminished after 4 weeks (1.17±0.98% vs. 1.07±1.02%, P>.05), when the transverse striation began to present in the mESCs-derived cardiomyocytes. In addition, mESCs differentiated into vimentin-positive cardiac fibroblasts in normal and infracted hearts. Our results indicated that transplanted mESCs cannot only survive but differentiate into cardiomyocytes in infarcted rat hearts. However, the infarcted cardiac microenvironment cannot selectively promote mESCs differentiation into cardiomyocytes.

  18. ASIC channel inhibition enhances excitotoxic neuronal death in an in vitro model of spinal cord injury.

    PubMed

    Mazzone, Graciela L; Veeraraghavan, Priyadharishini; Gonzalez-Inchauspe, Carlota; Nistri, Andrea; Uchitel, Osvaldo D

    2017-02-20

    In the spinal cord high extracellular glutamate evokes excitotoxic damage with neuronal loss and severe locomotor impairment. During the cell dysfunction process, extracellular pH becomes acid and may activate acid-sensing ion channels (ASICs) which could be important contributors to neurodegenerative pathologies. Our previous studies have shown that transient application of the glutamate analog kainate (KA) evokes delayed excitotoxic death of spinal neurons, while white matter is mainly spared. The present goal was to enquire if ASIC channels modulated KA damage in relation to locomotor network function and cell death. Mouse spinal cord slices were treated with KA (0.01 or 0.1mM) for 1h, and then washed out for 24h prior to analysis. RT-PCR results showed that KA (at 0.01mM concentration that is near-threshold for damage) increased mRNA expression of ASIC1a, ASIC1b, ASIC2 and ASIC3, an effect reversed by the ASIC inhibitor 4',6-diamidino-2-phenylindole (DAPI). A KA neurotoxic dose (0.1mM) reduced ASIC1a and ASIC2 expression. Cell viability assays demonstrated KA-induced large damage in spinal slices from mice with ASIC1a gene ablation. Likewise, immunohistochemistry indicated significant neuronal loss when KA was followed by the ASIC inhibitors DAPI or amiloride. Electrophysiological recording from ventral roots of isolated spinal cords showed that alternating oscillatory cycles were slowed down by 0.01mMKA, and intensely inhibited by subsequently applied DAPI or amiloride. Our data suggest that early rise in ASIC expression and function counteracted deleterious effects on spinal networks by raising the excitotoxicity threshold, a result with potential implications for improving neuroprotection.

  19. Development of propidium iodide as a fluorescence probe for the on-line screening of non-specific DNA-intercalators in Fufang Banbianlian Injection.

    PubMed

    Niu, Yanyan; Li, Sensen; Lin, Zongtao; Liu, Meixian; Wang, Daidong; Wang, Hong; Chen, Shizhong

    2016-09-09

    Fufang Banbianlian Injection (FBI) has been widely used as an anti-inflammatory and anti-tumor prescription. To understand the relationships between its bioactive ingredients and pharmacological efficacies, our previous study has been successfully identified some DNA-binding compounds in FBI using an established on-line screening system, in which 4',6-diamidino-2-phenylindole (DAPI) was developed as a probe. However, DAPI can be only used to screen ATT-specific DNA minor groove binders, leaving the potential active intercalators unknown in FBI. As a continuation of our studies on FBI, here we present a sensitive analytical method for rapid identification and evaluation of DNA-intercalators using propidium iodide (PI) as a fluorescent probe. We have firstly established the technique of high-performance liquid chromatography-diode-array detector-multistage mass spectrometry-deoxyribonucleic acid-propidium iodide-fluorescence detector (HPLC-DAD-MS(n)-DNA-PI-FLD) system. As a result, 38 of 58 previously identified compounds in FBI were DNA-intercalation active. Interestingly, all previously reported DNA-binders also showed intercalative activities, suggesting they are dual-mode DNA-binders. Quantitative study showed that flavonoid glycosides and chlorogenic acids were the main active compounds in FBI, and displayed similar DNA-binding ability using either DAPI or PI. In addition, 13 active compounds were used to establish the structure-activity relationships. In this study, PI was developed into an on-line method for identifying DNA-intercalators for the first time, and thus it will be a useful high-throughput screening technique for other related samples.

  20. Unraveling the karyotype structure of the spurges Euphorbia hirta Linnaeus, 1753 and E. hyssopifolia Linnaeus, 1753 (Euphorbiaceae) using genome size estimation and heterochromatin differentiation.

    PubMed

    Santana, Karla C B; Pinangé, Diego S B; Vasconcelos, Santelmo; Oliveira, Ana R; Brasileiro-Vidal, Ana C; Alves, Marccus V; Benko-Iseppon, Ana M

    2016-01-01

    Euphorbia Linnaeus, 1753 (Euphorbiaceae) is one of the most diverse and complex genera among the angiosperms, showing a huge diversity in morphologic traits and ecologic patterns. In order to improve the knowledge of the karyotype organization of Euphorbia hirta (2n = 18) and Euphorbia hyssopifolia (2n = 12), cytogenetic studies were performed by means of conventional staining with Giemsa, genome size estimations with flow cytometry, heterochromatin differentiation with chromomycin A3 (CMA) and 4',6-diamidino-2-phenylindole (DAPI) and Giemsa C-banding, fluorescent in situ hybridization (FISH) with 45S and 5S rDNA probes, and impregnation with silver nitrate (AgNO3). Our results revealed small metacentric chromosomes, CMA(+)/DAPI(0) heterochromatin in the pericentromeric regions of all chromosomes and CMA(+)/DAPI(-) in the distal part of chromosome arms carriers of nucleolar organizing regions (NORs). The DNA content measurements revealed small genomes for both species: Euphorbia hirta with 2C = 0.77 pg and Euphorbia hyssopifolia with 2C = 1.41 pg. After FISH procedures, Euphorbia hirta, and Euphorbia hyssopifolia presented three and four pairs of terminal 45S rDNA sites, respectively, colocalizing with CMA(+) heterochromatic blocks, besides only one interstitial pair of 5S rDNA signals. Additionally, the maximum number of active NORs agreed with the total number of observed 45S rDNA sites. This work represents the first analysis using FISH in the subfamily Euphorbioideae, revealing a significant number of chromosomal markers, which may be very helpful to understand evolutionary patterns among Euphorbia species.

  1. Human mitochondria and mitochondrial genome function as a single dynamic cellular unit

    PubMed Central

    1994-01-01

    rho 0 HeLa cells entirely lacking mitochondrial DNA (mtDNA) and mitochondrial transfection techniques were used to examine intermitochondrial interactions between mitochondria with and without mtDNA, and also between those with wild-type (wt) and mutant-type mtDNA in living human cells. First, unambiguous evidence was obtained that the DNA-binding dyes ethidium bromide (EtBr) and 4',6-diamidino-2- phenylindole (DAPI) exclusively stained mitochondria containing mtDNA in living human cells. Then, using EtBr or DAPI fluorescence as a probe, mtDNA was shown to spread rapidly to all rho 0 HeLa mitochondria when EtBr- or DAPI-stained HeLa mitochondria were introduced into rho 0 HeLa cells. Moreover, coexisting wt-mtDNA and mutant mtDNA with a large deletion (delta-mtDNA) were shown to mix homogeneously throughout mitochondria, not to remain segregated by use of electron microscopic analysis of cytochrome c oxidase activities of individual mitochondria as a probe to identify mitochondria with predominantly wt- or delta- mtDNA in single cells. This rapid diffusion of mtDNA and the resultant homogeneous distribution of the heteroplasmic wt- and delta-mtDNA molecules throughout mitochondria in a cell suggest that the mitochondria in living human cells have lost their individuality. Thus, the actual number of mitochondria per cell is not of crucial importance, and mitochondria in a cell should be considered as a virtually single dynamic unit. PMID:8138574

  2. Nonviral gene therapy in vivo with PAM-RG4/apoptin as a potential brain tumor therapeutic

    PubMed Central

    An, Songhie; Nam, Kihoon; Choi, Sunghyun; Bai, Cheng Z; Lee, Yan; Park, Jong-Sang

    2013-01-01

    Background Glioma is still one of the most complicated forms of brain tumor to remove completely due to its location and the lack of an efficient means to specifically eliminate tumor cells. For these reasons, this study has examined the effectiveness of a nonviral gene therapy approach utilizing a tumor-selective killer gene on a brain tumor xenograft model. Methods and results The therapeutic apoptin gene was recombined into the JDK plasmid and delivered into human brain tumor cells (U87MG) by using a polyamidoamine dendrimer with an arginine surface (PAM-RG4). Studies in vitro showed that the PAM-RG4/apoptin plasmid polyplex exhibited a particularly high transfection activity of .40%. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, 4′,6-Diamidino-2-phenylindole (DAPI) TUNEL assay, DAPI staining, and caspase-3 activity assay verified that the tumor cells had undergone apoptosis induced by apoptin. For in vivo studies, the polyplex was injected into tumors, which were induced by injecting U87MG cells intradermally into nude mice. Based on hematoxylin and eosin staining, epidermal growth factor receptor immunohistochemistry results and tumor volume measurement results, tumor growth was effectively inhibited and no specific edema, irritation, or other harm to the skin was observed after polyplex injection. The in vivo expression of apoptin and the induction of apoptosis were verified by reverse-transcription polymerase chain reaction analysis, TUNEL assay, and DAPI staining. Conclusion The PAM-RG4/apoptin gene polyplex is a strong candidate for brain tumor therapeutics because of the synergistic effect of the carrier’s high transfection efficiency (35%–40%) in glioma cells and the selective apoptosis-inducing activity of apoptin in tumor cells. PMID:23589689

  3. Targeting of chemical mutagens to differentiating B-lymphocytes in vivo: detection by direct DNA labeling and sister chromatid exchange induction

    SciTech Connect

    Bloom, S.E.; Nanna, U.C.; Dietert, R.R.

    1987-01-01

    In vivo systems for analyzing mutagen interactions with a specific differentiating cell population are rare. Taking advantage of the unique anatomical features of the bursa of Fabricius in the chicken, the authors explored the possibility of targeting chemical mutagens to a defined differentiating cell population in the animal, namely, the B-lymphocytes series. Such cells are known to be the targets for the oncogene-activating avian leukosis virus. Targeting of chemicals to cells of the bursa was demonstrated by application of the DNA-specific fluorochrome 4'-6-diamidino-2-phenylindole (DAPI) to the anal lips of neonatal chicks. Bright nuclear fluorescence of cells in the bursa demonstrated to occur within minutes after the application of 500..mu..l of DAPI. DAPI labeling of nuclei was detected up to several days after a single application. No nuclear labeling was exhibited in cells of neighboring tissues. Methyl methanesulfonate (MMS)(10..mu..l) was applied to the anal lips of day-old chicks to study dose-response kinetics for mutagen targeting to DNA of dividing B-lymphocytes in the bursa. Since the mitotic index was found to be quite high (25-30%) in the bursa, chromosome analysis was used to assay for genome damage. Sister chromatid exchange frequencies of 3.9, 7.3, and 9.0 (baseline 2.5) per cell were obtained at MMS dosages per animal of 50 ..mu..g, 100..mu..g, and 200..mu..g, respectively. These results indicate the rapid and quantitative localization of DNA-binding chemicals to cells of the bursa, particularly the resident B-lymphocytes. The bursa should be a useful system for studying mutagen-DNA interactions in the differentiating B-lymphocyte and subsequent influences on the development of immunity and lymphoproliferative disease.

  4. [DNA quantification in nuclei of cultivated mushroom with DAPI staining].

    PubMed

    Pancheva, E V; Volkova, V N; Kamzolkina, O V

    2004-01-01

    Agaricus bisporus (Lange) Imbach is actively cultivated amphithallic basidiomycete, in which various strains are primary homothallic, heterothallic or secondary homothallic. Countings of relative nuclear DNA content by means of DAPI stain and its comparison in different strains can help to understand the mushroom's life cycle features. The authors for the first time observed change of nuclear phases in basidia of A. bisporus strains with different types of life cycle and revealed that DNA content in diploid nuclei is about 1.3 times higher than in haploid ones. The method is highly sensitive and can be used for quantitative measurings of nuclear DNA even in objects with nuclei of about 1 mkm in diameter.

  5. Efficacy of Trimetazidine Dihydrochloride for Relieving Chronic Tinnitus: A Randomized Double-Blind Study

    PubMed Central

    Kumral, Tolgar Lütfi; Yıldırım, Güven; Berkiten, Güler; Saltürk, Ziya; Ataç, Enes; Atar, Yavuz; Uyar, Yavuz

    2016-01-01

    Objectives. To evaluate the efficacy of trimetazidine dihydrochloride as a treatment for chronic tinnitus. Methods. A total of 97 chronic tinnitus patients were evaluated in this randomized, prospective, double-blind, placebo-controlled trial. After assessing for eligibility, 82 patients were randomly assigned into placebo or trimetazidine groups according to the medication. The trimetazidine group received 20×3 mg/day per oral trimetazidine dihydrochloride and the placebo group received 20×3 mg/day per oral placebo for 3 months. Tinnitus handicap inventory (THI), visual analogue scale (VAS) questionnaires and audiometric results were used to determine the effectiveness of trimetazidine treatment. Results. The study group comprised 82 tinnitus subjects, 42 (51%) of whom received trimetazidine dihydrochloride and 40 (49%) who received placebo. There was no significant difference between placebo and trimetazidine groups in THI grade and VAS (both pre- and posttreatment scores) (P>0.05) and no significant improvement was observed in subjective loudness score in either group (P>0.05). Additionally there was no significant difference between groups in pre- and posttreatment pure tone hearing thresholds at all measured frequencies (P>0.05). Conclusion. Trimetazidine dihydrochloride therapy was ineffective for relieving chronic tinnitus. PMID:27230273

  6. Synthesis, DNA binding, fluorescence measurements and antiparasitic activity of DAPI related diamidines.

    PubMed

    Farahat, Abdelbasset A; Kumar, Arvind; Say, Martial; Barghash, Alaa El-Din M; Goda, Fatma E; Eisa, Hassan M; Wenzler, Tanja; Brun, Reto; Liu, Yang; Mickelson, Leah; Wilson, W David; Boykin, David W

    2010-01-15

    A novel series of extended DAPI analogues were prepared by insertion of either a carbon-carbon triple bond (16a-d) or a phenyl group (21a,b and 24) at position-2. The new amidines were evaluated in vitro against both Trypanosoma brucei rhodesiense (T. b. r.) and Plasmodium falciparum (P. f.). Five compounds (16a, 16b, 16d, 21a, 21b) exhibited IC(50) values against T. b. r. of 9nM or less which is two to nine folds more effective than DAPI. The same five compounds exhibited IC(50) values against P. f. of 5.9nM or less which is comparable to that of DAPI. The fluorescence properties of these new molecules were recorded, however; they do not offer any advantage over those of DAPI.

  7. Organelles and chromatin fragmentation of human umbilical vein endothelial cell influence by the effects of zeta potential and size of silver nanoparticles in different manners.

    PubMed

    Tavakol, Shima; Hoveizi, Elham; Kharrazi, Sharmin; Tavakol, Behnaz; Karimi, Shabnam; Rezayat Sorkhabadi, Seyed Mahdi

    2017-06-01

    Recently, it has been disclosed that silver nanoparticles (AgNPs) have the potential to inhibit infection and cancerous cells and eventually penetrate through injected site into the capillary due to their small size. This study focuses on the effect of size and zeta potential of bare and citrate-coated AgNPs on human umbilical vein endothelial cells (HUVECs) as main capillary cells. AgNPs with high and low concentrations and no citrate coating were synthesized by using simple wet chemical method and named as AgNP/HC, AgNP/LC, and AgNP, respectively. Citrate coated particles showed larger zeta potential of -22 mV and AgNp/HC showed the smallest size of 13.2 nm. UV-Visible spectroscopy and dynamic light scattering (DLS) were performed to evaluate particle size and hydrodynamic diameter of NPs in water and cell culture media. Results indicated that higher concentrations of citrate decreased hydrodynamic diameter and NP agglomeration. reactive oxygen species (ROS) production of all AgNPs was similar at 28 ppm although it was significantly higher than control group. Their effects on cell membrane and chromosomal structure were studied using LDH measurement and 4',6-diamidino-2-phenylindole (DAPI) staining, as well. Results demonstrated that AgNP/LC was less toxic to cells owing to higher value of IC50, minimum inhibitory concentration (MIC), and less release of LDH. Cancerous (Human Caucasian neuroblastoma) and immortal cells (Mouse embryonic fibroblast cell line) were about twice more sensitive than HUVECs to toxic effects of AgNPs. DAPI staining results showed that AgNP and AgNP/HC induced highest and lowest breaking of chromosome. Overall results suggest that viability of HUVECs will be higher than 90% when viability of cancerous cells is 50% in AgNPs chemotherapy.

  8. Cytofluorometric determination of nuclear DNA in living and preserved algae.

    PubMed

    Hull, H M; Hoshaw, R W; Wang, J C

    1982-09-01

    Three DNA-localizing fluorochromes used in conjunction with epi (incident) UV illumination were examined for sensitivity and selectivity for the cytofluorometric determination of nuclear DNA in ten species of six algal genera: Mougeotia, Oedogonium, Sirogonium, Spirogyra and Zygnema among the green algae, and the marine red alga Polysiphonia boldii. In comparison with absorption photometry for the determination of nuclear DNA, the cytofluorometric procedure proved to be simpler and considerably more sensitive. Following staining with 4',6-diamidino-2-phenylindole (DAPI), nuclei fluoresce blue-white, the fluorescence intensity of the DNA-DAPI complex being considerably greater than that of the unbound dye molecule. Algal strains stained with 2,5-bis[4'-aminophenyl(1')]-1,3,4-oxadiazole (BAO) also showed brilliant blue-white nuclear fluorescence. Although the BAO schedule requires the use of freshly prepared dye and sulfite water, and careful control of hydrolysis, nuclear fluorescence of the stained specimens does not fade under irradiation of the UV beam as rapidly as it does with certain other fluorochrome procedures. A more useful fluorochrome was the fungal antibiotic mithramycin. Its staining schedule is simple and the bright orange-yellow fluorescence of the nuclei is associated with an exceptional degree of sensitivity and specificity for DNA. Forty-eight-year-old preserved filaments of Spirogyra jatobae, stained with either BAO or mithramycin, exhibited a fluorescence brilliance of nuclear and chloroplast DNA equal to that of fresh specimens of this species. The three schedules, but particularly the one with mithramycin, have proven useful in providing indirect evidence for variation in ploidy level in several of the above algal genera, and in verifying the assumed ploidy level of the gametophyte (haploid) and tetrasporophyte (diploid) of Polysiphonia boldii.

  9. Unraveling the karyotype structure of the spurges Euphorbia hirta Linnaeus, 1753 and E. hyssopifolia Linnaeus, 1753 (Euphorbiaceae) using genome size estimation and heterochromatin differentiation

    PubMed Central

    Santana, Karla C. B.; Pinangé, Diego S. B.; Vasconcelos, Santelmo; Oliveira, Ana R.; Brasileiro-Vidal, Ana C.; Alves, Marccus V.; Benko-Iseppon, Ana M.

    2016-01-01

    Abstract Euphorbia Linnaeus, 1753 (Euphorbiaceae) is one of the most diverse and complex genera among the angiosperms, showing a huge diversity in morphologic traits and ecologic patterns. In order to improve the knowledge of the karyotype organization of Euphorbia hirta (2n = 18) and Euphorbia hyssopifolia (2n = 12), cytogenetic studies were performed by means of conventional staining with Giemsa, genome size estimations with flow cytometry, heterochromatin differentiation with chromomycin A3 (CMA) and 4’,6-diamidino-2-phenylindole (DAPI) and Giemsa C-banding, fluorescent in situ hybridization (FISH) with 45S and 5S rDNA probes, and impregnation with silver nitrate (AgNO3). Our results revealed small metacentric chromosomes, CMA+/DAPI0 heterochromatin in the pericentromeric regions of all chromosomes and CMA+/DAPI− in the distal part of chromosome arms carriers of nucleolar organizing regions (NORs). The DNA content measurements revealed small genomes for both species: Euphorbia hirta with 2C = 0.77 pg and Euphorbia hyssopifolia with 2C = 1.41 pg. After FISH procedures, Euphorbia hirta, and Euphorbia hyssopifolia presented three and four pairs of terminal 45S rDNA sites, respectively, colocalizing with CMA+ heterochromatic blocks, besides only one interstitial pair of 5S rDNA signals. Additionally, the maximum number of active NORs agreed with the total number of observed 45S rDNA sites. This work represents the first analysis using FISH in the subfamily Euphorbioideae, revealing a significant number of chromosomal markers, which may be very helpful to understand evolutionary patterns among Euphorbia species. PMID:28123686

  10. Mobile phone radiation alters proliferation of hepatocarcinoma cells.

    PubMed

    Ozgur, Elcin; Guler, Goknur; Kismali, Gorkem; Seyhan, Nesrin

    2014-11-01

    This study investigated the effects of intermittent exposure (15 min on, 15 min off for 1, 2, 3, or 4 h, at a specific absorption rate of 2 W/kg) to enhanced data rates for global system for mobile communication evolution-modulated radiofrequency radiation (RFR) at 900- and 1,800-MHz frequencies on the viability of the Hepatocarcinoma cells (Hep G2). Hep G2 cell proliferation was measured by a colorimetric assay based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases in viable cells. Cell injury was evaluated by analyzing the levels of lactate dehydrogenase (LDH) and glucose released from lysed cells into the culture medium. Morphological observation of the nuclei was carried out by 4',6-diamidino-2-phenylindole (DAPI) staining using fluorescence microscopy. In addition, TUNEL assay was performed to confirm apoptotic cell death. It was observed that cell viability, correlated with the LDH and glucose levels, changed according to the frequency and duration of RFR exposure. Four-hour exposure produced more pronounced effects than the other exposure durations. 1,800-MHz RFR had a larger impact on cell viability and Hep G2 injury than the RFR at 900 MHz. Morphological observations also supported the biochemical results indicating that most of the cells showed irregular nuclei pattern determined by using the DAPI staining, as well as TUNEL assay which shows DNA damage especially in the cells after 4 h of exposure to 1,800-MHz RFR. Our results indicate that the applications of 900- and 1,800-MHz (2 W/kg) RFR cause to decrease in the proliferation of the Hep G2 cells after 4 h of exposure. Further studies will be conducted on other frequency bands of RFR and longer duration of exposure.

  11. Embryo quality and transcervical technique are not the limiting factors in donkey embryo transfer outcome.

    PubMed

    Panzani, D; Rota, A; Crisci, A; Kindahl, H; Govoni, N; Camillo, F

    2012-02-01

    Embryo transfer (ET) in the donkey resulted in a very low recipient pregnancy rates. The aim of these studies was to investigate if nonsurgical transfer techniques or donkey embryo quality affect donkey recipient pregnancy failure. In Study 1, the impact of transfer technique was investigated by evaluating if cervical catheterization is associated with prostaglandin release and suppression of luteal function and if donkey recipients would become pregnant after nonsurgical transfer of horse embryos. Four jennies, from 5 to 8 d after ovulation, were submitted to a sham transcervical ET and to evaluation of PGFM and progesterone plasma concentrations. Five 8 d horse embryos were nonsurgically transferred into synchronized donkey recipients (HD). Cervical stimulation caused a transient PGF(2α) release in two of four jennies in the absence of a significant decrease in progesterone plasma concentration. All transferred horse embryos resulted in pregnancies in the jenny recipients. In Study 2, donkey embryo viability was investigated by 1.2 meters, 6-diamidino-2-phenylindole (DAPI) staining of 10 embryos and by the transfer of 6 and 12 donkey embryos in synchronized mare (DH) and donkey (DD) recipients, respectively, of known fertility. The estimated proportion of dead cells in DAPI stained embryos was 0.9% (range 0-3.9%) and below what is considered normal (20%) for horse embryos. Three of six and six of 12 of the DH and DD ETs, respectively resulted in pregnancies at 14 and 25 d (50%), a higher pregnancy rate than previously reported after DD ET. The overall results of this study suggest that the transcervical technique for ET and donkey embryo viability are not the reasons for the low pregnancy rates that have previously been described in donkey recipients, and that nonsurgical ET in donkeys can result in acceptable results.

  12. Microorganisms, Organic Carbon, and Their Relationship with Oxidant Activity in Hyper-Arid Mars-Like Soils: Implications for Soil Habitability

    NASA Technical Reports Server (NTRS)

    Valdivia-Silva, Julio E.; Karouia, Fathi; Navarro-Gonzalez, Rafael; McKay, Christopher

    2016-01-01

    Soil samples from the hyper-arid region in the Atacama 23 Desert in Southern Peru (La Joya Desert) were analyzed for total and labile organic carbon (TOC & LOC), phospholipid fatty acids analysis (PLFA), quantitative real time polymerase chain reaction (qRT-PCR), 4',6- diamidino-2-phenylindole (DAPI)-fluorescent microscopy, culturable microorganisms, and oxidant activity, in order to understand the relationship between the presence of organic matter and microorganisms in these types of soils. TOC content levels were similar to the labile pool of carbon suggesting the absence of recalcitrant carbon in these soils. The range of LOC was from 2 to 60 micro-g/g of soil. PLFA analysis indicated a maximum of 2.3 x 10(exp 5) cell equivalents/g. Culturing of soil extracts yielded 1.1 x 10(exp 2)-3.7 x 10(exp 3) CFU/g. qRT-PCR showed between 1.0 x 10(exp 2) and 8 x 10(exp 3) cells/g; and DAPI fluorescent staining indicated bacteria counts up to 5 x 104 cells/g. Arid and semiarid samples (controls) showed values between 10(exp 7) and 10(exp 11) cells/g with all of the methods used. Importantly, the concentration of microorganisms in hyper-arid soils did not show any correlation with the organic carbon content; however, there was a significant dependence on the oxidant activity present in these soil samples evaluated as the capacity to decompose sodium formate in 10 hours. We suggest that the analysis of oxidant activity could be a useful indicator of the microbial habitability in hyper-arid soils, obviating the need to measure water activity over time. This approach could be useful in astrobiological studies on other worlds.

  13. Effect of growth conditions and staining procedure upon the subsurface transport and attachment behaviors of a groundwater protist

    USGS Publications Warehouse

    Harvey, R.W.; Mayberry, N.; Kinner, N.E.; Metge, D.W.; Novarino, F.

    2002-01-01

    The transport and attachment behaviors of Spumella guttula (Kent), a nanoflagellate (protist) found in contaminated and uncontaminated aquifer sediments in Cape Cod, Mass., were assessed in flowthrough and static columns and in a field injection-and-recovery transport experiment involving an array of multilevel samplers. Transport of S. guttula harvested from low-nutrient (10 mg of dissolved organic carbon per liter), slightly acidic, granular (porous) growth media was compared to earlier observations involving nanoflagellates grown in a traditional high-nutrient liquid broth. In contrast to the highly retarded (retardation factor of ???3) subsurface transport previously reported for S. guttula, the peak concentration of porous-medium-grown S. guttula traveled concomitantly with that of a conservative (bromide) tracer. About one-third of the porous-medium-grown nanoflagellates added to the aquifer were transported at least 2.8 m downgradient, compared to only ???2% of the broth-grown nanoflagellates. Flowthrough column studies revealed that a vital (hydroethidine [HE]) staining procedure resulted in considerably less attachment (more transport) of S. guttula in aquifer sediments than did a staining-and-fixation procedure involving 4???,6???-diamidino-2-phenylindole (DAPI) and glutaraldehyde. The calculated collision efficiency (???10-2. for porous-medium-grown, DAPI-stained nanoflagellates) was comparable to that observed earlier for the indigenous community of unattached ground-water bacteria that serve as prey. The attachment of HE-labeled S. guttula onto aquifer sediment grains was independent of pH (over the range from pH 3 to 9) suggesting a primary attachment mechanism that may be fundamentally different from that of their prey bacteria, which exhibit sharp decreases in fractional attachment with increasing pH. The high degree of mobility of S. guttula in the aquifer sediments has important ecological implications for the protistan community within the

  14. Cloning of DNA sequences localized on proximal fluorescent chromosome bands by microdissection in Pinus densiflora Sieb. & Zucc.

    PubMed

    Hizume, M; Shibata, F; Maruyama, Y; Kondo, T

    2001-09-01

    Japanese red pine, Pinus densiflora, has 2n=24 chromosomes, of which most carry chromomycin A3 (CMA) and 4',6-diamidino-2-phenylindole (DAPI) bands at their centromere-proximal regions. It was proposed that these regions contain highly repetitive DNA. The DNA localized in the proximal fluorescent bands was isolated and characterized. In P. densiflora, centromeric and neighboring segments of the somatic chromosomes were dissected with a manual micromanipulator. The centromeric DNA was amplified from the DNA contained in dissected centromeric segments by degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR) and a cloned DNA library was constructed. Thirty-one clones carrying highly repetitive DNA were selected by colony hybridization using Cot-1 DNA from this species as a probe, and their chromosomal localization was determined by fluorescent in situ hybridization (FISH). Clone PDCD501 was localized to the proximal CMA band of 20 chromosomes. This clone contained tandem repeats, comprising a 27 bp repeat unit, which was sufficient to provide the proximal FISH signal, with a 52.3% GC content. The repetitive sequence was named PCSR (proximal CMA band-specific repeat). Clone PDCD159 was 1700 bp in length, with a 61.7% AT content, and produced FISH signals at the proximal DAPI band of the remaining four chromosomes. Four clones hybridized strongly to the secondary constriction and gave weak signals at the centromeric region of several chromosomes. Clone PDCD537, one of the four clones, was homologous to the 26S rRNA gene. A PCR experiment using microdissected centromeric regions suggested that the centromeric region contains 18S and 26S rDNA. Another 24 clones hybridized to whole chromosome arms, with varying intensities and might represent dispersed repetitive DNA.

  15. Decellularization of porcine skeletal muscle extracellular matrix for the formulation of a matrix hydrogel: a preliminary study.

    PubMed

    Fu, Yuehe; Fan, Xuejiao; Tian, Chunxiang; Luo, Jingcong; Zhang, Yi; Deng, Li; Qin, Tingwu; Lv, Qing

    2016-04-01

    Extracellular matrix (ECM) hydrogels are used as scaffolds to facilitate the repair and reconstruction of tissues. This study aimed to optimize the decellularization process of porcine skeletal muscle ECM and to formulate a matrix hydrogel scaffold. Five multi-step methods (methods A-E) were used to generate acellular ECM from porcine skeletal muscle [rinsing in SDS, trypsin, ethylenediaminetetraacetic acid (EDTA), Triton X-100 and/or sodium deoxycholate at 4-37°C]. The resulting ECM was evaluated using haematoxylin and eosin, 4-6-diamidino-2-phenylindole (DAPI) staining, and DNA quantification. Acellular matrix was dissolved in pepsin and gelled at 37°C. Hydrogel response to temperature was observed in vivo and in vitro. ECM components were assessed by Masson, Sirius red, and alcian blue staining, and total protein content. Acellular porcine skeletal muscle exhibited a uniform translucent white appearance. No intact nuclear residue was detected by haematoxylin and eosin staining, while DAPI staining showed a few nuclei in the matrixes produced by methods B, C, and D. Method A generated a gel that was too thin for gelation. However, the matrix obtained by rinsing in 0.2% trypsin/0.1% EDTA, 0.5% Triton X-100, and 1% Triton X-100/0.2% sodium deoxycholate was nuclei-free and produced a viscous solution that formed a structurally stable white jelly-like hydrogel. The residual DNA content of this solution was 49.37 ± 0.72 ng/mg, significantly less than in fresh skeletal muscle, and decreased to 19.22 ± 0.85 ng/mg after gelation (P < 0.05). The acellular matrix was rich in collagen and glycosaminoglycan, with a total protein concentration of 64.8 ± 6.9%. An acellular ECM hydrogel from porcine skeletal muscle was efficiently produced.

  16. Chemical correction of pre-mRNA splicing defects associated with sequestration of muscleblind-like 1 protein by expanded r(CAG)-containing transcripts.

    PubMed

    Kumar, Amit; Parkesh, Raman; Sznajder, Lukasz J; Childs-Disney, Jessica L; Sobczak, Krzysztof; Disney, Matthew D

    2012-03-16

    Recently, it was reported that expanded r(CAG) triplet repeats (r(CAG)(exp)) associated with untreatable neurological diseases cause pre-mRNA mis-splicing likely due to sequestration of muscleblind-like 1 (MBNL1) splicing factor. Bioactive small molecules that bind the 5'CAG/3'GAC motif found in r(CAG)(exp) hairpin structure were identified by using RNA binding studies and virtual screening/chemical similarity searching. Specifically, a benzylguanidine-containing small molecule was found to improve pre-mRNA alternative splicing of MBNL1-sensitive exons in cells expressing the toxic r(CAG)(exp). The compound was identified by first studying the binding of RNA 1 × 1 nucleotide internal loops to small molecules known to have affinity for nucleic acids. Those studies identified 4',6-diamidino-2-phenylindole (DAPI) as a specific binder to RNAs with the 5'CAG/3'GAC motif. DAPI was then used as a query molecule in a shape- and chemistry alignment-based virtual screen to identify compounds with improved properties, which identified 4-guanidinophenyl 4-guanidinobenzoate, a small molecule that improves pre-mRNA splicing defects associated with the r(CAG)(exp)-MBNL1 complex. This compound may facilitate the development of therapeutics to treat diseases caused by r(CAG)(exp) and could serve as a useful chemical tool to dissect the mechanisms of r(CAG)(exp) toxicity. The approach used in these studies, defining the small RNA motifs that bind small molecules with known affinity for nucleic acids and then using virtual screening to optimize them for bioactivity, may be generally applicable for designing small molecules that target other RNAs in the human genomic sequence.

  17. Oxidized polyethylene films for orienting polar molecules for linear dichroism spectroscopy.

    PubMed

    Razmkhah, Kasra; Chmel, Nikola Paul; Gibson, Matthew I; Rodger, Alison

    2014-03-21

    Stretched polyethylene (PE) films have been used to orient small molecules for decades by depositing solutions on their surface and allowing the solvent to evaporate leaving the analyte absorbed on the polymer film. However, the non-polar hydrophobic nature of PE is an obstacle to aligning polar molecules and biological samples. In this work PE film was treated with oxygen plasma in order to increase surface hydrophilicity. Different treatment conditions were evaluated using contact angle measurement and X-ray photoelectron spectroscopy. Treated PE (PE(OX)) films are shown to be able to align molecules of different polarities including progesterone, 1-pyrenecarboxaldehyde, 4',6-diamidino-2-phenylindole (DAPI) and anthracene. The degree of alignment of each molecule was studied by running series of linear dichroism (LD) experiments and the polarizations of electronic transition moments were determined. For the first time optimal conditions (such as stretching factor and concentration of the sample) for stretched film LD were determined. PE(OX) aligning ability was compared to that of normal PE films. Progesterone showed a slightly better alignment on PE(OX) than PE. 1-Pyrenecarboxaldehyde oriented differently on the two different films which enabled transition moment assignment for this low symmetry molecule. DAPI (which does not align on PE) aligned well on PE(OX) and enabled us to obtain better LD data than had previously been collected with polyvinyl alcohol. Anthracene alignment and formation of dimers and higher order structures were studied in much more detail than previously possible, showing a variety of assemblies on PE and PE(OX) films.

  18. Polarized fluorescence correlation spectroscopy of DNA-DAPI complexes.

    PubMed

    Barcellona, Maria Luisa; Gammon, Seth; Hazlett, Theodore; Digman, Michelle A; Gratton, Enrico

    2004-11-01

    We discuss the use of fluorescence correlation spectroscopy for the measurement of relatively slow rotations of large macromolecules in solution or attached to other macromolecular structures. We present simulations and experimental results to illustrate the range of rotational correlation times and diffusion times that the technique can analyze. In particular, we examine various methods to analyze the polarization fluctuation data. We have found that by first constructing the polarization function and then calculating the autocorrelation function, we can obtain the rotational motion of the molecule with very little interference from the lateral diffusion of the macromolecule, as long as the rotational diffusion is significantly faster than the lateral diffusion. Surprisingly, for common fluorophores the autocorrelation of the polarization function is relatively unaffected by the photon statistics. In our instrument, two-photon excitation is used to define a small volume of illumination where a few molecules are present at any instant of time. The measurements of long DNA molecules labeled with the fluorescent probe DAPI show local rotational motions of the polymers in addition to translation motions of the entire polymer. For smaller molecules such as EGFP, the viscosity of the solution must be increased to bring the relaxation due to rotational motion into the measurable range. Overall, our results show that polarized fluorescence correlation spectroscopy can be used to detect fast and slow rotational motion in the time scale from microsecond to second, a range that cannot be easily reached by conventional fluorescence anisotropy decay methods.

  19. Formulation and evaluation of novel coated floating tablets of bergenin and cetirizine dihydrochloride for gastric delivery.

    PubMed

    He, Shuang; Li, Feng; Zhou, Dan; Du, Junrong; Huang, Yuan

    2012-10-01

    A novel coated gastric floating drug-delivery system (GFDDS) of bergenin (BN) and cetirizine dihydrochloride (CET) was developed. First, the pharmacodynamic studies were performed and the results revealed that the new compounds of bergenin/cetirizine dihydrochloride had comparative efficacy as commercial products (bergenin/chlorphenamine maleate) but with fewer side effects on central nervous system (CNS). Subsequently, bergenin was formulated as an extended-release core tablet while cetirizine dihydrochloride was incorporated into the gastric coating film for immediate release. The formulation of GFDDS was optimized by CET content uniformity test, in vitro buoyancy and drug release. Herein, the effects of sodium bicarbonate (effervescent), hydroxypropyl methylcellulose (HPMC, matrix polymer) and coating weight gain were investigated respectively. The optimized GFDDS exhibited good floating properties (buoyancy lag time < 2 min; floating duration > 10 h) and satisfactory drug-release profiles (immediate release of CET in 10 min and sustained release of BN for 12 h). In vivo gamma scintigraphy proved that the optimized GFDDS could retain in the stomach with a prolonged gastric retention time (GRT) of 5 h, and the coating layer showed no side effect for gastric retention. The novel coated gastric floating drug-delivery system offers a new approach to enhance BN's absorption at its absorption site and the efficacy of both CET and BN.

  20. The use of DAPI fluorescence lifetime imaging for investigating chromatin condensation in human chromosomes

    PubMed Central

    Estandarte, Ana Katrina; Botchway, Stanley; Lynch, Christophe; Yusuf, Mohammed; Robinson, Ian

    2016-01-01

    Chromatin undergoes dramatic condensation and decondensation as cells transition between the different phases of the cell cycle. The organization of chromatin in chromosomes is still one of the key challenges in structural biology. Fluorescence lifetime imaging (FLIM), a technique which utilizes a fluorophore’s fluorescence lifetime to probe changes in its environment, was used to investigate variations in chromatin compaction in fixed human chromosomes. Fixed human metaphase and interphase chromosomes were labeled with the DNA minor groove binder, DAPI, followed by measurement and imaging of the fluorescence lifetime using multiphoton excitation. DAPI lifetime variations in metaphase chromosome spreads allowed mapping of the differentially compacted regions of chromatin along the length of the chromosomes. The heteromorphic regions of chromosomes 1, 9, 15, 16, and Y, which consist of highly condensed constitutive heterochromatin, showed statistically significant shorter DAPI lifetime values than the rest of the chromosomes. Differences in the DAPI lifetimes for the heteromorphic regions suggest differences in the structures of these regions. DAPI lifetime variations across interphase nuclei showed variation in chromatin compaction in interphase and the formation of chromosome territories. The successful probing of differences in chromatin compaction suggests that FLIM has enormous potential for application in structural and diagnostic studies. PMID:27526631

  1. The use of DAPI fluorescence lifetime imaging for investigating chromatin condensation in human chromosomes.

    PubMed

    Estandarte, Ana Katrina; Botchway, Stanley; Lynch, Christophe; Yusuf, Mohammed; Robinson, Ian

    2016-08-16

    Chromatin undergoes dramatic condensation and decondensation as cells transition between the different phases of the cell cycle. The organization of chromatin in chromosomes is still one of the key challenges in structural biology. Fluorescence lifetime imaging (FLIM), a technique which utilizes a fluorophore's fluorescence lifetime to probe changes in its environment, was used to investigate variations in chromatin compaction in fixed human chromosomes. Fixed human metaphase and interphase chromosomes were labeled with the DNA minor groove binder, DAPI, followed by measurement and imaging of the fluorescence lifetime using multiphoton excitation. DAPI lifetime variations in metaphase chromosome spreads allowed mapping of the differentially compacted regions of chromatin along the length of the chromosomes. The heteromorphic regions of chromosomes 1, 9, 15, 16, and Y, which consist of highly condensed constitutive heterochromatin, showed statistically significant shorter DAPI lifetime values than the rest of the chromosomes. Differences in the DAPI lifetimes for the heteromorphic regions suggest differences in the structures of these regions. DAPI lifetime variations across interphase nuclei showed variation in chromatin compaction in interphase and the formation of chromosome territories. The successful probing of differences in chromatin compaction suggests that FLIM has enormous potential for application in structural and diagnostic studies.

  2. DAPI binding to the DNA minor groove: a continuum solvent analysis.

    PubMed

    De Castro, L F Pineda; Zacharias, M

    2002-01-01

    A continuum solvent model based on the generalized Born (GB) or finite-difference Poisson-Boltzmann (FDPB) approaches has been employed to compare the binding of 4'-6-diamidine-2-phenyl indole (DAPI) to the minor groove of various DNA sequences. Qualitative agreement between the results of GB and FDPB approaches as well as between calculated and experimentally observed trends regarding the sequence specificity of DAPI binding to B-DNA was obtained. Calculated binding energies were decomposed into various contributions to solvation and DNA-ligand interaction. DNA conformational adaptation was found to make a favorable contribution to the calculated total interaction energy but did not change the DAPI binding affinity ranking of different DNA sequences. The calculations indicate that closed complex formation is mainly driven by nonpolar contributions and was found to be disfavored electrostatically due to a desolvation penalty that outbalances the attractive Coulomb interaction. The calculated penalty was larger for DAPI binding to GC-rich sequences compared with AT-rich target sequences and generally larger for the FDPB vs the GB continuum model. A radial interaction profile for DAPI at different distances from the DNA minor groove revealed an electrostatic energy minimum a few Angstroms farther away from the closed binding geometry. The calculated electrostatic interaction up to this distance is attractive and it may stabilize a nonspecific binding arrangement.

  3. Exploration of DAPI analogues: Synthesis, antitrypanosomal activity, DNA binding and fluorescence properties.

    PubMed

    Farahat, Abdelbasset A; Kumar, Arvind; Say, Martial; Wenzler, Tanja; Brun, Reto; Paul, Ananya; Wilson, W David; Boykin, David W

    2017-03-10

    The DAPI structure has been modified by replacing the phenyl group with substituted phenyl or heteroaryl rings. Twelve amidines were synthesized and their DNA binding, fluorescence properties, in vitro and in vivo activities were evaluated. These compounds are shown to bind in the DNA minor groove with high affinity, and exhibit superior in vitro antitrypanosomal activity to that of DAPI. Six new diamidines (5b, 5c, 5d, 5e, 5f and 5j) exhibit superior in vivo activity to that of DAPI and four of these compounds provide 100% animal cure at a low dose of 4 × 5 mg/kg i.p. in T. b. rhodesiense infected mice. Generally, the fluorescence properties of the new analogues are inferior to that of DAPI with the exception of compound 5i which shows a moderate increase in efficacy while compound 5k is comparable to DAPI.

  4. Interferon-α and cyclooxygenase-2 inhibitor cooperatively mediates TRAIL-induced apoptosis in hepatocellular carcinoma

    SciTech Connect

    Zuo, Chaohui; Qiu, Xiaoxin; Liu, Nianli; Yang, Darong; Xia, Man; Liu, Jingshi; Wang, Xiaohong; and others

    2015-05-01

    Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide. Interferon-alpha (IFN-α) has recently been recognized to harbor therapeutic potential in the prevention and treatment of HCC, but it remains controversial as to whether IFN-α exerts direct cytotoxicity against HCC. Cyclooxygenase-2 (COX-2) is overexpressed in HCC and is considered to play a role in hepatocarcinogenesis. Therefore, we aimed to elucidate the combined effect of a COX-2 inhibitor, celecoxib, and IFN-α on in vitro growth suppression of HCC using the hepatoma cell line HLCZ01 and the in vivo nude mouse xenotransplantation model using HLCZ01 cells. Treatment with celecoxib and IFN-α synergistically inhibited cell proliferation in a dose- and time-dependent manner. Apoptosis was identified by 4',6-diamidino-2-phenylindole dihydrochloride and fluorescent staining. IFN-α upregulated the expression of TRAIL, while celecoxib increased the expression of TRAIL receptors. The combined regimen with celecoxib and IFN-α reduced the growth of xenotransplanted HCCs in nude mice. The regulation of IFN-α- and COX-2 inhibitor-induced cell death is impaired in a subset of TRAIL-resistant cells. The molecular mechanisms of HCC cells resistant to TRAIL-induced apoptosis were explored using molecular biological and immunological methods. Interferon-α and the COX-2 inhibitor celecoxib synergistically increased TRAIL-induced apoptosis in hepatocellular carcinoma. These data suggest that IFN-α and celecoxib may offer a novel role with important implications in designing new therapeutics for TRAIL-resistant tumors. - Highlights: ●The cytotoxic effect of TRAIL on a developed HCC HLCZ01 cells infected with HBV. ●IFN-α and celecoxib induced apoptosis in HLCZ01 cells infected with HBV. ●The combined regime reduced the growth of xenotransplanted HCCs in nude mice model.

  5. Hepatocellular apoptosis during Candida albicans colonization: involvement of TNF-alpha and infiltrating Fas-L positive lymphocytes.

    PubMed

    Renna, María S; Correa, Silvia G; Porporatto, Carina; Figueredo, Carlos M; Aoki, María P; Paraje, María G; Sotomayor, Claudia E

    2006-12-01

    The liver constitutes the first barrier in the control of hematogenous dissemination of Candida albicans of intestinal origin. In rats infected with C. albicans, this organ limits the growth of the yeast and mounts an efficient inflammatory reaction. However, in rats infected and exposed to chronic varied stress, the hepatic inflammatory reaction is compromised and the outcoming of the infection is more severe. Although in both groups the fungal burden is associated with hepatotoxicity, steatosis, increment of hepatic enzymes and lipid peroxidation, stress-related differences are clearly evident. Herein, we evaluated in infected and infected-stressed hosts the involvement of apoptosis and pro-apoptotic signals in the hepatic injury during the acute step of C. albicans infection. We studied in situ apoptosis by 4',6-diamidino-2-phenylindole dihydrochloride and terminal deoxynucleotidyl transferase dUTP nick-end labeling reactions, the levels of local tumor necrosis factor (TNF)-alpha mRNA by reverse transcription-PCR and the Fas/Fas-L expression by immunohistochemistry and western blot. We also purified intrahepatic lymphocytes (IHLs) to evaluate the dynamic of recruitment following the infection and to characterize the in vivo and in vitro interaction of C. albicans with this subset evaluating the kinetic of Fas-L and Toll-like receptor-2 (TLR-2) expression. This work shows, for the first time, the occurrence of in situ apoptosis of hepatocytes as well as the kinetic of IHL recruitment early during the C. albicans infection. Moreover, our results demonstrate the ability of the fungus to up-regulate the Fas-L and TLR-2 expression in this subset. In the scenario of early liver injury, the recruited IHLs and the modulated expression of TNF-alpha, Fas-L and TLR-2 molecules could act coordinately in delivering death signals.

  6. Sodium ascorbate inhibits growth via the induction of cell cycle arrest and apoptosis in human malignant melanoma A375.S2 cells.

    PubMed

    Lin, Shuw-Yuan; Lai, Wan-Wen; Chou, Chi-Chung; Kuo, Hsiu-Maan; Li, Te-Mao; Chung, Jing-Gung; Yang, Jen-Hung

    2006-12-01

    Vitamin C has been reported to be useful in the treatment and prevention of cancer. Inconsistent effects from growth stimulation to induction of apoptosis of malignant tumor cells, however, have been reported. Melanoma is an increasingly common and potentially lethal malignancy. It was reported that melanoma cells were more susceptible to ascorbate toxicity than any other tumor cells. The mechanisms accounting for ascorbate-induced apoptosis in human melanoma cells, however, have remained unclear. This study was undertaken to investigate the effect of sodium ascorbate on cytotoxicity and apoptosis in human malignant melanoma A375.S2 cells. A375.S2 cells were incubated with a certain range of concentrations of sodium ascorbate for various time periods. In order to examine the effects of sodium ascorbate on cell proliferation, cell cycle, apoptosis and necrosis, we performed 4,6-diamidino-2-phenylindole dihydrochloride assays and flow cytometry analysis. Polymerase chain reaction was used to examine the mRNA levels of p53, p21, p27, cyclin A, cyclin E, CDK2 and CDK4, which are associated with cell cycle S-phase arrest and apoptosis. Flow cytometric analysis showed that sodium ascorbate significantly induced cell cycle arrest and apoptosis in the A375.S2 cell line in a dose-dependent manner. The increased expressions of p53 and p21, and the decreased expressions of cyclin A, cyclin E, CDK2 and CDK4, indicated the cell cycle arrest at G1/S phase after the cells had been treated with sodium ascorbate. Induction of apoptosis involved an increase in the levels of p53, p21 and cellular Ca, and a decrease in mitochondrial membrane potential and activation of caspase 3 before culminating in apoptosis in sodium ascorbate-treated A375.S2 cells.

  7. 5-bromo-3-(3-hydroxyprop-1-ynyl)-2H-pyran-2-one induces apoptosis in T24 human bladder cancer cells through mitochondria-dependent signaling pathways

    PubMed Central

    Yu, Guo-Qiang; Dou, Zhong-Ling; Jia, Zhao-Hui

    2016-01-01

    The present study was performed to investigate the effect of 5-bromo-3-(3-hydroxyprop-1-ynyl)-2H-pyran-2-one (BHP) on the induction of apoptosis and cell cycle arrest in T24 human bladder carcinoma cells. An MTT assay was used to investigate the inhibition of cell proliferation. Flow cytometry was used to observe alterations in the cell cycle, generation of reactive oxygen species (ROS), alterations in mitochondrial membrane potential (MMP) and induction of apoptosis in the T24 cells following BHP treatment. Western blot analysis was performed for the determination of expression levels of apoptotic proteins, and 4,6-diamidino-2-phenylindole dihydrochloride staining was used to observe apoptosis and DNA damage. The results demonstrated that treatment of the bladder cancer cells with BHP enhanced the activation of caspases and increased the production of ROS. It also caused damage to DNA, reduced MMP, and increased the secretion of endonuclease G and apoptosis-inducing factor from the mitochondria. The expression levels of cyclin E and cell division cycle 25C were reduced, whereas the expression levels of p21 and phosphorylated p53 were increased in the BHP-treated cells. In addition, treatment with BHP caused cell cycle arrest at the G0/G1 phase, increased the expression levels of B cell lymphoma-2 (Bcl-2)-associated X protein and poly(ADP-ribose) polymerase, decreased the expression of Bcl-2 and ultimately induced apoptosis of the T24 cells. Thus, BHP inhibited the proliferation of bladder cancer cells by inducing cell apoptosis through the mitochondrial pathway. PMID:27922685

  8. Anti-cancer activities of Brassica juncea leaves in vitro

    PubMed Central

    Kwak, Youngeun; Lee, Jungjae; Ju, Jihyeung

    2016-01-01

    Mustard (Brassica juncea) leaves are commonly consumed in different Asian and African countries. Cancer is a major burden of disease worldwide, and the colorectal and lung cancers are the leading cause of morbidity and mortality among cancers. In the current study, we aimed to investigate the effects of ethanol extract of mustard leaf (MLE) on the growth, angiogenic, and metastatic potentials of HCT116 colorectal carcinoma and H1299 non-small cell lung carcinoma cells in vitro. Treatment of HCT116 and H1299 cells with MLE inhibited cell growth in a dose-dependent manner (in the range of 175-700 µg/ml, by 39-86 %) and anchorage-independent colonization (at 700 µg/ml, by 56-86 %). Induction of apoptosis by MLE was evidenced by heterogeneous and condensed nucleus morphology, increased 4′,6-diamidino-2-phenylindole dihydrochloride staining intensity, and elevated sub-G1 cell population. In both HCT116 and H1299 cells, treatment with MLE markedly suppressed the secretion of key pro-angiogenic factors, such as vascular endothelial cell growth factor (by >92 %) and basic fibroblast growth factor (by 73-94 %). MLE was also effective in inhibiting critical events during metastasis, such as invasion (by 18-33 % in HCT116 and H1299), migration (45-82 % in H1299), and adhesion (by 17-45 % in HCT116 and H1299). These results indicate that MLE possesses in vitro anti-cancer activities against colon and lung cancers. It needs to be verified whether similar effects are reproduced in vivo. PMID:28337101

  9. Compatibility of DAPl and silver staining for combined anterograde and retrograde tracing of neural connections.

    PubMed

    Rhoades, R W

    1980-11-10

    The geniculocortical and corticogeniculate pathways in hamster were used to test the compatibility of 4'6 diamidino-2 phenylindole 2HCl (DAPl)6,10 and anterograde degeneration techniques for tracing reciprocal connections in the brain. The two methods were compatible within a single brain and, with some loss of sensitivity in the retrograde labeling, within a single section.

  10. Development and Validation of a HPTLC Method for Simultaneous Quantitation of Flunarizine Dihydrochloride and Propranolol Hydrochloride in Capsule Dosage Form

    PubMed Central

    Shivarkar, N. A.; Dudhe, P. B.; Nagras, M. A.

    2013-01-01

    A simple, precise, accurate, and rapid high-performance thin layer chromatographic method has been developed and validated for the simultaneous quantitation of flunarizine dihydrochloride and propranolol hydrochloride in a combined capsule dosage form. The method was carried out on precoated silica gel 60 F254 TLC aluminum plate, (20×10 cm2). The solvent system was ethyl acetate:methanol:glacial acetic acid in the proportion of 8:1:1, (v/v/v). Rf value for flunarizine dihydrochloride and propranolol hydrochloride was found to be 0.62±0.02 and 0.18±0.02, respectively. The linearity regression analysis for calibration showed 0.999 and 0.999 for flunarizine dihydrochloride and propranolol hydrochloride with respect to peak area and height in the concentration range of 50-350 ng/spot and 500-3500 ng/spot, respectively. Accuracy of recovery studies was found to be 98-100.28 and 99.11-99.45% for flunarizine dihydrochloride and propranolol hydrochloride, respectively. The amounts of drug in marketed formulation were 100.5 and 101.25% of flunarizine dihydrochloride and propranolol hydrochloride, respectively. The method developed can be used for routine analysis in bulk drug and capsule dosage form. PMID:24082355

  11. Interaction of diamidino-2-phenylindole (DAPI) with natural and synthetic nucleic acids.

    PubMed Central

    Manzini, G; Barcellona, M L; Avitabile, M; Quadrifoglio, F

    1983-01-01

    The interaction of DAPI with natural and synthetic polydeoxynucleotides of different base content and sequences was studied with circular dichroism, ultracentrifugation, viscosity and calorimetry. All the polymers show two types of binding. The strength of the interaction and its resistance to ionic strength are related to the content of AT clusters in the chain. On the other hand, sedimentation measurements rule out an intercalation mechanism. A model of DAPI interaction with DNA, similar to that displayed by distamycin and netropsin, is proposed. PMID:6672773

  12. Molecular design, synthesis and biological evaluation of BP-O-DAPY and O-DAPY derivatives as non-nucleoside HIV-1 reverse transcriptase inhibitors.

    PubMed

    Yang, Shiqiong; Pannecouque, Christophe; Daelemans, Dirk; Ma, Xiao-Dong; Liu, Yang; Chen, Fen-Er; De Clercq, Erik

    2013-07-01

    This paper reports the synthesis and antiviral evaluation of a series of non-nucleoside reverse transcriptase inhibitors (NNRTIs) that combine the peculiar structural features of diarylpyrimidine derivatives (DAPYs) and benzophenone derivatives (BPs). The DAPY derivatives bearing benzoyl or alkoxyl substitutes on the A-ring showed the inhibitory activity against wild-type HIV-1 at the cellular level within the range of EC50 values from micromolar to nanomolar. Among these compounds, 1u exhibited the most potent anti-HIV-1 activity (EC50 = 0.06 ± 0.01 μM, SI > 6260), which were about 1.8-fold more active than nevirapine (NVP) and delavirdine (DLV). In addition, the binding modes with HIV-1 RT and the preliminary SAR studies of these derivatives were also considered for further investigation.

  13. Concentration and Detection of Cryptosporidium Oocysts in Surface Water Samples by Method 1622 Using Ultrafiltration and Capsule Filtration

    USGS Publications Warehouse

    Simmons, O. D.; Sobsey, M.D.; Heaney, C.D.; Schaefer, F. W.; Francy, D.S.

    2001-01-01

    The protozoan parasite Cryptosporidium parvum is known to occur widely in both source and drinking water and has caused waterborne outbreaks of gastroenteritis. To improve monitoring, the U.S. Environmental Protection Agency developed method 1622 for isolation and detection of Cryptosporidium oocysts in water. Method 1622 is performance based and involves filtration, concentration, immunomagnetic separation, fluorescent-antibody staining and 4???,6-diamidino-2-phenylindole (DAPI) counterstaining, and microscopic evaluation. The capsule filter system currently recommended for method 1622 was compared to a hollow-fiber ultrafilter system for primary concentration of C. parvum oocysts in seeded reagent water and untreated surface waters. Samples were otherwise processed according to method 1622. Rates of C. parvum oocyst recovery from seeded 10-liter volumes of reagent water in precision and recovery experiments with filter pairs were 42% (standard deviation [SD], 24%) and 46% (SD, 18%) for hollow-fiber ultrafilters and capsule filters, respectively. Mean oocyst recovery rates in experiments testing both filters on seeded surface water samples were 42% (SD, 27%) and 15% (SD, 12%) for hollow-fiber ultrafilters and capsule filters, respectively. Although C. parvum oocysts were recovered from surface waters by using the approved filter of method 1622, the recovery rates were significantly lower and more variable than those from reagent grade water. In contrast, the disposable hollow-fiber ultrafilter system was compatible with subsequent method 1622 processing steps, and it recovered C. parvum oocysts from seeded surface waters with significantly greater efficiency and reliability than the filter suggested for use in the version of method 1622 tested.

  14. INsPECT, an open-source and versatile software for automated quantification of (Leishmania) intracellular parasites.

    PubMed

    Yazdanparast, Ehsan; Dos Anjos, Antonio; Garcia, Deborah; Loeuillet, Corinne; Shahbazkia, Hamid Reza; Vergnes, Baptiste

    2014-05-01

    Intracellular protozoan parasites are causative agents of infectious diseases that constitute major health problems for developing countries. Leishmania sp., Trypanosoma cruzi or Toxoplasma gondii are all obligate intracellular protozoan parasites that reside and multiply within the host cells of mammals, including humans. Following up intracellular parasite proliferation is therefore an essential and a quotidian task for many laboratories working on primary screening of new natural and synthetic drugs, analyzing drug susceptibility or comparing virulence properties of natural and genetically modified strains. Nevertheless, laborious manual microscopic counting of intracellular parasites is still the most commonly used approach. Here, we present INsPECT (Intracellular ParasitE CounTer), an open-source and platform independent software dedicated to automate infection level measurement based on fluorescent DNA staining. It offers the possibility to choose between different types of analyses (fluorescent DNA acquisitions only or in combination with phase contrast image set to further separate intra- from extracellular parasites), and software running modes (automatic or custom). A proof-of-concept study with intracellular Leishmania infantum parasites stained with DAPI (4',6-diamidino-2-phenylindole) confirms a good correspondence between digital results and the "gold standard" microscopic counting method with Giemsa. Interestingly, this software is versatile enough to accurately detect intracellular T. gondii parasites on images acquired with High Content Screening (HCS) systems. In conclusion, INsPECT software is proposed as a new fast and simple alternative to the classical intracellular Leishmania quantification methods and can be adapted for mid to large-scale drug screening against different intracellular parasites.

  15. Minority cytotypes in European populations of the Gymnadenia conopsea complex (Orchidaceae) greatly increase intraspecific and intrapopulation diversity

    PubMed Central

    Trávníček, Pavel; Jersáková, Jana; Kubátová, Barbora; Krejčíková, Jana; Bateman, Richard M.; Lučanová, Magdalena; Krajníková, Eva; Těšitelová, Tamara; Štípková, Zuzana; Amardeilh, Jean-Pierre; Brzosko, Emilia; Jermakowicz, Edyta; Cabanne, Olivier; Durka, Walter; Efimov, Peter; Hedrén, Mikael; Hermosilla, Carlos E.; Kreutz, Karel; Kull, Tiiu; Tali, Kadri; Marchand, Olivier; Rey, Manel; Schiestl, Florian P.; Čurn, Vladislav; Suda, Jan

    2012-01-01

    Background and Aims Patterns of ploidy variation among and within populations can provide valuable insights into the evolutionary mechanisms shaping the dynamics of plant systems showing ploidy diversity. Whereas data on majority ploidies are, by definition, often sufficiently extensive, much less is known about the incidence and evolutionary role of minority cytotypes. Methods Ploidy and proportions of endoreplicated genome were determined using DAPI (4',6-diamidino-2-phenylindole) flow cytometry in 6150 Gymnadenia plants (fragrant orchids) collected from 141 populations in 17 European countries. All widely recognized European species, and several taxa of less certain taxonomic status were sampled within Gymnadenia conopsea sensu lato. Key Results Most Gymnadenia populations were taxonomically and/or ploidy heterogeneous. Two majority (2x and 4x) and three minority (3x, 5x and 6x) cytotypes were identified. Evolution largely proceeded at the diploid level, whereas tetraploids were much more geographically and taxonomically restricted. Although minority ploidies constituted <2 % of the individuals sampled, they were found in 35 % of populations across the entire area investigated. The amount of nuclear DNA, together with the level of progressively partial endoreplication, separated all Gymnadenia species currently widely recognized in Europe. Conclusions Despite their low frequency, minority cytotypes substantially increase intraspecific and intrapopulation ploidy diversity estimates for fragrant orchids. The cytogenetic structure of Gymnadenia populations is remarkably dynamic and shaped by multiple evolutionary mechanisms, including both the ongoing production of unreduced gametes and heteroploid hybridization. Overall, it is likely that the level of ploidy heterogeneity experienced by most plant species/populations is currently underestimated; intensive sampling is necessary to obtain a holistic picture. PMID:23002267

  16. Crenarchaeota and Their Role in the Nitrogen Cycle in a Subsurface Radioactive Thermal Spring in the Austrian Central Alps▿

    PubMed Central

    Weidler, Gerhard W.; Gerbl, Friedrich W.; Stan-Lotter, Helga

    2008-01-01

    Previous results from a 16S rRNA gene library analysis showed high diversity within the prokaryotic community of a subterranean radioactive thermal spring, the “Franz-Josef-Quelle” (FJQ) in Bad Gastein, Austria, as well as evidence for ammonia oxidation by crenarchaeota. This study reports further characterization of the community by denaturing gradient gel electrophoresis (DGGE) analysis, fluorescence in situ hybridization (FISH), and semiquantitative nitrification measurements. DGGE bands from three types of samples (filtered water, biofilms on glass slides, and naturally grown biofilms), including samples collected at two distinct times (January 2005 and July 2006), were analyzed. The archaeal community consisted mainly of Crenarchaeota of the soil-subsurface-freshwater group (group 1.1b) and showed a higher diversity than in the previous 16S rRNA gene library analysis, as was also found for crenarchaeal amoA genes. No bacterial amoA genes were detected. FISH analysis of biofilms indicated the presence of archaeal cells with an abundance of 5.3% (±4.5%) in the total 4′,6-diamidino-2-phenylindole (DAPI)-stained community. Microcosm experiments of several weeks in duration showed a decline of ammonium that correlated with an increase of nitrite, the presence of crenarchaeal amoA genes, and the absence of bacterial amoA genes. The data suggested that only ammonia-oxidizing archaea (AOA) perform the first step of nitrification in this 45°C environment. The crenarchaeal amoA gene sequences grouped within a novel cluster of amoA sequences from the database, originating from geothermally influenced environments, for which we propose the designation “thermal spring” cluster and which may be older than most AOA from soils on earth. PMID:18723663

  17. Deficient glucose and glutamine metabolism in Aralar/AGC1/Slc25a12 knockout mice contributes to altered visual function

    PubMed Central

    Ramirez, Laura; Du, Jianhai; Hurley, James B.; Satrústegui, Jorgina; de la Villa, Pedro

    2016-01-01

    Purpose To characterize the vision phenotype of mice lacking Aralar/AGC1/Slc25a12, the mitochondrial aspartate-glutamate carrier mutated in global cerebral hypomyelination (OMIM 612949). Methods We tested overnight dark-adapted control and aralar-deficient mice for the standard full electroretinogram (ERG) response. The metabolic stress of dark-adaptation was reduced by 5 min illumination after which the ERG response was monitored in darkness. We used the electrical response to two identical saturating light flashes (paired-flash stimulation) to isolate the inner retina and photoreceptor responses. Retinal morphology was examined with hematoxylin and eosin staining, immunohistochemistry of antibodies against retinal cells, and 4',6-diamidino-2-phenylindole (DAPI) labeling. Results Aralar plays a pivotal role in retina metabolism as aralar provides de novo synthesis pathway for glutamine, protects glutamate from oxidation, and is required for efficient glucose oxidative metabolism. Aralar-deficient mice are not blind as their retinas have light-evoked activity. However, we report an approximate 50% decrease in the ERG amplitude response in the light-evoked activity of dark-adapted retinas from aralar-deficient mice, in spite of normal retina histology. The defective response is partly reversed by exposure to a brief illumination period, which lowers the metabolic stress of dark-adaptation. The metabolic stress and ERG alteration takes place primarily in photoreceptors, but the response to two flashes applied in fast succession also revealed an alteration in synaptic transmission consistent with an imbalance of glutamate and an energy deficit in the inner retina neurons. Conclusions We propose that compromised glucose oxidation and altered glutamine and glutamate metabolism in the absence of aralar are responsible for the phenotype reported. PMID:27746674

  18. Evidence of intense archaeal and bacterial methanotrophic activity in the Black Sea water column.

    PubMed

    Durisch-Kaiser, Edith; Klauser, Lucia; Wehrli, Bernhard; Schubert, Carsten

    2005-12-01

    In the northwestern Black Sea, methane oxidation rates reveal that above shallow and deep gas seeps methane is removed from the water column as efficiently as it is at sites located off seeps. Hence, seeps should not have a significant impact on the estimated annual flux of approximately 4.1 x 10(9) mol methane to the atmosphere [W. S. Reeburgh, B. B. Ward, S. C. Wahlen, K. A. Sandbeck, K. A. Kilatrick, and L. J. Kerkhof, Deep-Sea Res. 38(Suppl. 2):S1189-S1210, 1991]. Both the stable carbon isotopic composition of dissolved methane and the microbial community structure analyzed by fluorescent in situ hybridization provide strong evidence that microbially mediated methane oxidation occurs. At the shelf, strong isotope fractionation was observed above high-intensity seeps. This effect was attributed to bacterial type I and II methanotrophs, which on average accounted for 2.5% of the DAPI (4',6'-diamidino-2-phenylindole)-stained cells in the whole oxic water column. At deep sites, in the oxic-anoxic transition zone, strong isotopic fractionation of methane overlapped with an increased abundance of Archaea and Bacteria, indicating that these organisms are involved in the oxidation of methane. In underlying anoxic water, we successfully identified the archaeal methanotrophs ANME-1 and ANME-2, eachof which accounted for 3 to 4% of the total cell counts. ANME-1 and ANME-2 appear as single cells in anoxicwater, compared to the sediment, where they may form cell aggregates with sulfate-reducing bacteria (A. Boetius, K. Ravenschlag, C. J. Schubert, D. Rickert, F. Widdel, A. Giesecke, R. Amann, B. B. Jørgensen, U. Witte, and O. Pfannkuche, Nature 407:623-626, 2000; V. J. Orphan, C. H. House, K.-U. Hinrichs, K. D. McKeegan, and E. F. DeLong, Proc. Natl. Acad. Sci. USA 99:7663-7668, 2002).

  19. Cytotype distribution at a diploid–hexaploid contact zone in Aster amellus (Asteraceae)

    PubMed Central

    Castro, S.; Loureiro, J.; Procházka, T.; Münzbergová, Z.

    2012-01-01

    Background and Aims The present study aims to assess the diversity and distribution of cytotypes of Aster amellus in central and eastern Europe, contributing with data to improve understanding of the evolutionary dynamics of the contact zone between diploids and hexaploids of this polyploid complex. Methods Large-scale cytotype screening of 4720 individuals collected in 229 populations was performed using 4′,6-diamidino-2-phenylindole (DAPI) flow cytometry. Fine-scale cytotype screening was performed in the mixed-ploidy population. Reproductive variables, such as number of florets per flower head, seed set and seedling emergence, as well as ploidy level of seeds and seedlings were recorded in this population. Key Results The diploid–hexaploid contact zone is large and complex, reaching the Czech Republic in the west, Austria in the south, Poland in the north-east and Romania in the extreme east of the surveyed areas. Most populations presented only one cytotype, either diploid or hexaploid. In several areas of the contact zone both cytotypes were found to grow in parapatry. One mixed-ploidy population of diploids and hexaploids was detected for the first time, but no signs of hybridization were detected. In this population, diploids had a significantly lower reproductive success, and significantly higher production of intercytotype offspring, being in reproductive disadvantage in comparison with hexaploids. Conclusions The contact zone of diploid and hexaploid A. amellus in central and eastern Europe seems to be highly dynamic and diffuse, with both primary and secondary contacts being possible. The obtained results suggest the origin of hexaploids through diploids, overall supporting previous hypotheses that this species is autopolyploid. Data from the only mixed-ploidy population detected so far suggest that the minority cytotype exclusion is an important evolutionary mechanisms driving the prevalence of single-cytotype populations, and thus contributing to

  20. Flow sorting of C-genome chromosomes from wild relatives of wheat Aegilops markgrafii, Ae. triuncialis and Ae. cylindrica, and their molecular organization

    PubMed Central

    Molnár, István; Vrána, Jan; Farkas, András; Kubaláková, Marie; Cseh, András; Molnár-Láng, Márta; Doležel, Jaroslav

    2015-01-01

    Background and Aims Aegilops markgrafii (CC) and its natural hybrids Ae. triuncialis (UtUtCtCt) and Ae. cylindrica (DcDcCcCc) represent a rich reservoir of useful genes for improvement of bread wheat (Triticum aestivum), but the limited information available on their genome structure and the shortage of molecular (cyto-) genetic tools hamper the utilization of the extant genetic diversity. This study provides the complete karyotypes in the three species obtained after fluorescent in situ hybridization (FISH) with repetitive DNA probes, and evaluates the potential of flow cytometric chromosome sorting. Methods The flow karyotypes obtained after the analysis of 4',6-diamidino-2-phenylindole (DAPI)-stained chromosomes were characterized and the chromosome content of the peaks on the flow karyotypes was determined by FISH. Twenty-nine conserved orthologous set (COS) markers covering all seven wheat homoeologous chromosome groups were used for PCR with DNA amplified from flow-sorted chromosomes and genomic DNA. Key Results FISH with repetitive DNA probes revealed that chromosomes 4C, 5C, 7Ct, T6UtS.6UtL-5CtL, 1Cc and 5Dc could be sorted with purities ranging from 66 to 91 %, while the remaining chromosomes could be sorted in groups of 2–5. This identified a partial wheat–C-genome homology for group 4 and 5 chromosomes. In addition, 1C chromosomes were homologous with group 1 of wheat; a small segment from group 2 indicated 1C–2C rearrangement. An extensively rearranged structure of chromosome 7C relative to wheat was also detected. Conclusions The possibility of purifying Aegilops chromosomes provides an attractive opportunity to investigate the structure and evolution of the Aegilops C genome and to develop molecular tools to facilitate the identification of alien chromatin and support alien introgression breeding in bread wheat. PMID:26043745

  1. Flow cytometry confirms reticulate evolution and reveals triploidy in Central European Diphasiastrum taxa (Lycopodiaceae, Lycophyta)

    PubMed Central

    Bennert, H. Wilfried; Horn, Karsten; Kauth, Marion; Fuchs, Jörg; Bisgaard Jakobsen, Iben Sophie; Øllgaard, Benjamin; Schnittler, Martin; Steinberg, Matthias; Viane, Ronnie

    2011-01-01

    Background and Aims Interspecific Diphasiastrum hybrids have been assumed to be homoploid and to produce well-formed spores serving sexual reproduction. If this were the case, forms intermediate between hybrids and parents or hybrid swarms should be expected. The purpose of this study was: (1) to check whether homoploidy consistently applies to the three hybrids throughout their Central European range; (2) to examine whether their genome sizes confirm their parentage as assumed by morphology; and (3) to perform a screening for detection of ploidy levels other than diploid and variation in DNA content due to backcrossing. Methods Flow cytometry was used first to measure the relative DNA values [with 4′,6-diamidino-2-phenylindole (DAPI) staining] and ploidy level as a general screening, and secondly to determine the absolute DNA 2C values [with propidium iodide (PI) staining] in a number of selected samples with the main focus on the hybrids. Key Results A considerable variation of DNA 2C values (5·26–7·52 pg) was detected between the three European Diphasiastrum species. The values of the diploid hybrids are highly constant without significant variation between regions. They are also intermediate between their assumed parents and agree closely with those calculated from their putative parents. This confirms their hybrid origin, assumed parentage and homoploid status. Considerably higher DNA amounts (9·48–10·30 pg) were obtained for three populations, suggesting that these represent triploid hybrids, an interpretation that is strongly supported by their morphology. Conclusions Diploid hybrids have retained their genetic and morphological identites throughout their Central European range, and thus no indications for diploid backcrossing were found. The triploid hybrids have probably originated from backcrossing between a diploid gametophyte of a hybrid (derived from a diplospore) and a haploid gametophyte of a diploid parental species. By repeated crossing

  2. Protective effects of statins on L-DOPA neurotoxicity due to the activation of phosphatidylinositol 3-kinase and free radical scavenging in PC12 cell culture.

    PubMed

    Koh, Seong-Ho; Park, Hyun-Hee; Choi, Na-Young; Lee, Kyu-Yong; Kim, Sangjae; Lee, Young Joo; Kim, Hee-Tae

    2011-01-25

    Neurotoxic effects have been suggested for l-3,4-dihydroxyphenylalanine (L-DOPA), while neuroprotective effects have been proposed for statins. We investigated whether certain statins (simvastatin or pitavastatin) could inhibit L-DOPA neurotoxicity. Neuronally-differentiated PC12 (nPC12) cells were treated with L-DOPA and/or statins for 24h, and their viabilities were measured using a cell counting kit, trypan blue staining, and 4',6-diamidino-2-phenylindole (DAPI) staining. Free radical and specific intracellular signaling protein levels were measured with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and Western blotting, respectively. High concentrations of l-DOPA reduced nPC12 cell viability, but combined treatment with statins restored viability. Treatment with 200 μM L-DOPA increased free radical and hydroxyl radical levels, but combined treatment with 5 μM statins decreased these levels. Survival-related signaling proteins were decreased in nPC12 cells treated with 200 μM L-DOPA, but combined treatment with 5μM statins significantly increased the levels of these proteins. Treatment with 200 μM L-DOPA significantly increased death-related signaling proteins, while combined treatment with 5 μM statins reduced the levels of these proteins. Pretreatment with LY294002, a phosphatidylinositol 3 kinase (PI3K) inhibitor, before combined treatment with statins and L-DOPA almost completely blocked the protective effects of statins. These results indicate that statins reduce L-DOPA neurotoxicity by lowering oxidative stress and by enhancing survival signals and inhibiting death signals via activation of the PI3K pathway.

  3. High resolution DNA content measurements of mammalian sperm

    SciTech Connect

    Pinkel, D.; Lake, S.; Gledhill, B.L.; Van Dilla, M.A.; Stephenson, D.; Watchmaker, G.

    1982-01-01

    The high condensation and flat shape of the mammalian sperm nucleus present unique difficulties to flow cytometric measurement of DNA content. Chromatin compactness makes quantitative fluorescent staining for DNA difficult and causes a high index of refraction. The refractive index makes optical measurements sensitive to sperm head orientation. We demonstrate that the optical problems can be overcome using the commercial ICP22 epiillumination flow cytometer (Ortho Instruments, Westwood, MA) or a specially built cell orientating flow cytometer (OFCM). The design and operation of the OFCM are described. Measurements of the angular dependence of fluorescence from acriflavine stained rabbit sperm show that it is capable of orienting flat sperm with a tolerance of +-7/sup 0/. Differences in the angular dependence for the similarly shaped bull and rabbit sperm allow discrimination of these cells. We show that DNA staining with 4-6 diamidino-2-phenylindole (DAPI) or an ethidium bromide mithramycin combination allows resolution of the X and Y populations in mouse sperm. They have also been successful with sperm from the bull, ram, rabbit, and boar. Reliable results with human sperm are not obtained. The accuracy of the staining and measurement techniques are verified by the correct determination of the relative content of these two populations in sperm from normal mice and those with the Cattanach (7 to X) translocation. Among the potential uses of these techniques are measurement of DNA content errors induced in sperm due to mutagen exposure, and assessment of the fractions of X and Y sperm in semen that may have one population artifically enriched.

  4. Estimates of nuclear DNA content in red algal lineages

    PubMed Central

    Kapraun, Donald F.; Freshwater, D. Wilson

    2012-01-01

    Background and aims The red algae are an evolutionarily ancient group of predominantly marine organisms with an estimated 6000 species. Consensus higher-level molecular phylogenies support a basal split between the unicellular Cyanidiophytina and morphologically diverse Rhodophytina, the later subphylum containing most red algal species. The Rhodophytina is divided into six classes, of which five represent early diverging lineages of generally uninucleate species, whose evolutionary relationships are poorly resolved. The remaining species compose the large (27 currently recognized orders), morphologically diverse and typically multinucleate Florideophyceae. Nuclear DNA content estimates have been published for <1 % of the described red algae. The present investigation summarizes the state of our knowledge and expands our coverage of DNA content information from 196 isolates of red algae. Methodology The DNA-localizing fluorochrome DAPI (4′,6-diamidino-2-phenylindole) and RBC (chicken erythrocytes) standards were used to estimate 2C values with static microspectrophotometry. Principal results Nuclear DNA contents are reported for 196 isolates of red algae, almost doubling the number of estimates available for these organisms. Present results also confirm the reported DNA content range of 0.1–2.8 pg, with species of Ceramiales, Nemaliales and Palmariales containing apparently polyploid genomes with 2C = 2.8, 2.3 and 2.8 pg, respectively. Conclusions Early diverging red algal lineages are characterized by relatively small 2C DNA contents while a wide range of 2C values is found within the derived Florideophyceae. An overall correlation between phylogenetic placement and 2C DNA content is not apparent; however, genome size data are available for only a small portion of red algae. Current data do support polyploidy and aneuploidy as pervasive features of red algal genome evolution. PMID:22479676

  5. A Simple and Sensitive High-Content Assay for the Characterization of Antiproliferative Therapeutic Antibodies

    PubMed Central

    Stengl, Andreas; Hörl, David; Leonhardt, Heinrich; Helma, Jonas

    2016-01-01

    Monoclonal antibodies (mAbs) have become a central class of therapeutic agents in particular as antiproliferative compounds. Their often complex modes of action require sensitive assays during early, functional characterization. Current cell-based proliferation assays often detect metabolites that are indicative of metabolic activity but do not directly account for cell proliferation. Measuring DNA replication by incorporation of base analogues such as 5-bromo-2′-deoxyuridine (BrdU) fills this analytical gap but was previously restricted to bulk effect characterization in enzyme-linked immunosorbent assay formats. Here, we describe a cell-based assay format for the characterization of antiproliferative mAbs regarding potency and mode of action in a single experiment. The assay makes use of single cell–based high-content-analysis (HCA) for the reliable quantification of replicating cells and DNA content via 5-ethynyl-2′-deoxyuridine (EdU) and 4′,6-diamidino-2-phenylindole (DAPI), respectively, as sensitive measures of antiproliferative mAb activity. We used trastuzumab, an antiproliferative therapeutic antibody interfering with HER2 cell surface receptor-mediated growth signal transduction, and HER2-overexpressing cell lines BT474 and SKBR3 to demonstrate up to 10-fold signal-to-background (S/B) ratios for treated versus untreated cells and a shift in cell cycle profiles indicating antibody-induced cell cycle arrest. The assay is simple, cost-effective, and sensitive, providing a cell-based format for preclinical characterization of therapeutic mAbs. PMID:27909235

  6. Use of fluorescent oligonucleotide probes for differentiation between Paracoccidioides brasiliensis and Paracoccidioides lutzii in yeast and mycelial phase

    PubMed Central

    Arantes, Thales Domingos; Theodoro, Raquel Cordeiro; Teixeira, Marcus de Melo; Bagagli, Eduardo

    2017-01-01

    BACKGROUND Fluorescence in situ hybridisation (FISH) associated with Tyramide Signal Amplification (TSA) using oligonucleotides labeled with non-radioactive fluorophores is a promising technique for detection and differentiation of fungal species in environmental or clinical samples, being suitable for microorganisms which are difficult or even impossible to culture. OBJECTIVE In this study, we aimed to standardise an in situ hybridisation technique for the differentiation between the pathogenic species Paracoccidioides brasiliensis and Paracoccidioides lutzii, by using species-specific DNA probes targeting the internal transcribed spacer-1 (ITS-1) of the rRNA gene. METHODS Yeast and mycelial phase of each Paracoccidioides species, were tested by two different detection/differentiation techniques: TSA-FISH for P. brasiliensis with HRP (Horseradish Peroxidase) linked to the probe 5’ end; and FISH for P. lutzii with the fluorophore TEXAS RED-X® also linked to the probe 5’ end. After testing different protocols, the optimised procedure for both techniques was accomplished without cross-positivity with other pathogenic fungi. FINDINGS The in silico and in vitro tests show no reaction with controls, like Candida and Cryptococcus (in silico) and Histoplasma capsulatum and Aspergillus spp. (in vitro). For both phases (mycelial and yeast) the in situ hybridisation showed dots of hybridisation, with no cross-reaction between them, with a lower signal for Texas Red probe than HRP-TSA probe. The dots of hybridisation was confirmed with genetic material marked with 4’,6-diamidino-2-phenylindole (DAPI), visualised in a different filter (WU) on fluorescent microscopic. MAIN CONCLUSION Our results indicated that TSA-FISH and/or FISH are suitable for in situ detection and differentiation of Paracoccidioides species. This approach has the potential for future application in clinical samples for the improvement of paracoccidioidomycosis patients prognosis. PMID:28177048

  7. Development of interspecies testicular germ-cell transplantation in flatfish.

    PubMed

    Pacchiarini, Tiziana; Sarasquete, Carmen; Cabrita, Elsa

    2014-06-01

    Interspecific testicular germ cell (TGC) transplantation was investigated in two commercial flatfish species. Testes from donor species (Senegalese sole) were evaluated using classical histological techniques (haematoxylin-eosin staining and haematoxylin-light green-orange G-acid fuchsine staining), in situ hybridisation and immunohistochemical analysis. Both Ssvasa1-2 mRNAs and SsVasa protein allowed the characterisation of TGCs, confirming the usefulness of the vasa gene in the detection of Senegalese sole TGCs. Xenogenic transplants were carried out using TGCs from one-year-old Senegalese sole into turbot larvae. Propidium iodide-SYBR-14 and 4',6'-diamidino-2-phenylindole (DAPI) staining showed that 87.98% of the extracted testicular cells were viable for microinjection and that 15.63% of the total recovered cells were spermatogonia. The vasa gene was characterised in turbot recipients using cDNA cloning. Smvasa mRNA was confirmed as a germ cell-specific molecular marker in this species. Smvasa expression analysis during turbot ontogeny was carried out before Senegalese sole TGC transplants into turbot larvae. Turbot larvae at 18 days after hatching (DAH) proved to be susceptible to manipulation procedures. High survival rates (83.75±15.90-100%) were obtained for turbot larvae at 27, 34 and 42 DAH. These data highlight the huge potential of this species for transplantation studies. Quantitative PCR was employed to detect Senegalese sole vasa mRNAs (Ssvasa1-2) in the recipient turbot larvae. The Ssvasa mRNAs showed a significant increase in relative expression in 42-DAH microinjected larvae three weeks after treatment, showing the proliferation of Senegalese sole spermatogonia in transplanted turbot larvae.

  8. Phosphorylated retinoblastoma protein (p-Rb) is involved in neuronal apoptosis after traumatic brain injury in adult rats.

    PubMed

    Liu, Wei; Liu, Xiaojuan; Yang, Huilin; Zhu, Xinhui; Yi, Hong; Zhu, Xuesong; Zhang, Jie

    2013-04-01

    Phosphorylated retinoblastoma protein (p-Rb), a well identified cell cycle related protein, is involved in regulating the biological functions of various cell types including neurons. One attractive biological function of p-Rb is releasing E2F transcription factor to induce S-phase entry and cellular proliferation of mitotic cells. However, some studies point out that the role of p-Rb in post-mitotic cells such as mature neurons is unique; it may induce cellular apoptosis rather than proliferation via regulating cell cycle reactivation. Up to now, the knowledge of p-Rb function in CNS is still limited. To investigate whether p-Rb is involved in CNS injury and repair, we performed a traumatic brain injury model in adult rats. Up-regulation of p-Rb was observed in the injured brain cortex by western blot analysis and immunohistochemistry staining. Terminal deoxynucleotidyl transferase deoxy-UTP-nick end labeling (TUNEL) and 4',6-diamidino-2-phenylindole (DAPI) staining suggested that p-Rb was relevant to neuronal apoptosis after brain injury. In addition, glutamate excitotoxic model of primary cortex neurons was introduced to further investigate the role of p-Rb in neuronal apoptosis; the result implied p-Rb was associated with cell cycle activation in the apoptotic neurons. Based on our data, we suggested that p-Rb might play an important role in neuronal apoptosis after traumatic brain injury in rat; which might also provide a basis for the further study on its role in regulating cell cycle re-entry in apoptotic neurons, and might gain a novel strategy for the clinical therapy for traumatic brain injury.

  9. Localization of single- and low-copy sequences on tomato synaptonemal complex spreads using fluorescence in situ hybridization (FISH).

    PubMed Central

    Peterson, D G; Lapitan, N L; Stack, S M

    1999-01-01

    Fluorescence in situ hybridization (FISH) is a powerful means by which single- and low-copy DNA sequences can be localized on chromosomes. Compared to the mitotic metaphase chromosomes that are normally used in FISH, synaptonemal complex (SC) spreads (hypotonically spread pachytene chromosomes) have several advantages. SC spreads (1) are comparatively free of debris that can interfere with probe penetration, (2) have relatively decondensed chromatin that is highly accessible to probes, and (3) are about ten times longer than their metaphase counterparts, which permits FISH mapping at higher resolution. To investigate the use of plant SC spreads as substrates for single-copy FISH, we probed spreads of tomato SCs with two single-copy sequences and one low-copy sequence (ca. 14 kb each) that are associated with restriction fragment length polymorphism (RFLP) markers on SC 11. Individual SCs were identified on the basis of relative length, arm ratio, and differential staining patterns after combined propidium iodide (PI) and 4', 6-diamidino-2-phenylindole (DAPI) staining. In this first report of single-copy FISH to SC spreads, the probe sequences were unambiguously mapped on the long arm of tomato SC 11. Coupled with data from earlier studies, we determined the distance in micrometers, the number of base pairs, and the rates of crossing over between these three FISH markers. We also observed that the order of two of the FISH markers is reversed in relation to their order on the molecular linkage map. SC-FISH mapping permits superimposition of markers from molecular linkage maps directly on pachytene chromosomes and thereby contributes to our understanding of the relationship between chromosome structure, gene activity, and recombination. PMID:10224272

  10. High resolution fluorescence microscopy evidence on the transport of immunoglobulins. Differences between mammalian IgG, F(ab')2 and avian IgY.

    PubMed

    Sevcik, Carlos; Salazar, Víctor; Díaz, Patricia; D'Suze, Gina; Vázquez, Hilda

    2013-03-01

    We describe the subcellular localization of horse F(ab')(2) and IgG, and ostrich IgY labeled with fluorescein isothiocyanate (FITC) administered IV to mice. We used wide field high sensitivity fluorescence microscopy deblurred by 3-dimensional blind deconvolution of kidney, liver, lungs and brain sections. Sections were obtained from mice sacrificed 15 min, 1 or 5 h after receiving FITC-immunoproteins, counter-stained with DAPI (4',6'-diamidino-2-phenylindole) and Evans blue. FITC-IgG and its fractions are rapidly taken up and extravasated by vascular endothelium. FITC-IgG and FITC-F(ab')(2) appear to be quickly secreted by glomeruli endothelium and to be reabsorbed along all nephron segments. FITC-IgG and FITC-F(ab')(2) appeared 15 min after IV injection within bronchial, alveolar and bile duct epithelium. Hepatocytes were loaded with fluorescence after 15 min of administration. Fluorescence was absent from brain slices, except for the endothelium of some vessels in brain ventricles which appeared intensely fluorescent. Fluorescence appeared in intracellular vesicles which conferred the tissues a glowing foamy aspect for up to 5 h after inoculation. Arterial elastic layers were intensely green after horse FITC-Ig inoculation. Ostrich FITC-IgY behaved completely differently to horse Ig's; only 1 h after injection it was possible to observe small brightly green scarce vesicles in vascular endothelium of arteries, interstitial kidney capillaries between nephron tubules and were also scarce in glomeruli endothelium; FITC-IgY appeared only in hepatic sinusoids in the liver. No IgY was seen in bronchial and alveolar endothelium, in bile ducts or in hepatocytes.

  11. The selective increase or decrease of organellar DNA in generative cells just after pollen mitosis one controls cytoplasmic inheritance.

    PubMed

    Nagata, N; Saito, C; Sakai, A; Kuroiwa, H; Kuroiwa, T

    1999-07-01

    Organellar DNA in mature pollen grains of eight angiosperm species (Actinidia deliciosa Lindl., Antirrhinum majus L., Arabidopsis thaliana (L.) Heynh., Medicago sativa L., Musa acuminata Colla, Pelargonium zonale (L.) L'Hér, Petunia hybrida Vilm. and Rhododendron mucronatum (Blume) G. Don, in which the modes of organellar inheritance have been determined genetically, was observed by fluorescence microscopy using Technovit 7100 resin sections double-stained with 4',6-diamidino-2-phenylindole (DAPI) and 3,3'-dihexyloxacarbocyanine iodide (DiOC(6)). The eight species were classified into four types, based on the presence or absence of organellar DNA in mature generative cells: namely (1) type "m+p+", which has both mitochondrial and plastid DNA (P. zonale), (2) type "m+p-", which only has mitochondrial DNA (M. acuminata), (3) type "m-p+", which only has plastid DNA (A. deliciosa, M. sativa, R. mucronatum), and (4) type "m-p-", which has neither mitochondrial nor plastid DNA (A. majus, A. thaliana, P. hybrida). This classification corresponded to the mode of organellar inheritance determined by genetic analysis. The presence or absence of mitochondrial and plastid DNA corresponded to paternal/biparental inheritance or maternal inheritance of the respective organelle, respectively. When organellar DNA was present in mature generative cells (m+ or p+), the DNA content of the organelles in the generative cells started to increase immediately after pollen mitosis one (PMI). In contrast, the DNA content of organelles in generative cells decreased rapidly after PMI when organellar DNA was absent from mature generative cells (m- or p-). These results indicate that the modes of inheritance (paternal/biparental inheritance or maternal inheritance) of mitochondria and plastids are determined independently of each other in young generative cells just after PMI.

  12. Non-Invasive Delivery of dsRNA into De-Waxed Tick Eggs by Electroporation.

    PubMed

    Ruiz, Newton; de Abreu, Leonardo Araujo; Parizi, Luís Fernando; Kim, Tae Kwon; Mulenga, Albert; Braz, Gloria Regina Cardoso; Vaz, Itabajara da Silva; Logullo, Carlos

    2015-01-01

    RNA interference-mediated gene silencing was shown to be an efficient tool for validation of targets that may become anti-tick vaccine components. Here, we demonstrate the application of this approach in the validation of components of molecular signaling cascades, such as the Protein Kinase B (AKT)/Glycogen Synthase Kinase (GSK) axis during tick embryogenesis. It was shown that heptane and hypochlorite treatment of tick eggs can remove wax, affecting corium integrity and but not embryo development. Evidence of AKT and GSK dsRNA delivery into de-waxed eggs of via electroporation is provided. Primers designed to amplify part of the dsRNA delivered into the electroporated eggs dsRNA confirmed its entry in eggs. In addition, it was shown that electroporation is able to deliver the fluorescent stain, 4',6-diamidino-2-phenylindole (DAPI). To confirm gene silencing, a second set of primers was designed outside the dsRNA sequence of target gene. In this assay, the suppression of AKT and GSK transcripts (approximately 50% reduction in both genes) was demonstrated in 7-day-old eggs. Interestingly, silencing of GSK in 7-day-old eggs caused 25% reduction in hatching. Additionally, the effect of silencing AKT and GSK on embryo energy metabolism was evaluated. As expected, knockdown of AKT, which down regulates GSK, the suppressor of glycogen synthesis, decreased glycogen content in electroporated eggs. These data demonstrate that electroporation of de-waxed R. microplus eggs could be used for gene silencing in tick embryos, and improve the knowledge about arthropod embryogenesis.

  13. The relationship between cell division and elongation during development of the nectar-yielding petal spur in Centranthus ruber (Valerianaceae)

    PubMed Central

    Mack, Jaimie-Lee K.; Davis, Arthur R.

    2015-01-01

    Background and Aims Floral spurs are hollow, tubular outgrowths that typically conceal nectar. By their involvement in specialized pollinator interactions, spurs have ecological and evolutionary significance, often leading to speciation. Despite their importance and diversity in shape and size among angiosperm taxa, detailed investigations of the mechanism of spur development have been conducted only recently. Methods Initiation and growth of the nectar-yielding petal spur of Centranthus ruber ‘Snowcloud’ was investigated throughout seven stages, based on bud size and developmental events. The determination of the frequency of cell division, quantified for the first time in spurs, was conducted by confocal microscopy following 4',6-diamidino-2-phenylindole (DAPI) staining of mitotic figures. Moreover, using scanning electron microscospy of the outer petal spur surface unobstructed by trichomes, morphometry of epidermal cells was determined throughout development in order to understand the ontogeny of this elongate, hollow tube. Key Results Spur growth from the corolla base initially included diffuse cell divisions identified among epidermal cells as the spur progressed through its early stages. However, cell divisions clearly diminished before a petal spur attained 30 % of its final length of 4·5 mm. Thereafter until anthesis, elongation of individual cells was primarily responsible for the spur’s own extension. Consequently, a prolonged period of anisotropy, wherein epidermal cells elongated almost uniformly in all regions along the petal spur’s longitudinal axis, contributed principally to the spur’s mature length. Conclusions This research demonstrates that anisotropic growth of epidermal cells – in the same orientation as spur elongation – chiefly explains petal spur extension in C. ruber. Representing the inaugural investigation of the cellular basis for spur ontogeny within the Euasterids II clade, this study complements the patterns in

  14. Crenarchaeota and their role in the nitrogen cycle in a subsurface radioactive thermal spring in the Austrian Central Alps.

    PubMed

    Weidler, Gerhard W; Gerbl, Friedrich W; Stan-Lotter, Helga

    2008-10-01

    Previous results from a 16S rRNA gene library analysis showed high diversity within the prokaryotic community of a subterranean radioactive thermal spring, the "Franz-Josef-Quelle" (FJQ) in Bad Gastein, Austria, as well as evidence for ammonia oxidation by crenarchaeota. This study reports further characterization of the community by denaturing gradient gel electrophoresis (DGGE) analysis, fluorescence in situ hybridization (FISH), and semiquantitative nitrification measurements. DGGE bands from three types of samples (filtered water, biofilms on glass slides, and naturally grown biofilms), including samples collected at two distinct times (January 2005 and July 2006), were analyzed. The archaeal community consisted mainly of Crenarchaeota of the soil-subsurface-freshwater group (group 1.1b) and showed a higher diversity than in the previous 16S rRNA gene library analysis, as was also found for crenarchaeal amoA genes. No bacterial amoA genes were detected. FISH analysis of biofilms indicated the presence of archaeal cells with an abundance of 5.3% (+/-4.5%) in the total 4',6-diamidino-2-phenylindole (DAPI)-stained community. Microcosm experiments of several weeks in duration showed a decline of ammonium that correlated with an increase of nitrite, the presence of crenarchaeal amoA genes, and the absence of bacterial amoA genes. The data suggested that only ammonia-oxidizing archaea (AOA) perform the first step of nitrification in this 45 degrees C environment. The crenarchaeal amoA gene sequences grouped within a novel cluster of amoA sequences from the database, originating from geothermally influenced environments, for which we propose the designation "thermal spring" cluster and which may be older than most AOA from soils on earth.

  15. Vertical distribution of ammonia-oxidizing crenarchaeota and methanogens in the epipelagic waters of Lake Kivu (Rwanda-Democratic Republic of the Congo).

    PubMed

    Llirós, Marc; Gich, Frederic; Plasencia, Anna; Auguet, Jean-Christophe; Darchambeau, François; Casamayor, Emilio O; Descy, Jean-Pierre; Borrego, Carles

    2010-10-01

    Four stratified basins in Lake Kivu (Rwanda-Democratic Republic of the Congo) were sampled in March 2007 to investigate the abundance, distribution, and potential biogeochemical role of planktonic archaea. We used fluorescence in situ hybridization with catalyzed-reported deposition microscopic counts (CARD-FISH), denaturing gradient gel electrophoresis (DGGE) fingerprinting, and quantitative PCR (qPCR) of signature genes for ammonia-oxidizing archaea (16S rRNA for marine Crenarchaeota group 1.1a [MCG1] and ammonia monooxygenase subunit A [amoA]). Abundance of archaea ranged from 1 to 4.5% of total DAPI (4',6-diamidino-2-phenylindole) counts with maximal concentrations at the oxic-anoxic transition zone (∼50-m depth). Phylogenetic analysis of the archaeal planktonic community revealed a higher level of richness of crenarchaeal 16S rRNA gene sequences (21 of the 28 operational taxonomic units [OTUs] identified [75%]) over euryarchaeotal ones (7 OTUs). Sequences affiliated with the kingdom Euryarchaeota were mainly recovered from the anoxic water compartment and mostly grouped into methanogenic lineages (Methanosarcinales and Methanocellales). In turn, crenarchaeal phylotypes were recovered throughout the sampled epipelagic waters (0- to 100-m depth), with clear phylogenetic segregation along the transition from oxic to anoxic water masses. Thus, whereas in the anoxic hypolimnion crenarchaeotal OTUs were mainly assigned to the miscellaneous crenarchaeotic group, the OTUs from the oxic-anoxic transition and above belonged to Crenarchaeota groups 1.1a and 1.1b, two lineages containing most of the ammonia-oxidizing representatives known so far. The concomitant vertical distribution of both nitrite and nitrate maxima and the copy numbers of both MCG1 16S rRNA and amoA genes suggest the potential implication of Crenarchaeota in nitrification processes occurring in the epilimnetic waters of the lake.

  16. Purple bamboo salt has anticancer activity in TCA8113 cells in vitro and preventive effects on buccal mucosa cancer in mice in vivo.

    PubMed

    Zhao, Xin; Deng, Xiaoxiao; Park, Kun-Young; Qiu, Lihua; Pang, Liang

    2013-02-01

    Bamboo salt is a traditional healthy salt known in Korea. The in vitro anticancer effects of the salt were evaluated using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay in TCA8113 human tongue carcinoma cells. At 1% concentration, the growth inhibitory rate of purple bamboo salt was 61% higher than that of sea salt (27%). Apoptosis analysis of the cancer cells was carried out using 4,6-diamidino-2-phenylindole (DAPI) staining to investigate the mechanism of the anticancer effects in tongue carcinoma cells. Purple bamboo salt induced a stronger apoptotic effect than sea salt. An Institute of Cancer Research (ICR) mouse buccal mucosa cancer model was established by injecting mice with U14 squamous cell carcinoma cells. Following injection, the wound at the injection site was smeared with salt samples. It was observed that the tumor volumes for the group treated with purple bamboo salt were smaller than those from the sea salt treatment and control groups. The sections of buccal mucosa cancer tissue showed that canceration in the purple bamboo salt group was weaker compared with that in the sea salt group. Similar results were observed in the lesion section of the cervical lymph. Using reverse transcription-polymerase chain reaction (RT-PCR) and western blotting, the purple bamboo salt group demonstrated an increase in Bcl-2-associated X protein (Bax) and a decrease in B cell lymphoma-2 (Bcl-2), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression, compared with the sea salt and control groups. The results demonstrated that purple bamboo salt had improved in vivo buccal mucosa cancer preventive activity compared with sea salt in mice.

  17. Antimutagenic activity and in vitro anticancer effects of bamboo salt on HepG2 human hepatoma cells.

    PubMed

    Zhao, Xin; Ju, Jaehyun; Kim, Hyung-Min; Park, Kun-Young

    2013-01-01

    Bamboo salt is a traditional Korean baked solar salt processed by packing the solar salt in bamboo joint cases and heating it several times to high temperatures. The antimutagenic activity and in vitro anticancer effects of bamboo salt on HepG2 human hepatoma cells were investigated and compared to those of other salt samples. Although solar salt and purified salt exhibited comutagenicity with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in the Salmonella typhimurium TA100 strain, bamboo salt was associated with a lower degree of comutagenicity or antimutagenic activity. Bamboo salt baked nine times (9×) showed a greater increase in antimutagenic activity than salts baked once (1×) or three times (3×). At a concentration of 1%, the growth rate of HepG2 cells treated with 9× bamboo salt determined by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MIT) assay was reduced by 65%; this rate of inhibition was higher than that achieved with 1× baked bamboo salt (40%). Purified and solar salts had relatively lower inhibitory effects on growth rate (25% and 29%, respectively). Compared to the other salt samples, 9× bamboo salt significantly (p<0.05) induced apoptosis as determined by 4,6-diamidino-2-phenylindole (DAPI) staining and flow cytometry analysis. It also upregulated the expression of Bax, caspase-9 and caspase-3; and downregulated Bcl-2 expression. The bamboo salts, especially 9× bamboo salt, also significantly (p<0.05) downregulated the expression of inflammation-related NF-κB, iNOS, and COX-2, and upregulated the gene expression of IκB-α compared to the other salt sample.

  18. The sperm chromatin dispersion test: a simple method for the determination of sperm DNA fragmentation.

    PubMed

    Fernández, Jose Luis; Muriel, Lourdes; Rivero, Maria Teresa; Goyanes, Vicente; Vazquez, Rosana; Alvarez, Juan G

    2003-01-01

    Sperm DNA fragmentation is being increasingly recognized as an important cause of infertility. We herein describe the Sperm Chromatin Dispersion (SCD) test, a novel assay for sperm DNA fragmentation in semen. The SCD test is based on the principle that sperm with fragmented DNA fail to produce the characteristic halo of dispersed DNA loops that is observed in sperm with non-fragmented DNA, following acid denaturation and removal of nuclear proteins. This was confirmed by the analysis of DNA fragmentation using the specific DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) assay, which allows the detection of DNA breaks in lysed sperm nuclei. Sperm suspensions either prepared from semen or isolated from semen by gradient centrifugation were embedded in an agarose microgel on slides and treated with 0.08 N HCl and lysing solutions containing 0.8 M dithiothreitol (DTT), 1% sodium dodecyl sulfate (SDS), and 2 M NaCl. Then, the slides were sequentially stained with DAPI (4',6-diamidino-2-phenylindole) and/or the Diff-Quik reagent, and the percentages of sperm with nondispersed and dispersed chromatin loops were monitored by fluorescence and brightfield microscopy, respectively. The results indicate that all sperm with nondispersed chromatin displayed DNA fragmentation, as measured by DBD-FISH. Conversely, all sperm with dispersed chromatin had very low to undetectable DBD-FISH labeling. SCD test values were significantly higher in patients being screened for infertility than in normozoospermic sperm donors who had participated in a donor insemination program. The coefficient of variation obtained using 2 different observers, either by digital image analysis (DIA) or by brightfield microscopy scoring, was less than 3%. In conclusion, the SCD test is a simple, accurate, highly reproducible, and inexpensive method for the analysis of sperm DNA fragmentation in semen and processed sperm. Therefore, the SCD test could potentially be used as a routine test

  19. Decellularized human amniotic membrane: more is needed for an efficient dressing for protection of burns against antibiotic-resistant bacteria isolated from burn patients.

    PubMed

    Gholipourmalekabadi, M; Bandehpour, M; Mozafari, M; Hashemi, A; Ghanbarian, H; Sameni, M; Salimi, M; Gholami, M; Samadikuchaksaraei, A

    2015-11-01

    Human amniotic membranes (HAMs) have attracted the attention of burn surgeons for decades due to favorable properties such as their antibacterial activity and promising support of cell proliferation. On the other hand, as a major implication in the health of burn patients, the prevalence of bacteria resistant to multiple antibiotics is increasing due to overuse of antibiotics. The aim of this study was to investigate whether HAMs (both fresh and acellular) are an effective antibacterial agent against antibiotic-resistant bacteria isolated from burn patients. Therefore, a HAM was decellularized and tested for its antibacterial activity. Decellularization of the tissue was confirmed by hematoxylin and eosin (H&E) and 4,6-diamidino-2-phenylindole (DAPI) staining. In addition, the cyto-biocompatibility of the acellular HAM was proven by the cell viability test (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, MTT) and scanning electron microscopy (SEM). The resistant bacteria were isolated from burns, identified, and tested for their susceptibility to antibiotics using both the antibiogram and polymerase chain reaction (PCR) techniques. Among the isolated bacteria, three blaIMP gene-positive Pseudomonas aeruginosa strains were chosen for their high resistance to the tested antibiotics. The antibacterial activity of the HAM was also tested for Klebsiella pneumoniae (American Type Culture Collection (ATCC) 700603) as a resistant ATCC bacterium; Staphylococcus aureus (mecA positive); and three standard strains of ATCC bacteria including Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27833), and S. aureus (ATCC 25923). Antibacterial assay revealed that only the latter three bacteria were susceptible to the HAM. All the data obtained from this study suggest that an alternative strategy is required to complement HAM grafting in order to fully protect burns from nosocomial infections.

  20. Non-Invasive Delivery of dsRNA into De-Waxed Tick Eggs by Electroporation

    PubMed Central

    Ruiz, Newton; de Abreu, Leonardo Araujo; Parizi, Luís Fernando; Kim, Tae Kwon; Mulenga, Albert; Braz, Gloria Regina Cardoso; Vaz, Itabajara da Silva; Logullo, Carlos

    2015-01-01

    RNA interference-mediated gene silencing was shown to be an efficient tool for validation of targets that may become anti-tick vaccine components. Here, we demonstrate the application of this approach in the validation of components of molecular signaling cascades, such as the Protein Kinase B (AKT) / Glycogen Synthase Kinase (GSK) axis during tick embryogenesis. It was shown that heptane and hypochlorite treatment of tick eggs can remove wax, affecting corium integrity and but not embryo development. Evidence of AKT and GSK dsRNA delivery into de-waxed eggs of via electroporation is provided. Primers designed to amplify part of the dsRNA delivered into the electroporated eggs dsRNA confirmed its entry in eggs. In addition, it was shown that electroporation is able to deliver the fluorescent stain, 4',6-diamidino-2-phenylindole (DAPI). To confirm gene silencing, a second set of primers was designed outside the dsRNA sequence of target gene. In this assay, the suppression of AKT and GSK transcripts (approximately 50% reduction in both genes) was demonstrated in 7-day-old eggs. Interestingly, silencing of GSK in 7-day-old eggs caused 25% reduction in hatching. Additionally, the effect of silencing AKT and GSK on embryo energy metabolism was evaluated. As expected, knockdown of AKT, which down regulates GSK, the suppressor of glycogen synthesis, decreased glycogen content in electroporated eggs. These data demonstrate that electroporation of de-waxed R. microplus eggs could be used for gene silencing in tick embryos, and improve the knowledge about arthropod embryogenesis. PMID:26091260

  1. Programmed cell death in kiwifruit stigmatic arms and its relationship to the effective pollination period and the progamic phase

    PubMed Central

    Ferradás, Yolanda; López, Marián; Rey, Manuel; González, Ma Victoria

    2014-01-01

    Background and Aims Kiwifruit is a crop with a highly successful reproductive performance, which is impaired by the short effective pollination period of female flowers. This study investigates whether the degenerative processes observed in both pollinated and non-pollinated flowers after anthesis may be considered to be programmed cell death (PCD). Methods Features of PCD in kiwifruit, Actinidia chinensis var. deliciosa, were studied in both non-pollinated and pollinated stigmatic arms using transmission electron microscopy, DAPI (4′,6-diamidino-2-phenylindole) staining, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) assays, DNA gel electrophoresis and caspase-like activity assays. Key Results In the secretory tissues of the stigmatic arms, cell organelles disintegrated sequentially while progressive vacuolization was detected. At the same time, chromatin condensation, nuclear deformation, and DNA fragmentation and degradation were observed. These features were detected in both non-pollinated and pollinated stigmatic arms; they were evident in the stigmas of pollinated flowers by the second day after anthesis but only by 4 d after anthesis in non-pollinated flowers. In addition, in pollinated stigmatic arms, these features were first initiated in the stigma and gradually progressed through the style, consistent with pollen tube growth. This timing of events was also observed in both non-pollinated and pollinated stigmatic arms for caspase-3-like activity. Conclusions The data provide evidence to support the hypothesis that PCD processes occurring in the secretory tissue of non-pollinated kiwifruit stigmatic arms could be the origin for the observed short effective pollination period. The results obtained in the secretory tissue of pollinated kiwifruit stigmatic arms upon pollination support the idea that PCD might be accelerated by pollination, pointing to the involvement of PCD during the progamic phase. PMID:24782437

  2. Hyperthermia sensitizes Rhizopus oryzae to posaconazole and itraconazole action through apoptosis.

    PubMed

    Shirazi, Fazal; Pontikos, Michael A; Walsh, Thomas J; Albert, Nathaniel; Lewis, Russell E; Kontoyiannis, Dimitrios P

    2013-09-01

    The high mortality rate of mucormycosis with currently available monotherapy has created interest in studying novel strategies for antifungal agents. With the exception of amphotericin B (AMB), the triazoles (posaconazole [PCZ] and itraconazole [ICZ]) are fungistatic in vitro against Rhizopus oryzae . We hypothesized that growth at a high temperature (42°C) results in fungicidal activity of PCZ and ICZ that is mediated through apoptosis. R. oryzae had high MIC values for PCZ and ICZ (16 to 64 μg/ml) at 25°C; in contrast, the MICs for PCZ and ICZ were significantly lower at 37°C (8 to 16 μg/ml) and 42°C (0.25 to 1 μg/ml). Furthermore, PCZ and ICZ dose-dependent inhibition of germination was more pronounced at 42°C than at 37°C. In addition, intracellular reactive oxygen species (ROS) increased significantly when fungi were exposed to antifungals at 42°C. Characteristic cellular changes of apoptosis in R. oryzae were induced by the accumulation of intracellular reactive oxygen species. Cells treated with PCZ or ICZ in combination with hyperthermia (42°C) exhibited characteristic markers of early apoptosis: phosphatidylserine externalization visualized by annexin V staining, membrane depolarization visualized by bis-[1,3-dibutylbarbituric acid] trimethine oxonol (DiBAC) staining, and increased metacaspase activity. Moreover, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay and DAPI (4',6-diamidino-2-phenylindole) staining demonstrated DNA fragmentation and condensation, respectively. The addition of N-acetylcysteine increased fungal survival, prevented apoptosis, reduced ROS accumulation, and decreased metacaspase activation. We concluded that hyperthermia, either alone or in the presence of PCZ or ICZ, induces apoptosis in R. oryzae. Local thermal delivery could be a therapeutically useful adjunct strategy for these refractory infections.

  3. Image-Based High-Throughput Drug Screening Targeting the Intracellular Stage of Trypanosoma cruzi, the Agent of Chagas' Disease▿ ‡

    PubMed Central

    Engel, Juan C.; Ang, Kenny K. H.; Chen, Steven; Arkin, Michelle R.; McKerrow, James H.; Doyle, Patricia S.

    2010-01-01

    Chagas' disease, caused by infection with the parasite Trypanosoma cruzi, is the major cause of heart failure in Latin America. Classic clinical manifestations result from the infection of heart muscle cells leading to progressive cardiomyopathy. To ameliorate disease, chemotherapy must eradicate the parasite. Current drugs are ineffective and toxic, and new therapy is a critical need. To expedite drug screening for this neglected disease, we have developed and validated a cell-based, high-throughput assay that can be used with a variety of untransfected T. cruzi isolates and host cells and that simultaneously measures efficacy against the intracellular amastigote stage and toxicity to host cells. T. cruzi-infected muscle cells were incubated in 96-well plates with test compounds. Assay plates were automatically imaged and analyzed based on size differences between the DAPI (4′,6-diamidino-2-phenylindole)-stained host cell nuclei and parasite kinetoplasts. A reduction in the ratio of T. cruzi per host cell provided a quantitative measure of parasite growth inhibition, while a decrease in count of the host nuclei indicated compound toxicity. The assay was used to screen a library of clinically approved drugs and identified 55 compounds with activity against T. cruzi. The flexible assay design allows the use of various parasite strains, including clinical isolates with different biological characteristics (e.g., tissue tropism and drug sensitivity), and a broad range of host cells and may even be adapted to screen for inhibitors against other intracellular pathogens. This high-throughput assay will have an important impact in antiparasitic drug discovery. PMID:20547819

  4. Hyperthermia Sensitizes Rhizopus oryzae to Posaconazole and Itraconazole Action through Apoptosis

    PubMed Central

    Shirazi, Fazal; Pontikos, Michael A.; Walsh, Thomas J.; Albert, Nathaniel; Lewis, Russell E.

    2013-01-01

    The high mortality rate of mucormycosis with currently available monotherapy has created interest in studying novel strategies for antifungal agents. With the exception of amphotericin B (AMB), the triazoles (posaconazole [PCZ] and itraconazole [ICZ]) are fungistatic in vitro against Rhizopus oryzae . We hypothesized that growth at a high temperature (42°C) results in fungicidal activity of PCZ and ICZ that is mediated through apoptosis. R. oryzae had high MIC values for PCZ and ICZ (16 to 64 μg/ml) at 25°C; in contrast, the MICs for PCZ and ICZ were significantly lower at 37°C (8 to 16 μg/ml) and 42°C (0.25 to 1 μg/ml). Furthermore, PCZ and ICZ dose-dependent inhibition of germination was more pronounced at 42°C than at 37°C. In addition, intracellular reactive oxygen species (ROS) increased significantly when fungi were exposed to antifungals at 42°C. Characteristic cellular changes of apoptosis in R. oryzae were induced by the accumulation of intracellular reactive oxygen species. Cells treated with PCZ or ICZ in combination with hyperthermia (42°C) exhibited characteristic markers of early apoptosis: phosphatidylserine externalization visualized by annexin V staining, membrane depolarization visualized by bis-[1,3-dibutylbarbituric acid] trimethine oxonol (DiBAC) staining, and increased metacaspase activity. Moreover, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay and DAPI (4′,6-diamidino-2-phenylindole) staining demonstrated DNA fragmentation and condensation, respectively. The addition of N-acetylcysteine increased fungal survival, prevented apoptosis, reduced ROS accumulation, and decreased metacaspase activation. We concluded that hyperthermia, either alone or in the presence of PCZ or ICZ, induces apoptosis in R. oryzae. Local thermal delivery could be a therapeutically useful adjunct strategy for these refractory infections. PMID:23817366

  5. Particle size tailoring of ursolic acid nanosuspensions for improved anticancer activity by controlled antisolvent precipitation.

    PubMed

    Wang, Yancai; Song, Ju; Chow, Shing Fung; Chow, Albert H L; Zheng, Ying

    2015-10-15

    The present study was aimed at tailoring the particle size of ursolic acid (UA) nanosuspension for improved anticancer activity. UA nanosuspensions were prepared by antisolvent precipitation using a four-stream multi-inlet vortex mixer (MIVM) under defined conditions of varying solvent composition, drug feeding concentration or stream flow rate. The resulting products were characterized for particle size and polydispersity. Two of the UA nanosuspensions with mean particle sizes of 100 and 300 nm were further assessed for their in-vitro activity against MCF-7 breast cancer cells using fluorescence microscopy with 4',6-diamidino-2-phenylindole (DAPI) staining, as well as flow cytometry with propidium (PI) staining and with double staining by fluorescein isothiocyanate. It was revealed that the solvent composition, drug feeding concentration and stream flow rate were critical parameters for particle size control of the UA nanosuspensions generated with the MIVM. Specifically, decreasing the UA feeding concentration or increasing the stream flow rate or ethanol content resulted in a reduction of particle size. Excellent reproducibility for nanosuspension production was demonstrated for the 100 and 300 nm UA preparations with a deviation of not more than 5% in particle size from the mean value of three independent batches. Fluorescence microscopy and flow cytometry revealed that these two different sized UA nanosuspensions, particularly the 300 nm sample, exhibited a higher anti-proliferation activity against the MCF-7 cells and afforded a larger population of these cells in both early and late apoptotic phases. In conclusion, MIVM is a robust and pragmatic tool for tailoring the particle size of the UA nanosuspension. Particle size appears to be a critical determinant of the anticancer activity of the UA nanoparticles.

  6. Intracerebral and Intravenous Transplantation Represents a Favorable Approach for Application of Human Umbilical Cord Mesenchymal Stromal Cells in Intracerebral Hemorrhage Rats

    PubMed Central

    Xie, Jiang; Wang, Bin; Wang, Lian; Dong, Fang; Bai, Gang; Liu, Yongjun

    2016-01-01

    Background Intracerebral hemorrhage (ICH) is one severe subtype of stroke, with a very complex pathology. Stem cell-based therapy holds promising potential in the treatment of neurological disorders. Human umbilical cord-derived mesenchymal stem cells (UC-MSCs) have a therapeutic effect in recovery from brain damage following ICH. The aim of this study was to identify an effective and convenient way of using UC-MSCs in the ICH rat model. Material/Methods CM-DiI-labeled human UC-MSCs were transplanted intracerebrally or intravenously into collagenase VII-induced ICH rat models. Neurological function was evaluated before ICH and at 0, 7, 14, 21, and 28 days after treatment. ICH rats were sacrificed to evaluate the injury volume. Neurogenesis and angiogenesis and vascular areas were investigated using microtubule-associated protein 2 (MAP2), glial fibrillary acidic protein (GFAP), and 4′,6-diamidino-2-phenylindole (DAPI) immunohistochemistry at two weeks after transplantation. Results The intracerebral and intravenous administration of UC-MSCs both resulted in significant improvement in neurological function and decrease in injury volume of ICH rats. Transplanted UC-MSCs were chemotactic in vivo and showed a predominant distribution around the ICH region. In addition, UC-MSCs could integrate into the cerebral vasculature in both groups. Conclusions Both intracerebral and intravenous administration of UC-MSCs could have a favorable effect on recovery of neurological function in ICH rats, although the fundamental mechanisms may be different between the two groups. Our data suggest that intravenous implantation of UC-MSCs could serve as a favorable approach for cell-based therapy in central nervous system (CNS) diseases according to clinical needs. PMID:27703134

  7. Influence of uranium (VI) on the metabolic activity of stable multispecies biofilms studied by oxygen microsensors and fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Krawczyk-Bärsch, Evelyn; Grossmann, Kay; Arnold, Thuro; Hofmann, Susann; Wobus, Axel

    2008-11-01

    The effect of uranium added in ecologically relevant concentrations (1 × 10 -5 and 1 × 10 -6 M) to stable multispecies biofilms was studied by electrochemical oxygen microsensors with tip diameters of 10 μm and by confocal laser fluorescence microscopy (CLSM). The microsensor profile measurements in the stable multispecies biofilms exposed to uranium showed that the oxygen concentration decreased faster with increasing biofilm depth compared to the uranium free biofilms. In the uranium containing biofilms, the oxygen consumption, calculated from the steady-state microprofiles, showed high consumption rates of up to 61.7 nmol cm -3 s -1 in the top layer (0-70 μm) and much lower consumption rates in the lower zone of the biofilms. Staining experiments with 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and 4,6-diamidino-2-phenylindole (DAPI) confirmed the high respiratory activities of the bacteria in the upper layer. Analysis of the amplified 16S rRNA gene fragments showed that the addition of uranium in ecologically relevant concentrations did not change the bacterial diversity in the stable multispecies biofilms and is therefore not responsible for the different oxygen profiles in the biofilms. The fast decrease in the oxygen concentrations in the biofilm profiles showed that the bacteria in the top region of the biofilms, i.e., the metabolically most active biofilm zone, battle the toxic effects of aqueous uranium with an increased respiratory activity. This increased respiratory activity results in O 2 depleted zones closer to the biofilm/air interface which may trigger uranium redox processes, since suitable redox partners, e.g., extracellular polymeric substance (EPS) and other organics (e.g., metabolites), are sufficiently available in the biofilm porewaters. Such redox reactions may lead to precipitation of uranium (IV) solids and consequently to a removal of uranium from the aqueous phase.

  8. Primo vascular system in the subarachnoid space of the spinal cord of a pig.

    PubMed

    Moon, Sang-Ho; Cha, Richard; Lee, MinSun; Kim, Sungchul; Soh, Kwang-Sup

    2012-10-01

    The primo vascular system was recently observed in the central nervous systems of rabbits and rats, but no investigations in large animals have been reported. In the present work we found a putative primo vascular system in the spinal cord of a pig. We obtained spines from four healthy pigs and fixed them with paraformaldehyde. The primo vessels were expected to lie in the subarachnoid space between the pia mater and the arachnoid mater. The composite of three membranes (the pia, the arachnoid, and the dura maters) wrapping the spinal cord was peeled off, isolated from the spine, and put on a slide glass. This composite was stained with 4',6'-Diamidino-2-phenylindole (DAPI) and phalloidin to show the nuclei and the f-actin, respectively, in the cells of the primo vessels. We observed eleven pieces of the putative primo vessels in the subarachnoid space of the spines at the thoracic spinal nerve area. They had the typical rod-shaped nuclei distributed in a broken line, and f-actin signals around nuclei. The lengths of the nuclei were 12-15 μm, and the thicknesses of the primo vessels were 8∼20 μm, which were consistent with other primo vessels that had been observed in the various organs of rabbits, rats, and mice. In addition, we observed branching of the primo vessels, which is again an expected result from previous works. In conclusion, a primo vessel was observed in the subarachnoid space of the spinal cord of a pig. This was the first observation of a primo vessel in a large animal, and the staining method used to observe the primo vessel in a fixed sample was newly developed in this work.

  9. Intestinal ammonia transport in freshwater and seawater acclimated rainbow trout (Oncorhynchus mykiss): evidence for a Na+ coupled uptake mechanism.

    PubMed

    Rubino, Julian G; Zimmer, Alex M; Wood, Chris M

    2015-05-01

    In vitro gut sac experiments were performed on freshwater and 60% seawater acclimated trout (Oncorhynchus mykiss) under treatments designed to discern possible mechanisms of intestinal ammonia transport. Seawater acclimation increased ammonia flux rate into the serosal saline (Jsamm) in the anterior intestine, however it did not alter Jsamm in the mid- or posterior intestine suggesting similar mechanisms of ammonia handling in freshwater and seawater fish. Both fluid transport rate (FTR) and Jsamm were inhibited in response to basolateral ouabain treatment, suggesting a linkage of ammonia uptake to active transport, possibly coupled to fluid transport processes via solvent drag. Furthermore, decreases in FTR and Jsamm caused by low Na(+) treatment indicated a Na(+) linked transport mechanism. Mucosal bumetanide (10(-4) M) had no impact on FTR, yet decreased Jsamm in the anterior and mid-intestine, suggesting NH4(+) substitution for K(+) on an apical NKCC, and at least a partial uncoupling of ammonia transport from fluid transport. Additional treatments (amiloride, 5-(N-ethyl-N-isopropyl)amiloride (EIPA), phenamil, bafilomycin, 4',6-diamidino-2-phenylindole (DAPI), high sodium) intended to disrupt alternative routes of Na(+) uptake yielded no change in FTR or Jsamm, suggesting the absence of direct competition between Na(+) and ammonia for transport. Finally, [(14)C]methylamine permeability (PMA) measurements indicated the likely presence of an intestinal Rh-mediated ammonia transport system, as increasing NH4Cl (0, 1, 5 mmol l(-1)) concentrations reduced PMA, suggesting competition for transport through Rh proteins. Overall, the data presented in this paper provide some of the first insights into mechanisms of teleost intestinal ammonia transport.

  10. TGFβ2 Induces the Formation of Cross-Linked Actin Networks (CLANs) in Human Trabecular Meshwork Cells Through the Smad and Non-Smad Dependent Pathways

    PubMed Central

    Montecchi-Palmer, Michela; Bermudez, Jaclyn Y.; Webber, Hannah C.; Patel, Gaurang C.; Clark, Abbot F.; Mao, Weiming

    2017-01-01

    Purpose Increased intraocular pressure results from increased aqueous humor (AH) outflow resistance at the trabecular meshwork (TM) due to pathologic changes including the formation of cross-linked actin networks (CLANs). Transforming growth factor β2 (TGFβ2) is elevated in the AH and TM of primary open angle glaucoma (POAG) patients and induces POAG-associated TM changes, including CLANs. We determined the role of individual TGFβ2 signaling pathways in CLAN formation. Methods Cultured nonglaucomatous human TM (NTM) cells were treated with control or TGFβ2, with or without the inhibitors of TGFβ receptor, Smad3, c-Jun N-terminal kinases (JNK), extracellular signal regulated kinase (ERK), P38, or Rho-associated protein kinase (ROCK). NTM cells were cotreated with TGFβ2 plus inhibitors for 10 days or pretreated with TGFβ2 for 10 days followed by 1-hour inhibitor treatment. NTM cells were immunostained with phalloidin-Alexa-488 and 4′,6-diamidino-2-phenylindole (DAPI). Data were analyzed using 1-way ANOVA and Dunnett's post hoc test. Results TGFβ2 significantly induced CLAN formation (n = 6 to 12, P < 0.05), which was completely inhibited by TGFβ receptor, Smad3, and ERK inhibitors, as well as completely or partially inhibited by JNK, P38, and ROCK inhibitors, depending on cell strains. One-hour exposure to ROCK inhibitor completely resolved formed CLANs (P < 0.05), whereas TGFβ receptor, Smad3 inhibitor, and ERK inhibitors resulted in partial or complete resolution. The JNK and P38 inhibitors showed partial or no resolution. Among these inhibitors, the ROCK inhibitor was the most disruptive to the actin stress fibers, whereas ERK inhibition showed the least disruption. Conclusions TGFβ2-induced CLANs in NTM cells were prevented and resolved using various pathway inhibitors. Apart from CLAN inhibition, some of these inhibitors also had different effects on actin stress fibers. PMID:28241317

  11. How Do Deep Saline Aquifer Microbial Communities Respond to Supercritical CO2 Injection?

    NASA Astrophysics Data System (ADS)

    Mu, A.; Billman-Jacobe, H.; Boreham, C.; Schacht, U.; Moreau, J. W.

    2011-12-01

    Carbon Capture and Storage (CCS) is currently seen as a viable strategy for mitigating anthropogenic carbon dioxide pollution. The Cooperative Research Centre for Greenhouse Gas Technologies (CO2CRC) is currently conducting a field experiment in the Otway Basin (Australia) studying residual gas saturation in the water-saturated reservoir of the Paaratte Formation. As part of this study, a suite of pre-CO2 injection water samples were collected from approximately 1400 meters depth (60°C, 13.8 MPa) via an in situ sampling system. The in situ sampling system isolates aquifer water from sources of contamination while maintaining the formation pressure. Whole community DNA was extracted from these samples to investigate the prokaryotic biodiversity of the saline Paaratte aquifer (EC = 1509.6 uS/cm). Bioinformatic analysis of preliminary 16S ribosomal gene data revealed Thermincola, Acinetobacter, Sphingobium, and Dechloromonas amongst the closest related genera to environmental clone sequences obtained from a subset of pre-CO2 injection groundwater samples. Epifluorescent microscopy with 4',6-diamidino-2-phenylindole (DAPI) highlighted an abundance of filamentous cells ranging from 5 to 45 μM. Efforts are currently directed towards utilising a high throughput sequencing approach to capture an exhaustive profile of the microbial diversity of the Paaratte aquifer CO2 injection site, and to understand better the response of in situ microbial populations to the injection of large volumes (e.g. many kilotonnes) of supercritical CO2 (sc-CO2). Sequencing results will be used to direct cultivation efforts towards enrichment of a CO2-tolerant microorganism. Understanding the microbial response to sc-CO2 is an integral aspect of carbon dioxide storage, for which very little information exists in the literature. This study aims to elucidate molecular mechanisms, through genomic and cultivation-based methods, for CO2 tolerance with the prospect of engineering biofilms to enhance

  12. Bacteria Community in the Terrestrial Deep Subsurface Microbiology Research of the Chinese Continent Scientific Drilling

    NASA Astrophysics Data System (ADS)

    Wang, Y.; Xia, Y.; Dong, H.; Dong, X.; Yang, K.; Dong, Z.; Huang, L.

    2005-12-01

    Microbial communities in the deep drill cores from the Chinese Continent Scientific Drilling were analyzed with culture-independent and dependent techniques. Genomic DNA was extracted from two metamorphic rocks: S1 from 430 and S13 from 1033 meters below the ground surface. The 16S rRNA gene was amplified by polymerase chain reaction (PCR) followed by cloning and sequencing. The total cell number was counted using the 4',6-diamidino-2-phenylindole (DAPI) staining and biomass of two specific bacteria were quantified using real-time PCR. Enrichment was set up for a rock from 3911 meters below the surface in medium for authotrophic methanogens (i.e., CO2 + H2). The total cell number in S13 was 1.0 × 104 cells per gram of rock. 16S rRNA gene analysis indicated that low G + C Gram positive sequences were dominant (50 percent of all 54 clone sequenced) followed by the alpha-, beta, and gamma-Proteobacteria. Within the low G + C Gram positive bacteria, most clone sequences were similar to species of Bacillus from various natural environments (deserts, rivers etc.). Within the Proteobacteria, our clone sequences were similar to species of Acinetobacter, Acidovorax, and Aeromonas. The RT-RCP results showed that biomass of two particular clone sequences (CCSD1305, similar to Aeromonas caviae and CCSD1307, similar to Acidovorax facilis) was 95 and 1258 cells/g, respectively. A bacterial isolate was obtained from the 3911-m rock in methanogenic medium. It was Gram negative with no flagella, immobile, and facultative anaerobic, and grows optimally at 65oC. Phylogenetic analysis indicated that it was closely related to the genus of Bacillus. Physiological tests further revealed that it was a strain of Bacillus caldotenax.

  13. [Factors affecting the DAPI fluorescence direct count in the tidal river sediment].

    PubMed

    Chen, Chen; Huang, Shan; Wu, Qun-he; Li, Rui-yi; Zhang, Ren-duo

    2010-08-01

    The factors affecting the DAPI (4', 6-diamidino-2-phenylidole) fluorescence direct count in the tidal river sediment were examined. Sediment samples were collected from the Guangzhou section of the Pearl River. Besides sediment texture and organic matter, an improved staining procedure and the involved parameters were analyzed. Results showed that the procedure with the sediment with 2000 fold dilution and ultrasonic water bath for 10 min, and with a final DAPI concentration of 10 microg x mL(-1) and staining time for more than 30 min produced the optimum results of DAPI direct count in the sediment. The total bacterial number was correlated to the proportion of the non-nucleoid-containing cells to the total bacterial number (r = 0.587, p = 0.004). The organic matter content also correlated to the ration. The clay content had a strong correlation with the organic matter, through which the clay content also affected the ratio. A multiple regression analysis between the ration versus the organic matter, the total bacterial number, and the clay content showed that the regression equation fit the measure values satisfactorily (r = 0.694). These results indicated that the above factors needed to be considered in the applications of the DAPI fluorescence direct counting method to the tidal river sediment.

  14. Relationship between DAPI-fluorescence fading and nuclear DNA content: An alternative method to DNA quantification?

    PubMed

    Gallardo-Escárate, Cristian; Alvarez-Borrego, Josué; Von Brand, Elisabeth; Dupré, Enrique; Del Río-Portilla, Miguel Angel

    2007-01-01

    In observations by confocal or conventional fluorescence microscopy, important factors should be considered in order to obtain accurate images. One of them, such as the fluorescence bleaching from highest intensity to lowest signal of fluorescence is a common problem with several DNA fluorochromes and especially for DAPI stain. The fluorescence of DAPI fades rapidly when it is exposed to UV light, under optimal conditions of observation. Although the fading process can be retarded using a mounting medium with antifading reagents, the photochemical process underlying the fluorescence decay has not yet been fully explained. In addition, no relationship between fluorescence fading and nuclear DNA content has been tested. In order to test this relationship, we measured by means of image analysis the DAPI-fluorescence intensity in several cellular types (spermatozoa, erythrocytes and haemocytes) during their fluorescence bleaching. An algorithm specifically built in MATLAB software was used for this approach. The correlation coefficient between nuclear DNA content and DAPI-fluorescence fading was found equal to 99%. This study demonstrates the feasibility to measure nuclear DNA content by fluorescence fading quantification, as an alternative method concurrently with image analysis procedures.

  15. [Nuclei in the plasmodium of Intoshia variabili (Orthonectida) as revealed by DAPI staining].

    PubMed

    Sliusarev, G S; Manylov, O G; Cherkasov, A S

    2002-01-01

    DAPI staining of wholeamounts was used to reveal the parasitic plasmodium of the orthonectid Intoshia variabili in its host, the turbellarian Macrorhynchus crocea. The nuclei of the parasite differ drastically from those of the host in size, morphology, and the estimated DNA content. Our findings indirectly support the idea that the orthonectid plasmodium is a distinct parasitic organism, rather than modified host cells.

  16. Solid Microneedles for Transdermal Delivery of Amantadine Hydrochloride and Pramipexole Dihydrochloride

    PubMed Central

    Hoang, Mylien T.; Ita, Kevin B.; Bair, Daniel A.

    2015-01-01

    The aim of this project was to study the influence of microneedles on transdermal delivery of amantadine hydrochloride and pramipexole dihydrochloride across porcine ear skin in vitro. Microchannel visualization studies were carried out and characterization of the microchannel depth was performed using confocal laser scanning microscopy (CLSM) to demonstrate microchannel formation following microneedle roller application. We also report, for the first time, the use of TA.XT Plus Texture Analyzer to characterize burst force in pig skin for transdermal drug delivery experiments. This is the force required to rupture pig skin. The mean passive flux of amantadine hydrochloride, determined using a developed LC–MS/MS technique, was 22.38 ± 4.73 µg/cm2/h, while the mean flux following the use of a stainless steel microneedle roller was 49.04 ± 19.77 µg/cm2/h. The mean passive flux of pramipexole dihydrochloride was 134.83 ± 13.66 µg/cm2/h, while the flux following the use of a stainless steel microneedle roller was 134.04 ± 0.98 µg/cm2/h. For both drugs, the difference in flux values following the use of solid stainless steel microneedle roller was not statistically significantly (p > 0.05). Statistical analysis was carried out using the Mann–Whitney Rank sum test. PMID:26426039

  17. Pharmaceutical and pharmacokinetic evaluation of a novel fast dissolving film formulation of flupentixol dihydrochloride.

    PubMed

    Abdelbary, Ahmed; Bendas, Ehab R; Ramadan, Afaf A; Mostafa, Dalia A

    2014-12-01

    The objective of the present study was to develop fast dissolving oral film of the antipsychotic drug, flupentixol dihydrochloride, to enhance its bioavailability, optimize its therapeutic effect when used to treat depression with anxiety, and increase the convenience and compliance by the mentally ill, developmentally disable, elderly, and pediatric patients. Six formulae were prepared with different concentrations of water-soluble polymers vis. hydroxypropyl methylcellulose (HPMC E5) and carboxymethyl cellulose (CMC) by solvent casting technique. The prepared films were subjected to characterization for folding endurance, weight variations, thickness, disintegration time, drug release pattern, and drug content. Physical compatibility between the drug and excipients was guaranteed in the selected formulation (2% HPMC) by means of differential scanning calorimetry analysis and Fourier-transform infrared spectroscopy. This formulation revealed high stability after testing according to the International Conference on Harmonisation guidelines. In vivo studies based on single phase parallel design were carried out for the optimized formulation in healthy human volunteers. The concentration of flupentixol dihydrochloride in plasma samples was analyzed by a developed validated LC-MS/MS assay method and the pharmacokinetic parameters of the established formulation were compared with the commercially available oral tablets. Faster rate of absorption of flupentixol could be obtained from the oral film formulation and the relative bioavailability was found to be 151.06% compared to the marketed product.

  18. Chitosan-based Floating Microspheres of Trimetazidin Dihydrochloride; Preparation and In vitro Characterization

    PubMed Central

    El-Nahas, H. M.; Hosny, K. M.

    2011-01-01

    The aim of present study involves preparation and characterization of floating microspheres using trimetazidin dihydrochloride as a model drug to increase the residence time in the stomach without contact with the mucosa, Floating microspheres were prepared by the capillary extrusion technique using chitosan as polymer and sodium lauryl sulphate as cross linking agent. The surface morphology of the prepared microspheres was characterized by the optical microscopic method. The effect of the stirring rate during preparation, polymer concentration and cross linking concentration on the percent yield, in vitro floating behavior, physical state of the incorporated drug, drug loading and in vitro drug release were studied. The prepared microspheres exhibited prolonged drug release (12 h) and remained buoyant for more than 11 h. The microspheres were found to be regular in shape and highly porous. The trimetazidin dihydrochloride release rate was higher in the case of microspheres prepared at a higher agitation speed and decreased with increasing the polymer and cross linking agent concentration. All formulations demonstrated favorable in vitro floating characteristics. The drug entrapment increased from 65.13 to 85.3% with increasing polymer to drug ratio. Diffusion was found to be the main release mechanism. Thus, the prepared floating microspheres may prove to be potential candidates for multiple-unit delivery devices adaptable to any intragastric conditions. PMID:22707823

  19. Preparation and evaluation of itraconazole dihydrochloride for the solubility and dissolution rate enhancement.

    PubMed

    Tao, Tao; Zhao, Yan; Wu, Jinjin; Zhou, Beiyi

    2009-02-09

    The purpose of this work was to explore the feasibility of preparing itraconazole hydrochloride to improve the solubility and dissolution rate. Itraconazole dihydrochloride was synthesized by bubbling anhydrous hydrogen chloride gas into the acetone suspension of itraconazole. Results of the elementary analysis gave the molecular formula of C(35)H(38)Cl(2)N(8)O(4).2HCl and its structure was confirmed by Fourier transform infrared (FTIR), thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). Powder X-Ray diffraction (PXRD) suggested that a new crystalline form of the salt was formed. The morphology and mean size distribution study by scanning electron microscopy (SEM) and dynamic light scattering (DLS) confirmed that the salt was dispersable nanoparticle aggregation. Aqueous solubility measurements showed that the solubility of the salt, its 1:1, 1:2 and 1:3 (w/w) physical mixtures with beta-cyclodextrin (beta-CD) was 6, 99, 236 and 388 times greater than itraconazole. More than 94% of itraconazole was dissolved out of the salt/beta-CD 1/3 physical mixture after 60min. The stability studies indicated that the physical mixture remained stable for 24 months in assay, the related substances and dissolution. Based on the present results, it is concluded that hydrochloride formation can significantly increase solubility and dissolution rate of itraconazole, and the formulation of itraconazole dihydrochloride/beta-CD (1/3) would be an environment-friendly, economic and practical alternative to the commercially available itraconazole capsules (Sporanox)

  20. Biowaiver monographs for immediate release solid oral dosage forms: ethambutol dihydrochloride.

    PubMed

    Becker, C; Dressman, J B; Amidon, G L; Junginger, H E; Kopp, S; Midha, K K; Shah, V P; Stavchansky, S; Barends, D M

    2008-04-01

    Literature data relevant to the decision to allow a waiver of in vivo bioequivalence (BE) testing for the approval of immediate release (IR) solid oral dosage forms containing ethambutol dihydrochloride as the only active pharmaceutical ingredient (API) are reviewed. Ethambutol dihydrochloride is a Biopharmaceutics Classification System (BCS) Class III drug with permeability properties approaching the border between BCS Class I and III. BE problems of ethambutol formulations containing different excipients and different dosages forms have not been reported and hence the risk of bioinequivalence caused by excipients is low. Ethambutol has a narrow therapeutic index related to ocular toxicity. However, as long as the prescribers' information of the test product stipulates the need for regular monitoring of ocular toxicity, the additional patient risk is deemed acceptable. It is concluded that a biowaiver can be recommended for IR solid oral dosage forms provided that the test product (a) contains only excipients present in ethambutol IR solid oral drug products approved in ICH or associated countries, for instance as presented in this paper, (b) complies with the criteria for "very rapidly dissolving" and (c) has a prescribers' information indicating the need for testing the patient's vision prior to initiating ethambutol therapy and regularly during therapy.

  1. Dose- and duration-dependent effects of betahistine dihydrochloride treatment on histamine turnover in the cat.

    PubMed

    Tighilet, Brahim; Trottier, Suzanne; Lacour, Michel

    2005-10-31

    Drugs interacting with the histaminergic system are currently used for vertigo treatment and it was shown in animal models that structural analogues of histamine like betahistine improved the recovery process after vestibular lesion. This study was aimed at determining the possible dose and duration effects of betahistine treatment on histamine turnover in normal adult cats, as judged by the level of messenger RNA for histidine decarboxylase (enzyme synthesizing histamine) in the tuberomammillary nuclei. Experiments were conducted on betahistine-treated cats receiving daily doses of 2, 5, 10, or 50 mg/kg during 1 week, 3 weeks, 2 months, or 3 months. The 1-week, 3-week, and 2- and 3-month treatments correspond to the acute, compensatory, and sustained compensatory stages of vestibular compensation, respectively. The lowest dose (2 mg/kg) given the longest time (3 months) was close to the dosage for vestibular defective patients. Data from the experimental groups were compared to control, untreated cats and to placebo-treated animals. The results clearly show that betahistine dihydrochloride administered orally in the normal cat interferes with histamine turnover by increasing the basal expression level of histidine decarboxylase mRNA of neurons located in the tuberomammillary nuclei of the posterior hypothalamus. The effects were both dose- and time-dependent. In conclusion, compensation of both static and dynamic deficits is subtended by long-term adaptive mechanisms that could be facilitated pharmacologically using betahistine dihydrochloride.

  2. 99mTc-labeled HYNIC-DAPI causes plasmid DNA damage with high efficiency.

    PubMed

    Kotzerke, Joerg; Punzet, Robert; Runge, Roswitha; Ferl, Sandra; Oehme, Liane; Wunderlich, Gerd; Freudenberg, Robert

    2014-01-01

    (99m)Tc is the standard radionuclide used for nuclear medicine imaging. In addition to gamma irradiation, (99m)Tc emits low-energy Auger and conversion electrons that deposit their energy within nanometers of the decay site. To study the potential for DNA damage, direct DNA binding is required. Plasmid DNA enables the investigation of the unprotected interactions between molecules and DNA that result in single-strand breaks (SSBs) or double-strand breaks (DSBs); the resulting DNA fragments can be separated by gel electrophoresis and quantified by fluorescent staining. This study aimed to compare the plasmid DNA damage potential of a (99m)Tc-labeled HYNIC-DAPI compound with that of (99m)Tc pertechnetate ((99m)TcO4(-)). pUC19 plasmid DNA was irradiated for 2 or 24 hours. Direct and radical-induced DNA damage were evaluated in the presence or absence of the radical scavenger DMSO. For both compounds, an increase in applied activity enhanced plasmid DNA damage, which was evidenced by an increase in the open circular and linear DNA fractions and a reduction in the supercoiled DNA fraction. The number of SSBs elicited by 99mTc-HYNIC-DAPI (1.03) was twice that caused by (99m)TcO4(-) (0.51), and the number of DSBs increased fivefold in the (99m)Tc-HYNIC-DAPI-treated sample compared with the (99m)TcO4(-) treated sample (0.02 to 0.10). In the presence of DMSO, the numbers of SSBs and DSBs decreased to 0.03 and 0.00, respectively, in the (99m)TcO4(-) treated samples, whereas the numbers of SSBs and DSBs were slightly reduced to 0.95 and 0.06, respectively, in the (99m)Tc-HYNIC-DAPI-treated samples. These results indicated that (99m)Tc-HYNIC-DAPI induced SSBs and DSBs via a direct interaction of the (99m)Tc-labeled compound with DNA. In contrast to these results, (99m)TcO4(-) induced SSBs via radical formation, and DSBs were formed by two nearby SSBs. The biological effectiveness of (99m)Tc-HYNIC-DAPI increased by approximately 4-fold in terms of inducing SSBs and by

  3. 99mTc-Labeled HYNIC-DAPI Causes Plasmid DNA Damage with High Efficiency

    PubMed Central

    Kotzerke, Joerg; Punzet, Robert; Runge, Roswitha; Ferl, Sandra; Oehme, Liane; Wunderlich, Gerd; Freudenberg, Robert

    2014-01-01

    99mTc is the standard radionuclide used for nuclear medicine imaging. In addition to gamma irradiation, 99mTc emits low-energy Auger and conversion electrons that deposit their energy within nanometers of the decay site. To study the potential for DNA damage, direct DNA binding is required. Plasmid DNA enables the investigation of the unprotected interactions between molecules and DNA that result in single-strand breaks (SSBs) or double-strand breaks (DSBs); the resulting DNA fragments can be separated by gel electrophoresis and quantified by fluorescent staining. This study aimed to compare the plasmid DNA damage potential of a 99mTc-labeled HYNIC-DAPI compound with that of 99mTc pertechnetate (99mTcO4−). pUC19 plasmid DNA was irradiated for 2 or 24 hours. Direct and radical-induced DNA damage were evaluated in the presence or absence of the radical scavenger DMSO. For both compounds, an increase in applied activity enhanced plasmid DNA damage, which was evidenced by an increase in the open circular and linear DNA fractions and a reduction in the supercoiled DNA fraction. The number of SSBs elicited by 99mTc-HYNIC-DAPI (1.03) was twice that caused by 99mTcO4− (0.51), and the number of DSBs increased fivefold in the 99mTc-HYNIC-DAPI-treated sample compared with the 99mTcO4− treated sample (0.02 to 0.10). In the presence of DMSO, the numbers of SSBs and DSBs decreased to 0.03 and 0.00, respectively, in the 99mTcO4– treated samples, whereas the numbers of SSBs and DSBs were slightly reduced to 0.95 and 0.06, respectively, in the 99mTc-HYNIC-DAPI-treated samples. These results indicated that 99mTc-HYNIC-DAPI induced SSBs and DSBs via a direct interaction of the 99mTc-labeled compound with DNA. In contrast to these results, 99mTcO4− induced SSBs via radical formation, and DSBs were formed by two nearby SSBs. The biological effectiveness of 99mTc-HYNIC-DAPI increased by approximately 4-fold in terms of inducing SSBs and by approximately 10-fold in terms of

  4. HU-GFP and DAPI co-localize on the Escherichia coli nucleoid.

    PubMed

    Wery, M; Woldringh, C L; Rouviere-Yaniv, J

    2001-02-01

    The heterodimeric HU protein, one of the most abundant DNA binding proteins, plays a pleiotropic role in bacteria. Among others, HU was shown to contribute to the maintenance of DNA superhelical density in Escherichia coli. By its properties HU shares some traits with histones and HMG proteins. More recently, its specific binding to DNA recombination and repair intermediates suggests that HU should be considered as a DNA damage sensor. For all these reasons, it will be of interest to follow the localization of HU within the living bacterial cells. To this end, we constructed HU-GFP fusion proteins and compared by microscopy the GFP green fluorescence with images of the nucleoid after DAPI staining. We show that DAPI and HU-GFP colocalize on the E. coli nucleoid. HU, therefore, can be considered as a natural tracer of DNA in the living bacterial cell.

  5. Pretreatment cognitive and neural differences between sapropterin dihydrochloride responders and non-responders with phenylketonuria.

    PubMed

    Hawks, Zoë; Shimony, Joshua; Rutlin, Jerrel; Grange, Dorothy K; Christ, Shawn E; White, Desirée A

    2017-09-01

    Sapropterin dihydrochloride (BH4) reduces phenylalanine (Phe) levels and improves white matter integrity in a subset of individuals with phenylketonuria (PKU) known as "responders." Although prior research has identified biochemical and genotypic differences between BH4 responders and non-responders, cognitive and neural differences remain largely unexplored. To this end, we compared intelligence and white matter integrity prior to treatment with BH4 in 13 subsequent BH4 responders with PKU, 16 subsequent BH4 non-responders with PKU, and 12 healthy controls. Results indicated poorer intelligence and white matter integrity in non-responders compared to responders prior to treatment. In addition, poorer white matter integrity was associated with greater variability in Phe across the lifetime in non-responders but not in responders. These results underscore the importance of considering PKU as a multi-faceted, multi-dimensional disorder and point to the need for additional research to delineate characteristics that predict response to treatment with BH4.

  6. Contact dermatitis due to 2,2'-azobis(2-amidinopropane) dihydrochloride: an outbreak in production workers.

    PubMed

    Takiwaki, H; Arase, S; Nakayama, H

    1998-07-01

    2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) is an azo compound which has been used as a radical chain initiator. The purpose of this study was to confirm contact sensitivity to AAPH in individuals who were engaged in the production of AAPH, and presented with prolonged eczematous eruptions mainly on exposed areas. Patch testing was carried out with AAPH (1 and 5% aq.) on 8 patients and 6 healthy volunteers who had never been exposed to this chemical. All patients showed a strong positive patch test reaction to this agent, while all control subjects showed negative results. Because this chemical has recently been used for studies on the oxidation of biological materials, not only production workers in the chemical industry but also medical researchers should avoid prolonged exposure to this agent.

  7. C-Banding/DAPI and in situ hybridization reflect karyotype structure and sex chromosome differentiation in Humulus japonicus Siebold & Zucc.

    PubMed

    Grabowska-Joachimiak, A; Mosiolek, M; Lech, A; Góralski, G

    2011-01-01

    Japanese hop (Humulus japonicus Siebold & Zucc.) was karyotyped by chromosome measurements, fluorescence in situ hybridization with rDNA and telomeric probes, and C-banding/DAPI. The karyotype of this species consists of sex chromosomes (XX in female and XY1Y2 in male plants) and 14 autosomes difficult to distinguish by morphology. The chromosome complement also shows a rather monotonous terminal distribution of telomeric repeats, with the exception of a pair of autosomes possessing an additional cluster of telomeric sequences located within the shorter arm. Using C-banding/DAPI staining and 5S and 45S rDNA probes we constructed a fluorescent karyotype that can be used to distinguish all autosome pairs of this species except for the 2 largest autosome pairs, lacking rDNA signals and having similar size and DAPI-banding patterns. Sex chromosomes of H. japonicus display a unique banding pattern and different DAPI fluorescence intensity. The X chromosome possesses only one brightly stained AT-rich terminal segment, the Y1 has 2 such segments, and the Y2 is completely devoid of DAPI signal. After C-banding/DAPI, both Y chromosomes can be easily distinguished from the rest of the chromosome complement by the increased fluorescence of their arms. We discuss the utility of these methods for studying karyotype and sex chromosome evolution in hops.

  8. Investigating antimalarial drug interactions of emetine dihydrochloride hydrate using CalcuSyn-based interactivity calculations

    PubMed Central

    Matthews, Holly; Deakin, Jon; Rajab, May; Idris-Usman, Maryam

    2017-01-01

    The widespread introduction of artemisinin-based combination therapy has contributed to recent reductions in malaria mortality. Combination therapies have a range of advantages, including synergism, toxicity reduction, and delaying the onset of resistance acquisition. Unfortunately, antimalarial combination therapy is limited by the depleting repertoire of effective drugs with distinct target pathways. To fast-track antimalarial drug discovery, we have previously employed drug-repositioning to identify the anti-amoebic drug, emetine dihydrochloride hydrate, as a potential candidate for repositioned use against malaria. Despite its 1000-fold increase in in vitro antimalarial potency (ED50 47 nM) compared with its anti-amoebic potency (ED50 26–32 uM), practical use of the compound has been limited by dose-dependent toxicity (emesis and cardiotoxicity). Identification of a synergistic partner drug would present an opportunity for dose-reduction, thus increasing the therapeutic window. The lack of reliable and standardised methodology to enable the in vitro definition of synergistic potential for antimalarials is a major drawback. Here we use isobologram and combination-index data generated by CalcuSyn software analyses (Biosoft v2.1) to define drug interactivity in an objective, automated manner. The method, based on the median effect principle proposed by Chou and Talalay, was initially validated for antimalarial application using the known synergistic combination (atovaquone-proguanil). The combination was used to further understand the relationship between SYBR Green viability and cytocidal versus cytostatic effects of drugs at higher levels of inhibition. We report here the use of the optimised Chou Talalay method to define synergistic antimalarial drug interactivity between emetine dihydrochloride hydrate and atovaquone. The novel findings present a potential route to harness the nanomolar antimalarial efficacy of this affordable natural product. PMID:28257497

  9. Tandem repeat DNA localizing on the proximal DAPI bands of chromosomes in Larix, Pinaceae.

    PubMed

    Hizume, Masahiro; Shibata, Fukashi; Matsumoto, Ayako; Maruyama, Yukie; Hayashi, Eiji; Kondo, Teiji; Kondo, Katsuhiko; Zhang, Shozo; Hong, Deyuan

    2002-08-01

    Repetitive DNA was cloned from HindIII-digested genomic DNA of Larix leptolepis. The repetitive DNA was about 170 bp long, had an AT content of 67%, and was organized tandemly in the genome. Using fluorescence in situ hybridization and subsequent DAPI banding, the repetitive DNA was localized in DAPI bands at the proximal region of one arm of chromosomes in L. leptolepis and Larix chinensis. Southern blot hybridization to genomic DNA of seven species and five varieties probed with cloned repetitive DNA showed that the repetitive DNA family was present in a tandem organization in genomes of all Larix taxa examined. In addition to the 170-bp sequence, a 220-bp sequence belonging to the same DNA family was also present in 10 taxa. The 220-bp repeat unit was a partial duplication of the 170-bp repeat unit. The 220-bp repeat unit was more abundant in L. chinensis and Larix potaninii var. macrocarpa than in other taxa. The repetitive DNA composed 2.0-3.4% of the genome in most taxa and 0.3 and 0.5% of the genome in L. chinensis and L. potaninii var. macrocarpa, respectively. The unique distribution of the 220-bp repeat unit in Larix indicates the close relationship of these two species. In the family Pinaceae, the LPD (Larix proximal DAPI band specific repeat sequence family) family sequence is widely distributed, but their amount is very small except in the genus Larix. The abundant LPD family in Larix will occur after its speciation.

  10. DNA interaction with DAPI fluorescent dye: Force spectroscopy decouples two different binding modes.

    PubMed

    Reis, L A; Rocha, M S

    2017-05-01

    In this work, we use force spectroscopy to investigate the interaction between the DAPI fluorescent dye and the λ-DNA molecule under high (174 mM) and low (34 mM) ionic strengths. Firstly, we have measured the changes on the mechanical properties (persistence and contour lengths) of the DNA-DAPI complexes as a function of the dye concentration in the sample. Then, we use recently developed models in order to connect the behavior of both mechanical properties to the physical chemistry of the interaction. Such analysis has allowed us to identify and to decouple two main binding modes, determining the relevant physicochemical (binding) parameters for each of these modes: minor groove binding, which saturates at very low DAPI concentrations ( CT ∼ 0.50 μM) and presents equilibrium binding constants of the order of ∼10(7) M(-1) for the two ionic strengths studied; and intercalation, which starts to play a significant role only after the saturation of the first mode, presenting much smaller equilibrium binding constants (∼10(5) M(-1) ).

  11. Light-Triggered Release of DNA from Plasmon-Resonant Nanoparticles

    NASA Astrophysics Data System (ADS)

    Huschka, Ryan

    Plasmon-resonant nanoparticle complexes show promising potential for lighttriggered, controllable delivery of deoxyribonucleic acids (DNA) for research and therapeutic purposes. For example, the approach of RNA interference (RNAi) . using antisense DNA or RNA oligonucleotides to silence activity of a specific pathogenic gene transcript and reduce expression of the encoded protein . is very useful in dissecting genetic function and holds promise as a molecular therapeutic. Herein, we investigate the mechanism and probe the in vitro therapeutic potential of DNA light-triggered release from plasmonic nanoparticles. First, we investigate the mechanism of light-triggered release by dehybridizing double-stranded (dsDNA) via laser illumination from two types of nanoparticle substrates: gold (Au) nanoshells and Au nanorods. Both light-triggered and thermally induced releases are distinctly observable from nanoshell-based complexes. Surprisingly, no analogous measurable light-triggered release was observable from nanorod-based complexes below the DNA melting temperature. These results suggest that a nonthermal mechanism may play a role in light-triggered DNA release. Second, we demonstrate the in vitro light-triggered release of molecules noncovalently attached within dsDNA bound to the Au nanoshell surface. DAPI (4',6- diamidino-2-phenylindole), a bright blue fluorescent molecule that binds reversibly to double-stranded DNA, was chosen to visualize this intracellular light-induced release process. Illumination through the cell membrane of the nanoshell-dsDNA-DAPI complexes dehybridizes the DNA and releases the DAPI molecules within living cells. The DAPI molecules diffuse to the nucleus and associate with the cell's endogenous DNA. This work could have future applications towards drug delivery of molecules that associate with dsDNA. Finally, we demonstrate an engineered Au nanoshell (AuNS)-based therapeutic oligonucleotide delivery vehicle, designed to release its cargo on

  12. Automated Quantification of DNA Demethylation Effects in Cells via 3D Mapping of Nuclear Signatures and Population Homogeneity Assessment1

    PubMed Central

    Gertych, Arkadiusz; Wawrowsky, Kolja A.; Lindsley, Erik; Vishnevsky, Eugene; Farkas, Daniel L.; Tajbakhsh, Jian

    2009-01-01

    Background Today’s advanced microscopic imaging applies to the preclinical stages of drug discovery that employ high-throughput and high-content three-dimensional (3D) analysis of cells to more efficiently screen candidate compounds. Drug efficacy can be assessed by measuring response homogeneity to treatment within a cell population. In this study topologically quantified nuclear patterns of methylated cytosine and global nuclear DNA are utilized as signatures of cellular response to the treatment of cultured cells with the demethylating anti-cancer agents: 5-azacytidine (5-AZA) and octreotide (OCT). Methods Mouse pituitary folliculostellate TtT-GF cells treated with 5-AZA and OCT for 48 hours, and untreated populations, were studied by immunofluorescence with a specific antibody against 5-methylcytosine (MeC), and 4,6-diamidino-2-phenylindole (DAPI) for delineation of methylated sites and global DNA in nuclei (n=163). Cell images were processed utilizing an automated 3D analysis software that we developed by combining seeded watershed segmentation to extract nuclear shells with measurements of Kullback-Leibler’s (K-L) divergence to analyze cell population homogeneity in the relative nuclear distribution patterns of MeC versus DAPI stained sites. Each cell was assigned to one of the four classes: similar, likely similar, unlikely similar and dissimilar. Results Evaluation of the different cell groups revealed a significantly higher number of cells with similar or likely similar MeC/DAPI patterns among untreated cells (~100%), 5-AZA-treated cells (90%), and a lower degree of same type of cells (64%) in the OCT-treated population. The latter group contained (28%) of unlikely similar or dissimilar (7%) cells. Conclusion Our approach was successful in the assessment of cellular behavior relevant to the biological impact of the applied drugs, i.e. the reorganization of MeC/DAPI distribution by demethylation. In a comparison with other metrics, K-L divergence has

  13. Interaction of DAPI with individual strands of trinucleotide repeats. Effects of replication in vitro of the AAT x ATT triplet.

    PubMed

    Trotta, Edoardo; Del Grosso, Nicoletta; Erba, Maura; Melino, Sonia; Cicero, Daniel; Paci, Maurizio

    2003-12-01

    The structural changes produced by the minor-groove binding ligand DAPI (4',6-diamidine-2-phenylindole) on individual strands of trinucleotide repeat sequences were detected by electrophoretic band-shift analysis and related to their effects on DNA replication in vitro. Among the 20 possible single-stranded trinucleotide repeats, only the T-rich strand of the AAT.ATT triplet exhibits an observable fluorescence band and a change in electrophoretic mobility due to the drug binding. This is attributable to the property of DAPI that favours folding of the random coil ATT strand into a fast-migrating hairpin structure by a minor-groove binding mechanism. Electrophoretic characteristics of AAT, ACT, AGT, ATG and ATC are unchanged by DAPI, suggesting the crucial role of T.T with respect to A.A, C.C and G.G mismatch, in favouring the binding properties and the structural features of the ATT-DAPI complexes. Primer extension experiments, using the Klenow fragment of DNA polymerase I, demonstrate that such a selective structural change at ATT targets presents a marked property to stall DNA replication in vitro in comparison with the complementary AAT and a random GC-rich sequence. The results suggest a novel molecular mechanism of action of the DNA minor-groove binding ligand DAPI.

  14. DAPI-fluorescent fading: a problem in microscopy or a way to measure nuclear DNA content?

    NASA Astrophysics Data System (ADS)

    Gallardo-Escárate, Cristian; Álvarez-Borrego, Josué; Kober, V.; del Río-Portilla, Miguel Á.

    2006-01-01

    In observation by confocal or conventional fluorescence microscopy, the retardation of the lost in fluorescence, from highest signal of fluorescence to lowest intensity are important factors in order to obtain accurate images. This problem is very common in fluorochromes for nuclear DNA and especially for DAPI stain. The fluorescence of DAPI is rapidly lost when it is exposure to excitation by ultra violet (UV) light, and especially under optimal condition of observation. Although the fading process could be retardate by using of mounting medium with antifading solutions, the photochemical process underlying the fluorescence decay has not yet been fully explained. In addiction, neither relationship has been tested between the fluorescence fading and nuclear DNA content. However, the capacity of the DNA to absorb UV light is knows. In order to test this relationship we measured by means of image analysis the fluorescence intensity in several nuclei types during a fading period. The analysis was performed by an algorithm specifically built in MATLAB software. The relationship between nuclear DNA content and DAPI-fluorescence fading was found equal to 99%. This study demonstrates the feasibility for estimates genome size by quantification of fluorescence fading. In this context, the present method allows to measure nuclear DNA content in several medical applications (cancer, HIV, organ transplants, etc). Nowadays, for measuring DNA content, flow cytometry is widely used; however, with the flow cytometry method it is not possible to select a specific group of cells, such as from a specific region of a tumor. Moreover, the using of image analysis allows automatizing diagnostics procedures.

  15. Influence of phosphate and disinfection on the composition of biofilms produced from drinking water, as measured by fluorescence in situ hybridization.

    PubMed

    Batté, M; Mathieu, L; Laurent, P; Prévost, M

    2003-12-01

    Biofilms were grown in annular reactors supplied with drinking water enriched with 235 microg C/L. Changes in the biofilms with ageing, disinfection, and phosphate treatment were monitored using fluorescence in situ hybridization. EUB338, BET42a, GAM42a, and ALF1b probes were used to target most bacteria and the alpha (alpha), beta (beta), and gamma (gamma) subclasses of Proteobacteria, respectively. The stability of biofilm composition was checked after the onset of colonization between T = 42 days and T = 113 days. From 56.0% to 75.9% of the cells detected through total direct counts with DAPI (4'-6-diamidino-2-phenylindole) were also detected with the EUB338 probe, which targets the 16S rRNA of most bacteria. Among these cells, 16.9%-24.7% were targeted with the BET42a probe, 1.8%-18.3% with the ALF1b probe, and <2.5% with the GAM42a probe. Phosphate treatment induced a significant enhancement to the proportion of gamma-Proteobacteria (detected with the GAM42a probe), a group that contains many health-related bacteria. Disinfection with monochloramine for 1 month or chlorine for 3 days induced a reduction in the percentage of DAPI-stained cells that hybridized with the EUB338 probe (as expressed by percentages of EUB338 counts/DAPI) and with any of the ALF1b, BET42a, and GAM42a probes. The percentage of cells detected by any of the three probes (ALF1b+BET42a+GAM42a) tended to decrease, and reached in total less than 30% of the EUB338-hybridized cells. Disinfection with chlorine for 7 days induced a reverse shift; an increase in the percentage of EUB338 counts targeted by any of these three probes was noted, which reached up to 87%. However, it should be noted that the global bacterial densities (heterotrophic plate counts and total direct counts) tended to decrease over the duration of the experiment. Therefore, those bacteria that could be considered to resist 7 days of chlorination constituted a small part of the initial biofilm community, up to the point at

  16. Measuring topology of low-intensity DNA methylation sites for high-throughput assessment of epigenetic drug-induced effects in cancer cells

    SciTech Connect

    Gertych, Arkadiusz; Farkas, Daniel L.; Tajbakhsh, Jian

    2010-11-15

    Epigenetic anti-cancer drugs with demethylating effects have shown to alter genome organization in mammalian cell nuclei. The interest in the development of novel epigenetic drugs has increased the demand for cell-based assays to evaluate drug performance in pre-clinical studies. An imaging-based cytometrical approach that can measure demethylation effects as changes in the spatial nuclear distributions of methylated cytosine and global DNA in cancer cells is introduced in this paper. The cells were studied by immunofluorescence with a specific antibody against 5-methylcytosine (MeC), and 4,6-diamidino-2-phenylindole (DAPI) for delineation of methylated sites and global DNA in nuclei. In the preprocessing step the segmentation of nuclei in three-dimensional images (3-D) is followed by an automated assessment of nuclear DAPI/MeC patterns to exclude dissimilar entities. Next, low-intensity MeC (LIM) and low-intensity DNA (LID) sites of similar nuclei are localized and processed to obtain specific nuclear density profiles. These profiles sampled at half of the total nuclear volume yielded two parameters: LIM{sub 0.5} and LID{sub 0.5}. The analysis shows that zebularine and 5-azacytidine-the two tested epigenetic drugs introduce changes in the spatial distribution of low-intensity DNA and MeC signals. LIM{sub 0.5} and LID{sub 0.5} were significantly different (p < 0.001) in 5-azacytidine treated (n = 660) and zebularine treated (n = 496) vs. untreated (n = 649) DU145 human prostate cancer cells. In the latter case the LIM sites were predominantly found at the nuclear border, whereas treated populations showed different degrees of increase in LIMs towards the interior nuclear space, in which a large portion of heterochromatin is located. The cell-by-cell evaluation of changes in the spatial reorganization of MeC/DAPI signals revealed that zebularine is a more gentle demethylating agent than 5-azacytidine. Measuring changes in the topology of low-intensity sites can

  17. Bis-biguanide dihydrochloride inhibits intracellular replication of M. tuberculosis and controls infection in mice

    PubMed Central

    Shen, Hongbo; Wang, Feifei; Zeng, Gucheng; Shen, Ling; Cheng, Han; Huang, Dan; Wang, Richard; Rong, Lijun; Chen, Zheng W.

    2016-01-01

    While there is an urgent need to develop new and effective drugs for treatment of tuberculosis (TB) and multi-drug resistant TB (MDR-TB), repurposing FDA (U.S. Food and Drug Administration) -approved drugs for development of anti-TB agents may decrease time and effort from bench to bedside. Here, we employed host cell-based high throughput screening (HTS) assay to screen and characterize FDA-approved, off-patent library drugs for anti-Mycobacterium tuberculosis (MTB) activities. The cell-based HTS allowed us to identify an anti-cancer drug of bis-biguanide dihydrochloride (BBD) as potent anti-mycobacteria agent. Further characterization showed that BBD could inhibit intracellular and extracellular growth of M. smegmatis and slow-growing M. bovis BCG. BBD also potently inhibited replication of clinically-isolated MTB and MDR-TB strains. The proof-of-concept study showed that BBD treatment of MTB-infected mice could significantly decrease CFU counts in the lung and spleen. Notably, comparative evaluation showed that MTB CFU counts in BBD-treated mice were lower than those in rifampicin-treated mice. No apparent BBD side effects were found in BBD-treated mice. Thus, our findings support further studies to develop BBD as a new and effective drug against TB and MDR-TB. PMID:27601302

  18. PCL/PHBV Microparticles as Innovative Carriers for Oral Controlled Release of Manidipine Dihydrochloride

    PubMed Central

    Barboza, Fernanda Malaquias; Machado, Willian Moreira; Padilha de Paula, Josiane; Zawadzki, Sônia Faria

    2014-01-01

    Microparticles of poly(ε-caprolactone) (PCL) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) containing manidipine dihydrochloride (MAN) were successfully prepared by the simple emulsion/solvent evaporation method. All formulations showed loading efficiency rates greater than 80% and average particle size less than 8 μm. Formulations had spherical shape with smooth and porous surface for PCL and PHBV, respectively. According to Fourier-transform infrared spectroscopy, initial components were not chemically modified during microencapsulation. X-ray diffraction patterns and differential scanning calorimetry demonstrated that this process led to drug amorphization. In vitro dissolution studies showed that all microparticles prolonged MAN release, mainly which one obtained using PCL that contained 5% of drug loaded (PCL-M5). Animal studies demonstrated that formulation PCL-M5 was able to keep the variation of mean arterial pressure after phenylephrine administration up to 24 hours. These data confirmed the sustained antihypertensive effect of the investigated microparticles. Results provided an experimental basis for using formulation PCL-M5 as a feasible carrier for oral controlled release of MAN intended for treating high blood pressure. PMID:24550699

  19. Study on the interaction of levocetirizine dihydrochloride with human serum albumin by molecular spectroscopy

    NASA Astrophysics Data System (ADS)

    Liu, Xiangping; Du, Yingxiang; Sun, Wen; Kou, Junping; Yu, Boyang

    2009-12-01

    The interaction between cetirizine dihydrochloride and human serum albumin (HSA) has been examined by the spectroscopic techniques first. According to Stern-Volmer equation at different temperatures and the UV-vis spectra examination it was demonstrated that HSA fluorescence quenching initiated by levocetirizine was static. The values of binding constant ( KA) and the number of binding sites ( n) for levocetirizine and HSA were smaller than those for cetirizine and HSA, which meant that the transport of drug was regulated by the stereoselectivity of HSA to the enantiomer. The effect of the non-enzymatic glycosylation (NEG) on the interaction between levocetirizine and HSA signified that the administration of levocetirizine for diabetes should be different from the normal. The positive Δ S° and negative Δ H° indicated that ionic interaction played a major role between levocetirizine and HSA. Circular dichroism (CD) measurement showed that the secondary structure of HSA has changed in the presence of levocetirizine, and α-helical content decreased from 63.1% for free HSA to 54.9% for combined HSA, and accordingly the other secondary structure (β-strand, β-turns and others) contents increased to some extent. Finally, by the competitive binding experiments it was deduced that levocetirizine specifically bound to HSA in the region of site II, which meant the curative effect of levocetirizine should be reconsidered when it was administrated together with other site II drugs.

  20. Inactivation of Acinetobacter baumannii Biofilms on Polystyrene, Stainless Steel, and Urinary Catheters by Octenidine Dihydrochloride

    PubMed Central

    Narayanan, Amoolya; Nair, Meera S.; Karumathil, Deepti P.; Baskaran, Sangeetha A.; Venkitanarayanan, Kumar; Amalaradjou, Mary Anne Roshni

    2016-01-01

    Acinetobacter baumannii is a major nosocomial pathogen causing human infections with significant mortality rates. In most cases, infections are acquired through exposure to A. baumannii biofilms that persist on contaminated hospital equipment and surfaces. Thus, it is imperative to develop effective measures for controlling A. baumannii biofilms in nosocomial settings. This study investigated the efficacy of octenidine dihydrochloride (OH), a new generation disinfectant for reducing A. baumannii biofilms on polystyrene, stainless steel and catheters. OH at 0.3% (5 mM), 0.6% (10 mM), and 0.9% (15 mM) was effective in significantly inactivating A. baumannii biofilms on all tested surfaces (P < 0.05). Furthermore, OH was equally effective in inactivating biofilms of multidrug resistant and drug susceptible A. baumannii isolates. In addition, confocal imaging revealed the predominance of dead cells in the OH-treated samples in comparison to the control. Further, scanning electron microscopy of biofilms formed on catheters revealed that OH treatment significantly reduced A. baumannii biofilm populations in corroboration with our antibiofilm assay. These data underscore the efficacy of OH in inactivating A. baumannii biofilms, thereby suggesting its potential use as a disinfectant or a catheter lock solution to control A. baumannii infections. PMID:27375572

  1. PCL/PHBV microparticles as innovative carriers for oral controlled release of manidipine dihydrochloride.

    PubMed

    Barboza, Fernanda Malaquias; Machado, Willian Moreira; Olchanheski Junior, Luiz Renato; Padilha de Paula, Josiane; Zawadzki, Sônia Faria; Fernandes, Daniel; Farago, Paulo Vitor

    2014-01-01

    Microparticles of poly(ε-caprolactone) (PCL) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) containing manidipine dihydrochloride (MAN) were successfully prepared by the simple emulsion/solvent evaporation method. All formulations showed loading efficiency rates greater than 80% and average particle size less than 8 μm. Formulations had spherical shape with smooth and porous surface for PCL and PHBV, respectively. According to Fourier-transform infrared spectroscopy, initial components were not chemically modified during microencapsulation. X-ray diffraction patterns and differential scanning calorimetry demonstrated that this process led to drug amorphization. In vitro dissolution studies showed that all microparticles prolonged MAN release, mainly which one obtained using PCL that contained 5% of drug loaded (PCL-M5). Animal studies demonstrated that formulation PCL-M5 was able to keep the variation of mean arterial pressure after phenylephrine administration up to 24 hours. These data confirmed the sustained antihypertensive effect of the investigated microparticles. Results provided an experimental basis for using formulation PCL-M5 as a feasible carrier for oral controlled release of MAN intended for treating high blood pressure.

  2. Analysis of 2,2'-azobis (2-amidinopropane) dihydrochloride degradation and hydrolysis in aqueous solutions.

    PubMed

    Werber, Jay; Wang, Y John; Milligan, Michael; Li, Xiaohua; Ji, Junyan A

    2011-08-01

    2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH), a free radical-generating azo compound, is gaining prominence as a model oxidant in small molecule and protein therapeutics, namely for its ability to initiate oxidation reactions via both nucleophilic and free radical mechanisms. To better understand its degradation pathways, AAPH was degraded at 40°C in aqueous solutions over a wide pH range. Samples were analyzed via liquid chromatography-ultraviolet spectroscopy and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The thermal decomposition rate of AAPH to form radical species averaged 2.1 × 10(-6) s(-1) and did not vary significantly with pH. The hydrolysis rate increased exponentially with pH, showing hydroxide ion dependence. A mechanism for AAPH hydrolysis is proposed. The LC-MS/MS results provided evidence that the alkoxyl radical is a major radical species in solution. The LC-MS/MS results also showed a radical disproportionation reaction and enabled the generation of an overall reaction scheme showing the various side and termination products of AAPH degradation.

  3. Ultrastructural aspects of acute pancreatitis induced by 2, 2'-azobis (2-amidinopropane) dihydrochloride (AAPH) in rats.

    PubMed

    Tukaj, C; Olewniak-Adamowska, A; Pirski, M I; Woźniak, M

    2012-08-01

    Pathophysiology of acute pancreatitis (AP) has not been clearly established; nevertheless, accumulating evidence implicates highly reactive oxygen species (ROS) as important mediators of exocrine tissue damage. In this study, we used a water-soluble radical initiator, 2,2 -azobis-(2-amidinopropane) dihydrochloride (AAPH), to investigate the consequences of oxidative stress insult to the rat pancreas. The detailed characterisation of acini ultrastructural changes in the early course (3, 6, 12, 24 h) of AAPH-induced pancreatitis (40 mg/1 kg body weight) was performed. Considerable damage to the mitochondria in acinar cells manifested by increased translucence of the matrix, partial destruction of cristae, and formation of myelin figures were noted. At the same time, focal dilation, degranulation of rough endoplasmic reticulum, and reduced number of zymogen granules was observed. The most prominent ultrastructural feature was accumulation of highly polymorphic cytoplasmic vacuoles in acinar cells. Double membrane-bound autophagosomes, different in size and shape, with sequestered organelles, autophagolysosomes, and large, empty, single-membrane-bound vacuoles were observed within the cytoplasm. The results indicate that intensive and impaired autophagy mediates pathological accumulation of vacuoles in acinar cells. The rat model of acute pancreatitis induced by AAPH is useful to investigate the early events of oxidative stress insult to the pancreas.

  4. Protection by estrogens of biological damage by 2,2'-azobis(2-amidinopropane) dihydrochloride.

    PubMed

    Muraoka, Sanae; Miura, Toshiaki

    2002-11-01

    We examined by using 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) as a radical generator the ability of estrogens to scavenge carbon-centered and peroxyl radicals. Electron spin resonance signals of carbon-centered radicals from AAPH were diminished by catecholestrogens but not by phenolic estrogens, showing that catecholestrogens efficiently scavenged carbon-centered radicals. However, fluorescent decomposition of R-phycoerythrin by AAPH-derived peroxyl radicals was inhibited by catecholestrogens and phenolic estrogens. Evidently, peroxyl radicals were scavenged by catecholestrogens and by phenolic estrogens. However, the scavenging ability of 4-hydroxyestradiol was less than 2-hydroxyestradiol. Strand break of DNA induced by AAPH was inhibited by catecholestrogens, but not by phenolic estrogens under aerobic and anaerobic conditions. Inactivation of lysozyme induced by AAPH was completely blocked by 2-hydroxyestradiol under aerobic and anaerobic conditions, and by 4-hyroxyestradiol only under anaerobic conditions. Peroxidation of arachidonic acid by AAPH was strongly inhibited by catecholestrogens at low concentrations. Only large amounts of phenolic estrogens markedly inhibited lipid peroxidation. These results show that catecholestrogens were antioxidant against AAPH-induced damage to biological molecules through scavenging both carbon-centered and peroxyl radicals, but phenolic estrogens partially inhibited AAPH-induced damage because they scavenged only peroxyl radicals.

  5. Antioxidative activity of green tea treated with radical initiator 2, 2'-azobis(2-amidinopropane) dihydrochloride.

    PubMed

    Yokozawa, T; Cho, E J; Hara, Y; Kitani, K

    2000-10-01

    This study investigated the antioxidative activity of green tea extract, and a green tea tannin mixture and its components, under conditions of radical generation using the hydrophilic azo compound, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) to generate peroxyl radicals at a constant and measurable rate in the cultured renal epithelial cell line, LLC-PK(1), which is susceptible to oxidative damage. Treatment with AAPH decreased cell viability and increased the formation of thiobarbituric acid-reactive substances. However, green tea extract, and the tannin mixture and its components, comprising (-)-epigallocatechin 3-O-gallate (EGCg), (-)-gallocatechin 3-O-gallate (GCg), (-)-epicatechin 3-O-gallate (ECg), (-)-epigallocatechin (EGC), (+)-gallocatechin (GC), (-)-epicatechin (EC), and (+)-catechin (C), showed protective activity against AAPH-induced cellular damage. The tannin mixture and its components exhibited higher antioxidative activity than the green tea extract. Furthermore, EGCg and GCg had higher activity than EGC and GC, respectively. In particular, EGCg exerted the most significant cellular protective activity against AAPH. These results indicate that green tea tannin may inhibit cellular loss and lipid peroxidation resulting from the peroxyl radical generated by AAPH, and that the chemical structure of tannin is also involved in the activity, suggesting that the O-dihydroxy structure in the B ring and the galloyl groups are important determinants for radical scavenging and antioxidative potential.

  6. Oxidative modification of glutamine synthetase by 2,2'-azobis(2- amidinopropane) dihydrochloride.

    PubMed

    Ma, Y S; Chao, C C; Stadtman, E R

    1999-03-01

    In the present study, we examined the pattern of protein modification elicited by alkylperoxyl radicals and alkylperoxides. To this end, we exposed glutamine synthetase (GS) and the peptide melittin to solutions containing 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), which is known to decompose in aqueous, aerobic solutions to yield alkyl radicals and alkylperoxides. Under our conditions, pH 7.4, 37 degrees C, the AAPH-dependent formation of alkylhydroperoxide increased linearly with time and led to 40% inactivation of GS in 1 h and to complete inactivation in 4 h. Complete inactivation was associated with the loss of 2 of 16 histidine residues, 6 of 17 tyrosine residues, 5 of 16 methionine residues, and all of the tryptophan residues (2 residues) per subunit. Inactivation of GS was associated also with some protein fragmentation and the formation of some higher molecular weight aggregates. Exposure of GS to AAPH led also to the generation of protein carbonyl derivatives (0.34 mol/mol subunit) and to formation of a significant amount (0.038 mol/mol subunits) of quinoprotein derivatives. To investigate the mechanism of tryptophan modification, the 26-amino-acid peptide, melittin, which contains one tryptophan but no histidine, tyrosine, or methionine residues, was treated with AAPH. N-Formylkynurenine was identified as the major product of tryptophan oxidation in melittin.

  7. Betahistine Dihydrochloride With and Without Early Vestibular Rehabilitation for the Management of Patients With Balance Disorders Following Head Trauma: A Preliminary Randomized Clinical Trial

    PubMed Central

    Naguib, Maged B.; Madian, Yasser T.

    2014-01-01

    Objective The purpose of this study was to compare the effect of betahistine dihydrochloride alone and in combination with vestibular rehabilitation for the management of patients with balance disorders following head trauma. Methods In this preliminary clinical trial, a group of patients with head trauma was referred to our university-based tertiary care balance unit over a 1-year period. The study included 60 patients with balance disorder following head trauma. Patients were randomly divided into 3 groups with 20 patients each. The first group was treated by betahistine dihydrochloride tablets 48 mg/d alone. The second group was treated with a standard vestibular rehabilitation program. The third group was given betahistine dihydrochloride tablets (48 mg/d) in addition to the early standard vestibular rehabilitation program. Videonystagmography was used in the diagnosis, characterization, and monitoring of all patients with balance disorders, with improvement of the pretreatment objective results taken as a marker for treatment progress. Results Recovery time was within the first 3 months following head trauma in 57 (95%) of the patients. Recovery was faster after mild head trauma than after moderate and severe traumas. Patients who underwent vestibular rehabilitation immediately after the onset of head trauma (with or without addition of betahistine dihydrochloride) recovered earlier than those treated with betahistine dihydrochloride alone. Conclusion Based on these preliminary findings in a small group of patients, early vestibular rehabilitation with the concomitant use of betahistine dihydrochloride showed better results than the other 2 treatments alone in patients with balance disorders following head trauma. Early vestibular rehabilitation seemed to improve recovery that was enhanced by the use of betahistine dihydrochloride, and may have depressed the associated adverse effects such as nausea and vomiting. PMID:24711780

  8. [Physical localization of ribosomal genes and chromosome DAPI banding by in situ hybridization in Medicago sativa L].

    PubMed

    Chen, Jian-Min; Hong, Yi-Huan; Wang, You-Ping; Bowley, Steve; Wan, Jian-Min

    2006-02-01

    Physical localization of ribosomal genes in diploid and tetraploid alfalfa (Medicago. sativa) was studied using fluorescent in situ hybridization (FISH). It was revealed that 45s gene was only located at nucleolus organizer region (NOR) with a single locus in both diploid and tetraploid alfalfa, while 5s gene had 2-3 loci on chromosomes. Using the genomic DNA from M. coerulea and M. falcata as probe to hybridize with tetraploid species in alfalfa, both diploid species were successfully hybridized with tetraploid chromosomes, only showing the difference in hybridization signals in different numbers of chromosomes. Chromosomes of alfalfa exhibited DAPI banding by FISH analysis. In general, the patterns of distribution of DAPI banding were consistent with C-banding for M. coerulea. The possible origination of tetraploid alfalfa was discussed based on DAPI banding patterns and FISH analysis for ribosomal genes .

  9. High diversity in CMA3/DAPI-banding patterns in Heteropterans.

    PubMed

    Bardella, V B; Grazia, J; Fernandes, J A M; Vanzela, A L L

    2014-01-01

    Heteroptera is the most numerous and diverse suborder of Hemiptera, with about 38,000 species. This diversity also involves cytogenetic features, including chromosome number and a sex determining system. Information about heterochromatin occurrence and distribution is scarce in heteropterans, but still, there is some evidence of variability. We determined the chromosome number and CMA3/DAPI-banding pattern of 179 individuals of 25 heteropteran species from Brazil. Eight species of Pentatomidae exhibited a constant chromosome number (2n = 12 + XY), but in Coreidae (12 species), Largidae (1 species), Rhopalidae (1 species), and Pyrrhocoridae (3 species), the numbers ranged from 2n = 10 + 2m + X0 to 2n = 24 + 2m + X0. Although there were no large differences in the chromosome size between species, the CMA3/DAPI-banding patterns differed markedly. Among the genera, species of Edessa, Spartocera, Hypselonotus, Phtia,Holhymenia and Euryophthalmus showed a large accumulation of heterochromatin, while the other species exhibited few or no heterochromatic bands. In general, when heterochromatin was more accumulated, this occurred preferentially at terminal positions, except in Holhymenia histrio, which exhibited intercalary bands. This study made it possible to identify some chromosome rearrangements and to enhance our knowledge of the evolutionary mechanisms that determine karyotype differentiation in Heteroptera.

  10. High dose sapropterin dihydrochloride therapy improves monoamine neurotransmitter turnover in murine phenylketonuria (PKU).

    PubMed

    Winn, Shelley R; Scherer, Tanja; Thöny, Beat; Harding, Cary O

    2016-01-01

    Central nervous system (CNS) deficiencies of the monoamine neurotransmitters, dopamine and serotonin, have been implicated in the pathophysiology of neuropsychiatric dysfunction in phenylketonuria (PKU). Increased brain phenylalanine concentration likely competitively inhibits the activities of tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH), the rate limiting steps in dopamine and serotonin synthesis respectively. Tetrahydrobiopterin (BH4) is a required cofactor for TH and TPH activity. Our hypothesis was that treatment of hyperphenylalaninemic Pah(enu2/enu2) mice, a model of human PKU, with sapropterin dihydrochloride, a synthetic form of BH4, would stimulate TH and TPH activities leading to improved dopamine and serotonin synthesis despite persistently elevated brain phenylalanine. Sapropterin (20, 40, or 100mg/kg body weight in 1% ascorbic acid) was administered daily for 4 days by oral gavage to Pah(enu2/enu2) mice followed by measurement of brain biopterin, phenylalanine, tyrosine, tryptophan and monoamine neurotransmitter content. A significant increase in brain biopterin content was detected only in mice that had received the highest sapropterin dose, 100mg/kg. Blood and brain phenylalanine concentrations were unchanged by sapropterin therapy. Sapropterin therapy also did not alter the absolute amounts of dopamine and serotonin in brain but was associated with increased homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA), dopamine and serotonin metabolites respectively, in both wild type and Pah(enu2/enu2) mice. Oral sapropterin therapy likely does not directly affect central nervous system monoamine synthesis in either wild type or hyperphenylalaninemic mice but may stimulate synaptic neurotransmitter release and subsequent metabolism.

  11. Identification of all pachytene bivalents in the common shrew using DAPI-staining of synaptonemal complex spreads.

    PubMed

    Belonogova, N M; Karamysheva, T V; Biltueva, L S; Perepelov, E A; Minina, J M; Polyakov, A V; Zhdanova, N S; Rubtsov, N B; Searle, J B; Borodin, P M

    2006-01-01

    A major problem in studies of synaptonemal complexes (SC) is the difficulty in distinguishing individual chromosomes. This problem can be solved combining SC immunostaining with FISH of chromosome-specific sequences. However, this procedure is expensive, time-consuming and applicable only to a very limited number of species. In this paper we show how a combination of SC immunostaining and DAPI staining can allow identification of all chromosome arms in surface-spreads of the SC of the common shrew (Sorex araneus L.). Enhancement of brightness and contrast of the images with photo editing software allowed us to reveal clear DAPI-positive and negative bands with relative sizes and positions similar to DAPI landmarks on mitotic metaphase chromosomes. Using FISH with DNA probes prepared from chromosome arms m and n we demonstrated correct recognition of the chromosomes mp and hn on the basis of their DAPI pattern. We show that the approach we describe here may be applied to other species and can provide an important tool for identification of individual bivalents in pachytene surface-spreads.

  12. Bacterial biomass and activity in the marginal ice zone of the northern Barents Sea

    NASA Astrophysics Data System (ADS)

    Tammert, Helen; Olli, Kalle; Sturluson, Maria; Hodal, Helene

    2008-10-01

    Bacteria in the Arctic Waters are well adapted to low temperatures and play a key role in the transformation of organic matter. However, the activity of planktonic bacteria at cellular level remains poorly understood. In this study, we use fluorescent markers (4',6'-diamidino-2-phenylindole (DAPI), 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), Live/Dead BacLight viability kit) to discriminate between bacterial cells with a variety of physiological activities in the 0-200 m water column and sinking particles. During two field studies (July 2003 and 2004), we covered nine stations in the northern Barents Sea. The median bacterial abundance (DAPI staining) in the upper 50 m layer was 0.9×10 6 cells ml -1 (range 0.2-3.2×10 6 cells ml -1) in 2003 and 0.5×10 6 cells ml -1 (range 0.2-1.0×10 6 cells ml -1) in 2004. Bacteria with sufficient electron transport activity to be stained with CTC were on average 10% of the total count and ca. 20% of the total cells had intact cell membranes. In the water column, proxies of substrate availability (POC, PON, chlorophyll a, primary production) and bacterial production (thymidine and leucine uptake) correlated strongly with total bacterial count, CTC-stained cells and cells with 'leaky' membrane (stained with propidium iodine), but not with the concentration of cells with intact cell membrane. Contrary to expectations, the proportion of CTC-stained bacteria was not higher in the sinking particles (captured with sediment traps) compared to the ambient water. However, out of the bacteria with intact cell membranes, a higher proportion scored as CTC positive in the aggregates compared to the ambient water. Bacterial cells with 'leaky' cell membranes formed the largest part of total cell count in all samples, and accumulated in sites with high microbial activity (sinking aggregates, chlorophyll maxima, layers of high primary and bacterial production). We hypothesize that the source of the bacterial cells with 'leaky' cell membranes

  13. Biological Phosphorus Removal During High-Rate, Low-Temperature, Anaerobic Digestion of Wastewater

    PubMed Central

    Keating, Ciara; Chin, Jason P.; Hughes, Dermot; Manesiotis, Panagiotis; Cysneiros, Denise; Mahony, Therese; Smith, Cindy J.; McGrath, John W.; O’Flaherty, Vincent

    2016-01-01

    We report, for the first time, extensive biologically mediated phosphate removal from wastewater during high-rate anaerobic digestion (AD). A hybrid sludge bed/fixed-film (packed pumice stone) reactor was employed for low-temperature (12°C) anaerobic treatment of synthetic sewage wastewater. Successful phosphate removal from the wastewater (up to 78% of influent phosphate) was observed, mediated by biofilms in the reactor. Scanning electron microscopy and energy dispersive X-ray analysis revealed the accumulation of elemental phosphorus (∼2%) within the sludge bed and fixed-film biofilms. 4′, 6-diamidino-2-phenylindole (DAPI) staining indicated phosphorus accumulation was biological in nature and mediated through the formation of intracellular inorganic polyphosphate (polyP) granules within these biofilms. DAPI staining further indicated that polyP accumulation was rarely associated with free cells. Efficient and consistent chemical oxygen demand (COD) removal was recorded, throughout the 732-day trial, at applied organic loading rates between 0.4 and 1.5 kg COD m-3 d-1 and hydraulic retention times of 8–24 h, while phosphate removal efficiency ranged from 28 to 78% on average per phase. Analysis of protein hydrolysis kinetics and the methanogenic activity profiles of the biomass revealed the development, at 12°C, of active hydrolytic and methanogenic populations. Temporal microbial changes were monitored using Illumina MiSeq analysis of bacterial and archaeal 16S rRNA gene sequences. The dominant bacterial phyla present in the biomass at the conclusion of the trial were the Proteobacteria and Firmicutes and the dominant archaeal genus was Methanosaeta. Trichococcus and Flavobacterium populations, previously associated with low temperature protein degradation, developed in the reactor biomass. The presence of previously characterized polyphosphate accumulating organisms (PAOs) such as Rhodocyclus, Chromatiales, Actinobacter, and Acinetobacter was recorded

  14. Radiation Dosimetry via Automated Fluorescence Microscopy

    NASA Technical Reports Server (NTRS)

    Castleman, Kenneth R.; Schulze, Mark

    2005-01-01

    A developmental instrument for assessment of radiation-induced damage in human lymphocytes includes an automated fluorescence microscope equipped with a one or more chargecoupled- device (CCD) video camera(s) and circuitry to digitize the video output. The microscope is also equipped with a three-axis translation stage that includes a rotation stage, and a rotary tray that holds as many as thirty specimen slides. The figure depicts one version of the instrument. Once the slides have been prepared and loaded into the tray, the instrument can operate unattended. A computer controls the operation of the stage, tray, and microscope, and processes the digital fluorescence-image data to recognize and count chromosomes that have been broken, presumably by radiation. The design and method of operation of the instrument exploit fluorescence in situ hybridization (FISH) of metaphase chromosome spreads, which is a technique that has been found to be valuable for monitoring the radiation dose to circulating lymphocytes. In the specific FISH protocol used to prepare specimens for this instrument, metaphase lymphocyte cultures are chosen for high mitotic index and highly condensed chromosomes, then several of the largest chromosomes are labeled with three of four differently colored whole-chromosome-staining dyes. The three dyes, which are used both individually and in various combinations, are fluorescein isothiocyanate (FITC), Texas Red (or equivalent), and Cy5 (or equivalent); The fourth dye 4',6-diamidino- 2-phenylindole (DAPI) is used as a counterstain. Under control by the computer, the microscope is automatically focused on the cells and each slide is scanned while the computer analyzes the DAPI-fluorescence images to find the metaphases. Each metaphase field is recentered in the field of view and refocused. Then a four-color image (more precisely, a set of images of the same view in the fluorescent colors of the four dyes) is acquired. By use of pattern

  15. Biological Phosphorus Removal During High-Rate, Low-Temperature, Anaerobic Digestion of Wastewater.

    PubMed

    Keating, Ciara; Chin, Jason P; Hughes, Dermot; Manesiotis, Panagiotis; Cysneiros, Denise; Mahony, Therese; Smith, Cindy J; McGrath, John W; O'Flaherty, Vincent

    2016-01-01

    We report, for the first time, extensive biologically mediated phosphate removal from wastewater during high-rate anaerobic digestion (AD). A hybrid sludge bed/fixed-film (packed pumice stone) reactor was employed for low-temperature (12°C) anaerobic treatment of synthetic sewage wastewater. Successful phosphate removal from the wastewater (up to 78% of influent phosphate) was observed, mediated by biofilms in the reactor. Scanning electron microscopy and energy dispersive X-ray analysis revealed the accumulation of elemental phosphorus (∼2%) within the sludge bed and fixed-film biofilms. 4', 6-diamidino-2-phenylindole (DAPI) staining indicated phosphorus accumulation was biological in nature and mediated through the formation of intracellular inorganic polyphosphate (polyP) granules within these biofilms. DAPI staining further indicated that polyP accumulation was rarely associated with free cells. Efficient and consistent chemical oxygen demand (COD) removal was recorded, throughout the 732-day trial, at applied organic loading rates between 0.4 and 1.5 kg COD m(-3) d(-1) and hydraulic retention times of 8-24 h, while phosphate removal efficiency ranged from 28 to 78% on average per phase. Analysis of protein hydrolysis kinetics and the methanogenic activity profiles of the biomass revealed the development, at 12°C, of active hydrolytic and methanogenic populations. Temporal microbial changes were monitored using Illumina MiSeq analysis of bacterial and archaeal 16S rRNA gene sequences. The dominant bacterial phyla present in the biomass at the conclusion of the trial were the Proteobacteria and Firmicutes and the dominant archaeal genus was Methanosaeta. Trichococcus and Flavobacterium populations, previously associated with low temperature protein degradation, developed in the reactor biomass. The presence of previously characterized polyphosphate accumulating organisms (PAOs) such as Rhodocyclus, Chromatiales, Actinobacter, and Acinetobacter was recorded

  16. Bacterial Activity at -2 to -20 degrees C in Arctic wintertime sea ice.

    PubMed

    Junge, Karen; Eicken, Hajo; Deming, Jody W

    2004-01-01

    Arctic wintertime sea-ice cores, characterized by a temperature gradient of -2 to -20 degrees C, were investigated to better understand constraints on bacterial abundance, activity, and diversity at subzero temperatures. With the fluorescent stains 4',6'-diamidino-2-phenylindole 2HCl (DAPI) (for DNA) and 5-cyano-2,3-ditoyl tetrazolium chloride (CTC) (for O(2)-based respiration), the abundances of total, particle-associated (>3- micro m), free-living, and actively respiring bacteria were determined for ice-core samples melted at their in situ temperatures (-2 to -20 degrees C) and at the corresponding salinities of their brine inclusions (38 to 209 ppt). Fluorescence in situ hybridization was applied to determine the proportions of Bacteria, Cytophaga-Flavobacteria-Bacteroides (CFB), and Archaea. Microtome-prepared ice sections also were examined microscopically under in situ conditions to evaluate bacterial abundance (by DAPI staining) and particle associations within the brine-inclusion network of the ice. For both melted and intact ice sections, more than 50% of cells were found to be associated with particles or surfaces (sediment grains, detritus, and ice-crystal boundaries). CTC-active bacteria (0.5 to 4% of the total) and cells detectable by rRNA probes (18 to 86% of the total) were found in all ice samples, including the coldest (-20 degrees C), where virtually all active cells were particle associated. The percentage of active bacteria associated with particles increased with decreasing temperature, as did the percentages of CFB (16 to 82% of Bacteria) and Archaea (0.0 to 3.4% of total cells). These results, combined with correlation analyses between bacterial variables and measures of particulate matter in the ice as well as the increase in CFB at lower temperatures, confirm the importance of particle or surface association to bacterial activity at subzero temperatures. Measuring activity down to -20 degrees C adds to the concept that liquid inclusions in

  17. Automatic stage identification of Drosophila egg chamber based on DAPI images.

    PubMed

    Jia, Dongyu; Xu, Qiuping; Xie, Qian; Mio, Washington; Deng, Wu-Min

    2016-01-06

    The Drosophila egg chamber, whose development is divided into 14 stages, is a well-established model for developmental biology. However, visual stage determination can be a tedious, subjective and time-consuming task prone to errors. Our study presents an objective, reliable and repeatable automated method for quantifying cell features and classifying egg chamber stages based on DAPI images. The proposed approach is composed of two steps: 1) a feature extraction step and 2) a statistical modeling step. The egg chamber features used are egg chamber size, oocyte size, egg chamber ratio and distribution of follicle cells. Methods for determining the on-site of the polytene stage and centripetal migration are also discussed. The statistical model uses linear and ordinal regression to explore the stage-feature relationships and classify egg chamber stages. Combined with machine learning, our method has great potential to enable discovery of hidden developmental mechanisms.

  18. Pyrophosphate selective fluorescent chemosensors: cascade recognition of nuclear stain mimicking DAPI.

    PubMed

    Goswami, Shyamaprosad; Das, Avijit Kumar; Pakhira, Bholanath; Basu Roy, Sohini; Maity, Anup Kumar; Saha, Partha; Sarkar, Sabyasachi

    2014-09-07

    A new zinc(ii) complex with a condensed hydroxynaphthyl pyridine (SPHN) as the coordinated ligand has been synthesized for the selective recognition of pyrophosphate (PPi) over other anions including phosphate in a mixed aqueous solution. The fluorescence enhancement of SPHN in association with Zn(2+) ions is quenched in the presence of intracellular pyrophosphate. This phenomenon is utilized in the construction of a logic gate. The binding of SPHN with Zn(2+) and its displacement by PPi have been established by photophysical investigation and supported by the DFT level of studies. The development of blue fluorescence in the {} complex upon binding of zinc with is shown to be useful as a nucleus marker in a cell similar to the commercially available staining compound, DAPI (diamino-2-phenylindole).

  19. Automatic stage identification of Drosophila egg chamber based on DAPI images

    PubMed Central

    Jia, Dongyu; Xu, Qiuping; Xie, Qian; Mio, Washington; Deng, Wu-Min

    2016-01-01

    The Drosophila egg chamber, whose development is divided into 14 stages, is a well-established model for developmental biology. However, visual stage determination can be a tedious, subjective and time-consuming task prone to errors. Our study presents an objective, reliable and repeatable automated method for quantifying cell features and classifying egg chamber stages based on DAPI images. The proposed approach is composed of two steps: 1) a feature extraction step and 2) a statistical modeling step. The egg chamber features used are egg chamber size, oocyte size, egg chamber ratio and distribution of follicle cells. Methods for determining the on-site of the polytene stage and centripetal migration are also discussed. The statistical model uses linear and ordinal regression to explore the stage-feature relationships and classify egg chamber stages. Combined with machine learning, our method has great potential to enable discovery of hidden developmental mechanisms. PMID:26732176

  20. Fast Disintegrating Combination Tablet of Taste Masked Levocetrizine Dihydrochloride and Montelukast Sodium: Formulation Design, Development, and Characterization

    PubMed Central

    Gupta, M. M.; Gupta, Niraj; Chauhan, Bhupendra S.; Pandey, Shweta

    2014-01-01

    The aim of this study was to prepare fast disintegrating combination tablet of taste masked Levocetrizine dihydrochloride and Montelukast sodium by using direct compression method. To prevent bitter taste and unacceptable odour of the Levocetrizine dihydrochloride drug, the drug was taste masked with ion exchange resins like Kyron-T-104 and Tulsion-412. Among the two resins, Kyron-T-104 was selected for further studies because of high drug loading capacity, low cost, and better drug release profile. An ion exchange resin complex was prepared by the batch technique and various parameters; namely, resin activation, drug: resin ratio, pH, temperature, and stirring time, and swelling time were optimized to successfully formulate the tasteless drug resin complex (DRC). The tablets were prepared using microcrystalline cellulose (MCC) PH 102 as diluent along with crospovidone (CP), croscarmellose sodium (CCM), and sodium starch glycolate (SSG) as a superdisintegrants. The tablets were evaluated for weight variation, hardness, friability, wetting time, water absorption ratio, disintegration time (DT), and dissolution study and it was concluded that the tablet formulation prepared with 2% SSG + CCS showed better disintegration time in comparison with other formulation and good drug release. The stability studies were carried out for the optimized batch for three months and it showed acceptable results. PMID:26556198

  1. High-dosage betahistine dihydrochloride between 288 and 480 mg/day in patients with severe Menière's disease: a case series.

    PubMed

    Lezius, Franziska; Adrion, Christine; Mansmann, Ulrich; Jahn, Klaus; Strupp, Michael

    2011-08-01

    The objective of this study was to evaluate the clinical benefit and the side effects of high dosages of betahistine dihydrochloride (288-480 mg/day) in patients with severe Menière's disease (MD). In this case series 11 patients with MD who had not responded sufficiently to a dosage of 144 mg/day of betahistine dihydrochloride were treated on an individual basis with daily dosages between 288 and 480 mg of betahistine dihydrochloride. The number of attacks per month and the side effects were monitored. Non-parametric tests were used for statistical analysis. As a result, the frequency and the severity of vertigo were significantly reduced in all patients. The side effects were mild, self-limiting, and did not require any change in the treatment strategy. Despite the considerable limitations of an observational study--in particular in MD--high dosages of betahistine dihydrochloride between 288 and 480 mg/day seem to be effective in patients who do not sufficiently respond to lower dosages. Moreover, such dosages are well tolerated.

  2. Synthesis and biological evaluation of CHX-DAPYs as HIV-1 non-nucleoside reverse transcriptase inhibitors.

    PubMed

    Yan, Zi-Hong; Wu, Hai-Qiu; Chen, Wen-Xue; Wu, Yan; Piao, Hu-Ri; He, Qiu-Qin; Chen, Fen-Er; De Clercq, Erik; Pannecouque, Christophe

    2014-06-15

    A series of new diarylpyrimidines (DAPYs) characterized by a halogen atom on the methylene linker between wing I and the central pyrimidine ring was synthesized and evaluated for their anti-HIV activity in MT-4 cell cultures. The two most promising compounds 7f and 7g showed excellent activity against wild-type HIV-1 with low nanomolar EC50 values of 0.005 and 0.009 μM, respectively, which were comparable to or more potent than all the reference drugs zidovudine (AZT), lamivudine (3TC), nevirapine (NEV), efavirenz (EFV), delaviridine (DLV) and etravirine (ETV). In particular, 7g also displayed strong activity against the double mutant strain 103N + 181C with an EC50 value of 8.2 μM. The preliminary structure-activity relationship (SAR) and molecular docking analysis of this new series of CHX-DAPYs were also investigated.

  3. Derivatives of 1-naphthyl ethylenediamine dihydrochloride for standardization of direct-bilirubin assays done with the Technicon "SMAC".

    PubMed

    Furda, J; Morgenstern, S; Snyder, L R

    1976-07-01

    The Jendrassik--Groff assay for direct bilirubin was adapted for analysis rates of 150/h on the Technicon "SMAC" continuous-flow analyzer. This requires development of a standard that is compatible with the other 19 channels on this analyzer. N-1-Naphthyl ethylenediamine dihydrochloride has been used for standardization of direct bilirubin assays, but we found it to be unsuitable because values for potassium are falsely elevated when potassium is determined with a valinomycin ion-selective electrode. This interference can be eliminated by alkylating the aliphatic amine group in the standard. The resulting compounds undergo the coupling reaction in the same way as the original compound and function equally well as standards for the direct bilirubin reaction. The only limitation of these analogs is their decreased solubility at physiological pH in some cases. Thus only certain alkyl groups are suitable.

  4. Development of a strategy for biological monitoring in a chemical plant producing 3,3'-dichlorobenzidine dihydrochloride.

    PubMed

    Knoell, Kristian F; Will, Norbert; Leng, Gabriele; Selinski, Silvia; Hengstler, Jan G; Golka, Klaus; Bolt, Hermann M

    2012-01-01

    In a chemical plant in Germany producing 3,3'-dichlorobenzidine dihydrochloride for the manufacture of colorants, blood and urine samples were taken for biological monitoring. 3,3'-Dichlorobenzidine (DBZ) was analyzed in urine by thin-layer chromatography and subsequently further combined with analysis of adducts of 3,3'-DBZ in hemoglobin. Data highlight current ranges of industrial exposure to 3,3'-DBZ in Germany and demonstrate the applicability of biological monitoring to minimize this exposure. Effective biological monitoring was achieved by a combination of monitoring hemoglobin adducts with spot samplings of urinary 3,3'-DBZ excretion in cases of reported exposure periods. Data presented might help to identify biological guidance values (BGV/BAR) for 3,3'-DBZ-exposed individuals.

  5. Single molecule localization microscopy of the distribution of chromatin using Hoechst and DAPI fluorescent probes

    PubMed Central

    Szczurek, Aleksander T; Prakash, Kirti; Lee, Hyun-Keun; Żurek-Biesiada, Dominika J; Best, Gerrit; Hagmann, Martin; Dobrucki, Jurek W; Cremer, Christoph; Birk, Udo

    2014-01-01

    Several approaches have been described to fluorescently label and image DNA and chromatin in situ on the single-molecule level. These superresolution microscopy techniques are based on detecting optically isolated, fluorescently tagged anti-histone antibodies, fluorescently labeled DNA precursor analogs, or fluorescent dyes bound to DNA. Presently they suffer from various drawbacks such as low labeling efficiency or interference with DNA structure. In this report, we demonstrate that DNA minor groove binding dyes, such as Hoechst 33258, Hoechst 33342, and DAPI, can be effectively employed in single molecule localization microscopy (SMLM) with high optical and structural resolution. Upon illumination with low intensity 405 nm light, a small subpopulation of these molecules stochastically undergoes photoconversion from the original blue-emitting form to a green-emitting form. Using a 491 nm laser excitation, fluorescence of these green-emitting, optically isolated molecules was registered until “bleached”. This procedure facilitated substantially the optical isolation and localization of large numbers of individual dye molecules bound to DNA in situ, in nuclei of fixed mammalian cells, or in mitotic chromosomes, and enabled the reconstruction of high-quality DNA density maps. We anticipate that this approach will provide new insights into DNA replication, DNA repair, gene transcription, and other nuclear processes. PMID:25482122

  6. Single molecule localization microscopy of the distribution of chromatin using Hoechst and DAPI fluorescent probes.

    PubMed

    Szczurek, Aleksander T; Prakash, Kirti; Lee, Hyun-Keun; Zurek-Biesiada, Dominika J; Best, Gerrit; Hagmann, Martin; Dobrucki, Jurek W; Cremer, Christoph; Birk, Udo

    2014-01-01

    Several approaches have been described to fluorescently label and image DNA and chromatin in situ on the single-molecule level. These superresolution microscopy techniques are based on detecting optically isolated, fluorescently tagged anti-histone antibodies, fluorescently labeled DNA precursor analogs, or fluorescent dyes bound to DNA. Presently they suffer from various drawbacks such as low labeling efficiency or interference with DNA structure. In this report, we demonstrate that DNA minor groove binding dyes, such as Hoechst 33258, Hoechst 33342, and DAPI, can be effectively employed in single molecule localization microscopy (SMLM) with high optical and structural resolution. Upon illumination with low intensity 405 nm light, a small subpopulation of these molecules stochastically undergoes photoconversion from the original blue-emitting form to a green-emitting form. Using a 491 nm laser excitation, fluorescence of these green-emitting, optically isolated molecules was registered until "bleached". This procedure facilitated substantially the optical isolation and localization of large numbers of individual dye molecules bound to DNA in situ, in nuclei of fixed mammalian cells, or in mitotic chromosomes, and enabled the reconstruction of high-quality DNA density maps. We anticipate that this approach will provide new insights into DNA replication, DNA repair, gene transcription, and other nuclear processes.

  7. Analysis of the Trypanosoma brucei cell cycle by quantitative DAPI imaging.

    PubMed

    Siegel, T Nicolai; Hekstra, Doeke R; Cross, George A M

    2008-08-01

    Trypanosoma brucei has two DNA compartments: the nucleus and the kinetoplast. DNA replication of these two compartments only partially coincides. Woodward and Gull [Woodward R, Gull K. Timing of nuclear and kinetoplast DNA replication and early morphological events in the cell cycle of Trypanosoma brucei. J Cell Sci 1990;95:49-57] comprehensively studied the relative timing of the replication and segregation of nuclear DNA (nDNA) and kinetoplast DNA (kDNA). Others have since assumed the consistency of morphological indicators of cell-cycle stage among strains and conditions. We report the use of quantitative DAPI imaging to determine the cell-cycle stage of individual procyclic cells. Using this approach, we found that kinetoplast elongation occurs mainly during nuclear S phase and not during G2, as previously assumed. We confirmed this finding by sorting cells by DNA content, followed by fluorescence microscopy. In addition, simultaneous quantitative imaging at two wavelengths can be used to determine the abundance of cell-cycle-regulated proteins during the cell cycle. We demonstrate this technique by co-staining for the non-acetylated state of lysine 4 of histone H4 (H4K4), which is enriched during nuclear S phase.

  8. Analysis of the Trypanosoma brucei cell cycle by quantitative DAPI imaging

    PubMed Central

    Siegel, T. Nicolai; Hekstra, Doeke R.; Cross, George A. M.

    2008-01-01

    Trypanosoma brucei has two DNA compartments: the nucleus and the kinetoplast. DNA replication of these two compartments only partially coincides. Woodward and Gull [J Cell Sci 1990;95:49-57] comprehensively studied the relative timing of the replication and segregation of nuclear and kinetoplast DNA. Others have since assumed the consistency of morphological indicators of cell-cycle stage among strains and conditions. We report the use of quantitative DAPI imaging to determine the cell-cycle stage of individual procyclic cells. Using this approach, we found that kinetoplast elongation occurs mainly during nuclear S phase and not during G2, as previously assumed. We confirmed this finding by sorting cells by DNA content, followed by fluorescence microscopy. In addition, simultaneous quantitative imaging at two wavelengths can be used to determine the abundance of cell-cycle-regulated proteins during the cell cycle. We demonstrate this technique by co-staining for the non-acetylated state of lysine 4 of histone H4 (H4K4), which is enriched during nuclear S phase. PMID:18501977

  9. Oxidation kinetics of manganese (II) in seawater at nanomolar concentrations

    NASA Astrophysics Data System (ADS)

    von Langen, Peter J.; Johnson, Kenneth S.; Coale, Kenneth H.; Elrod, Virginia A.

    1997-12-01

    Manganese oxidation rates were determined at low (˜ 20 nM) concentrations in seawater by measuring dissolved manganese (Mn(II)) using flow injection analysis with chemiluminescence detection. Mn(II) was measured in samples that had been filtered (0.2 μm) and kept in the dark under controlled temperature and pH conditions for time periods up to 6 months. Eight 9 L carboys with mean pH values ranging from 8 to 8.7 were held at 25°C, another carboy (pH = 9.32) was kept at 5°C. Oxidation followed the Morgan (1967) homogeneous rate equation ( d[Mn(II)]/ dt = k1 [O 2][OH -] 2[Mn(II)]). The mean rate constant k1 = 1.7 ± 0.7 × 10 12 M -3 d -1 (95% CI), determined using hydroxide ion activities determined with pH measurements on the NBS scale, was in agreement with work by Morgan (1967; k1 = 4 × 10 12 M -3 d -1) and Davies and Morgan (1989; k1 = 1.1 × 10 12 M -3 d -1) in dilute solutions. The rate constant at 5°C was 1.3 ± 0.3 × 10 12 M -3 d -1. If free hydroxide concentrations (based on the free proton pH scale) are used, then the rate constant at 25°C was k 1∗ = 0.34 ± 0.14 × 10 12 M -3d -1. Autocatalytic increases in Mn(II) oxidation rates, as predicted by a heterogeneous reaction mechanism (Morgan, 1967) ( d[Mn(II)]/ dt= k2'[Mn(II)][MnO 2]) were not observed, indicating that the homogeneous reaction dominates Mn(II) oxidation at low nM concentrations in seawater. Bacteria were enumerated by 4',6-diamidino-2-phenylindole (DAPI) staining during the experiments. No significant correlation between bacterial concentrations and Mn(II) oxidation rates was found.

  10. Karyotypic similarity among Barycholos ternetzi and five species of the genus Eleutherodactylus from southeastern Brazil (Anura, Brachycephalidae).

    PubMed

    Campos, J R C; Ananias, Fernando; Haddad, Célio Fernando Baptista; Kasahara, Sanae

    2008-01-01

    Comparative cytogenetic analyses were carried out in six species of Brachycephalidae from southeastern Brazil. Barycholos ternetzi, Eleutherodactylus binotatus, Eleutherodactylus guentheri, Eleutherodactylus juipoca, Eleutherodactylus parvus and Eleutherodactylus sp. have 2n=22 karyotypes with a marked variation in the morphology of chromosome pairs 8, 10 and 11, which are of telocentric or metacentric types, resulting in FN=38, 40 and 44. Eleutherodactylus have a single chromosome pair bearing Ag-NOR, i.e. pair 1 in E. binotatus, pair 6 in E. guentheri and E. parvus, and pair 11 in E. juipoca and Eleutherodactylus sp. In contrast, B. ternetzi showed Ag-positive sites in the chromosome pairs 1, 4, 5, 9 and 11, and only one to three labelings per metaphase in each individual. Nevertheless, the main chromosome pair with Ag-NOR in the species seems to be the 11th, like in E. juipoca and Eleutherodactylus sp. The NOR site was confirmed by fluorescence in situ hybridization (FISH) technique in E. binotatus and in B. ternetzi, bearing 1p1p and 9p11p11p Ag-NOR pattern, respectively. All the species exhibited predominantly centromeric C-banding pattern, but interstitial bands have also been observed in some cases. In E. binotatus, there is an indication of geographical difference in the distribution of the interstitial C-bands. The fluorochromes GC-specific chromomycin A(3) (CMA(3)) and AT-specific 4',6-diamidino-2-phenylindole (DAPI), with distamycin A (DA) counterstaining, provided the molecular content of some repetitive regions in the karyotypes of the species. One male of E. binotatus presented an extensive heteromorphism, involving at least five different pairs, probably as a consequence of multiple reciprocal translocations. Such rearrangements might be responsible for the multivalent chain seen in the meiosis of this specimen, as well as in another male, although not exhibiting chromosome heteromorphism. The remaining males and those belonging to the other species

  11. Studies on the mechanism of action of antitumor bis(aminophenolate) ruthenium(III) complexes.

    PubMed

    Dömötör, Orsolya; de Almeida, Rodrigo F M; Côrte-Real, Leonor; Matos, Cristina P; Marques, Fernanda; Matos, António; Real, Carla; Kiss, Tamás; Enyedy, Éva Anna; Helena Garcia, M; Tomaz, Ana Isabel

    2017-03-01

    Two recently published Ru(III) complexes bearing (N2O2) tetradentate bis(aminophenolate) ligands, formulated as [Ru(III)(salan)(PPh3)Cl] (salan is the tetradentate ligand 6,6'-(1S,2S)-cyclohexane-1,2-diylbis(azanediyl)bis(methylene)bis(3-methoxyphenol) in complex 1, or 2,2'-(1S,2S)-cyclohexane-1,2-diylbis(azanediyl)bis(methylene)bis(4-methoxyphenol) in complex 2; PPh3 is triphenylphosphane) and found very active against ovarian and breast adenocarcinoma human cells were studied to outline their antitumor mode of action. The human cisplatin-sensitive ovarian adenocarcinoma line A2780 was used herein as the cell model. At a 24h challenge (similarly as found before for 72h) both complexes are active, their cytotoxicity being comparable to that of cisplatin in the same conditions. As a possible target in the cell for their action, the interaction of 1 and 2 with DNA was assessed through displacement of well-established DNA fluorescent probes (ethidium bromide, EB, and 4',6-diamidino-2-phenylindole, DAPI) through steady-state and time-resolved fluorescence spectroscopy. The whole emission spectra were analyzed globally for the binary DNA-probe and ternary DNA-probe-Ru(III) complex systems. Both Ru(III) complexes can displace EB and bind to DNA with similar and moderate strong affinity with conditional stability constants of logK'=(5.05±0.01) for 1 and logK'=(4.79±0.01) for 2. The analysis of time-domain fluorescence intensity decays confirmed both qualitatively and quantitatively the model used to describe the binding and competition processes. Cell studies indicated that apoptosis is the major mechanism of cell death for both complexes, with 2 (the more active complex) promoting that process more efficiently than 1. Transmission electron micrographs revealed clear alterations on intracellular organization consistent with the induction of programmed cell death processes.

  12. Glacier inputs influence organic matter composition and prokaryotic distribution in a high Arctic fjord (Kongsfjorden, Svalbard)

    NASA Astrophysics Data System (ADS)

    Bourgeois, Solveig; Kerhervé, Philippe; Calleja, Maria Ll.; Many, Gaël; Morata, Nathalie

    2016-12-01

    With climate change, the strong seasonality and tight pelagic-benthic coupling in the Arctic is expected to change in the next few decades. It is currently unclear how the benthos will be affected by changes of environmental conditions such as supplies of organic matter (OM) from the water column. In the last decade, Kongsfjorden (79°N), a high Arctic fjord in Svalbard influenced by several glaciers and Atlantic water inflow, has been a site of great interest owing to its high sensitivity to climate change, evidenced by a reduction in ice cover and an increase in melting freshwater. To investigate how spatial and seasonal changes in vertical fluxes can impact the benthic compartment of Kongsfjorden, we studied the organic matter characteristics (in terms of quantity and quality) and prokaryotic distribution in sediments from 3 stations along a transect extending from the glacier into the outer fjord in 4 different seasons (spring, summer, autumn and winter) in 2012-2013. The biochemical parameters used to describe the sedimentary organic matter were organic carbon (OC), total nitrogen, bulk stable isotope ratios, pigments (chorophyll-a and phaeopigments) and biopolymeric carbon (BPC), which is the sum of the main macromolecules, i.e. lipids, proteins and carbohydrates. Prokaryotic abundance and distribution were estimated by 4‧,6-diamidino-2-phenylindole (DAPI) staining. This study identifies a well-marked quantitative gradient of biogenic compounds throughout all seasons and also highlights a discrepancy between the quantity and quality of sedimentary organic matter within the fjord. The sediments near the glacier were organic-poor (< 0.3%OC), however the high primary productivity in the water column displayed during spring was reflected in summer sediments, and exhibited higher freshness of material at the inner station compared to the outer basin (means C-chlorophyll-a/OC 5 and 1.5%, respectively). However, sediments at the glacier front were depleted in BPC

  13. Bushen Zhuangjin decoction inhibits TM-induced chondrocyte apoptosis mediated by endoplasmic reticulum stress

    PubMed Central

    LIN, PINGDONG; WENG, XIAPING; LIU, FAYUAN; MA, YUHUAN; CHEN, HOUHUANG; SHAO, XIANG; ZHENG, WENWEI; LIU, XIANXIANG; YE, HONGZHI; LI, XIHAI

    2015-01-01

    Chondrocyte apoptosis triggered by endoplasmic reticulum (ER) stress plays a vital role in the pathogenesis of osteoarthritis (OA). Bushen Zhuangjin decoction (BZD) has been widely used in the treatment of OA. However, the cellular and molecular mechanisms responsible for the inhibitory effects of BZD on chondrocyte apoptosis remain to be elucidated. In the present study, we investigated the effects of BZD on ER stress-induced chondrocyte apoptosis using a chondrocyte in vitro model of OA. Chondrocytes obtained from the articular cartilage of the knee joints of Sprague Dawley (SD) rats were detected by immunohistochemical staining for type II collagen. The ER stress-mediated apoptosis of tunicamycin (TM)-stimulated chondrocytes was detected using 4-phenylbutyric acid (4-PBA). We found that 4-PBA inhibited TM-induced chondrocyte apoptosis, which confirmed the successful induction of chondrocyte apoptosis. BZD enhanced the viability of the TM-stimulated chondrocytes in a dose- and time-dependent manner, as shown by MTT assay. The apoptotic rate and the loss of mitochondrial membrane potential (ΔΨm) of the TM-stimulated chondrocytes treated with BZD was markedly decreased compared with those of chondrocytes not treated with BZD, as shown by 4′,6-diamidino-2-phenylindole (DAPI) staining, Annexin V-FITC binding assay and JC-1 assay. To further elucidate the mechanisms responsible for the inhibitory effects of BZD on TM-induced chondrocyte apoptosis mediated by ER stress, the mRNA and protein expression levels of binding immunoglobulin protein (Bip), X-box binding protein 1 (Xbp1), activating transcription factor 4 (Atf4), C/EBP-homologous protein (Chop), caspase-9, caspase-3, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) were measured by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. In the TM-stimulated chondrocytes treated with BZD, the mRNA and protein expression levels of Bip, Atf4, Chop, caspase-9, caspase-3

  14. Bushen Zhuangjin decoction inhibits TM-induced chondrocyte apoptosis mediated by endoplasmic reticulum stress.

    PubMed

    Lin, Pingdong; Weng, Xiaping; Liu, Fayuan; Ma, Yuhuan; Chen, Houhuang; Shao, Xiang; Zheng, Wenwei; Liu, Xianxiang; Ye, Hongzhi; Li, Xihai

    2015-12-01

    Chondrocyte apoptosis triggered by endoplasmic reticulum (ER) stress plays a vital role in the pathogenesis of osteoarthritis (OA). Bushen Zhuangjin decoction (BZD) has been widely used in the treatment of OA. However, the cellular and molecular mechanisms responsible for the inhibitory effects of BZD on chondrocyte apoptosis remain to be elucidated. In the present study, we investigated the effects of BZD on ER stress-induced chondrocyte apoptosis using a chondrocyte in vitro model of OA. Chondrocytes obtained from the articular cartilage of the knee joints of Sprague Dawley (SD) rats were detected by immunohistochemical staining for type Ⅱ collagen. The ER stress-mediated apoptosis of tunicamycin (TM)‑stimulated chondrocytes was detected using 4-phenylbutyric acid (4‑PBA). We found that 4‑PBA inhibited TM-induced chondrocyte apoptosis, which confirmed the successful induction of chondrocyte apoptosis. BZD enhanced the viability of the TM-stimulated chondrocytes in a dose- and time-dependent manner, as shown by MTT assay. The apoptotic rate and the loss of mitochondrial membrane potential (ΔΨm) of the TM-stimulated chondrocytes treated with BZD was markedly decreased compared with those of chondrocytes not treated with BZD, as shown by 4',6-diamidino-2-phenylindole (DAPI) staining, Annexin V-FITC binding assay and JC-1 assay. To further elucidate the mechanisms responsible for the inhibitory effects of BZD on TM‑induced chondrocyte apoptosis mediated by ER stress, the mRNA and protein expression levels of binding immunoglobulin protein (Bip), X‑box binding protein 1 (Xbp1), activating transcription factor 4 (Atf4), C/EBP‑homologous protein (Chop), caspase‑9, caspase-3, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) were measured by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. In the TM-stimulated chondrocytes treated with BZD, the mRNA and protein expression levels of Bip, Atf4, Chop, caspase

  15. Low energy proton beam induces tumor cell apoptosis through reactive oxygen species and activation of caspases

    PubMed Central

    Lee, Kheun Byeol; Lee, Jong-Soo; Park, Jin-Woo; Huh, Tae-Lin

    2008-01-01

    Proton beam is useful to target tumor tissue sparing normal cells by allowing precise dose only into tumor cells. However, the cellular and molecular mechanisms by which proton beam induces tumor cell death are still undefined. We irradiated three different tumor cells (LLC, HepG2, and Molt-4) with low energy proton beam (35 MeV) with spread out Bragg peak (SOBP) in vitro, and investigated cell death by MTT or CCK-8 assay at 24 h after irradiation. LLC and HepG2 cells were sensitive to proton beam at over 10 Gy to induce apoptosis whereas Molt-4 showed rather low sensitivity. Relative biological effectiveness (RBE) values for the death rate relative to γ-ray were ranged from 1.1 to 2.3 in LLC and HepG2 but from 0.3 to 0.7 in Molt-4 at 11 d after irradiation by colony formation assay. The typical apoptotic nuclear DNA morphological pattern was observed by staining with 4'-6-diamidino-2-phenylindole (DAPI). Tiny fragmented DNA was observed in HepG2 but not in Molt-4 by the treatment of proton in apoptotic DNA fragment assay. By FACS analysis after stained with FITC-Annexin-V, early as well as median apoptotic fractions were clearly increased by proton treatment. Proton beam-irradiated tumor cells induced a cleavage of poly (ADP-ribose) polymerase-1 (PARP-1) and procaspases-3 and -9. Activity of caspases was highly enhanced after proton beam irradiation. Reactive oxygen species (ROS) were significantly increased and N-acetyl cysteine pretreatment restored the apoptotic cell death induced by proton beam. Furthermore, p38 and JNK but not ERK were activated by proton and dominant negative mutants of p38 and JNK revived proton-induced apoptosis, suggesting that p38 and JNK pathway may be activated through ROS to activate apoptosis. In conclusion, our data clearly showed that single treatment of low energy proton beam with SOBP increased ROS and induced cell death of solid tumor cells (LLC and HepG2) in an apoptotic cell death program by the induction of caspases

  16. Ability of Catalonian donkey sperm to penetrate zona pellucida-free bovine oocytes matured in vitro.

    PubMed

    Taberner, E; Morató, R; Mogas, T; Miró, J

    2010-04-01

    An experiment was designed to study the interaction between fresh/frozen-thawed donkey spermatozoa and zona pellucida (ZP)-free bovine oocytes in an attempt to develop a model for assessing cryopreserved Catalonian donkey sperm function. Semen from five donkeys was collected using an artificial vagina. Sperm motility and viability were immediately assessed and the semen sample cryopreserved. Sperm viability and motility were then reassessed immediately after thawing. The motion characteristics of the fresh and frozen-thawed spermatozoa were determined using a computer-assisted sperm analysis system. In vitro-matured cow oocytes were inseminated with different percent live donkey sperm (high (>60%) or low (<40%) viability donkey sperm). After 18h of co-incubation, the oocytes were fixed, stained with 4',6-diamidino-2-phenylindole (DAPI) and examined for sperm penetration, the number of penetrated spermatozoa per oocyte, and male pronucleus formation. Frozen-thawed spermatozoa from high viability semen showed significantly lower VCL, VAP and mean ALH values than did high viability fresh spermatozoa. In contrast, frozen-thawed spermatozoa of low viability had significantly higher velocity values than fresh spermatozoa of low viability. A significant positive correlation (P<0.01) was detected between percentage fertilization and viability (r=0.84), and between percentage fertilization and certain CASA parameters (VAP, r=0.56; VCL, r=0.61 and mean ALH, r=0.68). Fresh or frozen-thawed high viability spermatozoa penetrated 90.1% and 85.4% of bovine oocytes respectively. Lower rates of penetration were observed for fresh and frozen-thawed low viability spermatozoa (34% and 22.5% respectively). The donkey spermatozoa were able to fuse with the oolema and even to decondense and form the male pronucleus (85-94%). Larger numbers of penetrated spermatozoa per oocyte were recorded when high viability sperm samples were used, whether fresh (3.02 vs. 1.12 for low viability sperm

  17. Characterization of Insertion Mutations in the Saccharomyces Cerevisiae Msh1 and Msh2 Genes: Evidence for Separate Mitochondrial and Nuclear Functions

    PubMed Central

    Reenan, RAG.; Kolodner, R. D.

    1992-01-01

    The MSH1 and MSH2 genes of Saccharomyces cerevisiae are predicted to encode proteins that are homologous to the Escherichia coli MutS and Streptococcus pneumoniae HexA proteins and their homologs. Disruption of the MSH1 gene caused a petite phenotype which was established rapidly. A functional MSH1 gene present on a single-copy centromere plasmid was incapable of rescuing the established msh1 petite phenotype. Analysis of msh1 strains demonstrated that mutagenesis and large-scale rearrangement of mitochondrial DNA had occurred. 4',6-Diamidino-2-phenylindole (DAPI) staining of msh1 yeast revealed an aberrant distribution of mtDNA. Haploid msh2 mutants displayed an increase of 85-fold in the rate of spontaneous mutation to canavanine resistance. Sporulation of homozygous msh2/msh2 diploids gave rise to a high level of lethality which was compounded during increased vegetative growth prior to sporulation. msh2 mutations also affected gene conversion of two HIS4 alleles. The his4x mutation, lying near the 5' end of the gene, was converted with equal frequency in both wild-type and msh2 strains. However, many of the events in the msh2 background were post-meiotic segregation (PMS) events (46.4%) while none (<0.25%) of the aberrant segregations in wild type were PMS events. The his4b allele, lying 1.6 kb downstream of his4x, was converted at a 10-fold higher frequency in the msh2 background than in the corresponding wild-type strain. Like the his4x allele, his4b showed a high level of PMS (30%) in the msh2 background compared to the corresponding wild-type strain where no (<0.26%) PMS events were observed. These results indicate that MSH1 plays a role in repair or stability of mtDNA and MSH2 plays a role in repair of 4-bp insertion/deletion mispairs in the nucleus. PMID:1334021

  18. HIV antiretroviral drug combination induces endothelial mitochondrial dysfunction and reactive oxygen species production, but not apoptosis

    SciTech Connect

    Jiang Bo; Hebert, Valeria Y.; Li, Yuchi; Mathis, J. Michael; Alexander, J. Steven; Dugas, Tammy R.

    2007-10-01

    ROS production culminate in apoptosis, we performed the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL), annexin V and 4',6-diamidino-2-phenylindole (DAPI) staining, and caspase-3 activity assays. However, none of these assays showed appreciable levels of ART-induced apoptosis. Our studies thus suggest that in endothelial cells, ART induces mitochondrial dysfunction with a concomitant increase in mitochondria-derived ROS. This compromised mitochondrial function may be one important factor culminating in endothelial dysfunction, without inducing an increase in apoptosis.

  19. Nuclear DNA Content Estimates in Green Algal Lineages: Chlorophyta and Streptophyta

    PubMed Central

    Kapraun, Donald F.

    2007-01-01

    Background and Aims Consensus higher-level molecular phylogenies present a compelling case that an ancient divergence separates eukaryotic green algae into two major monophyletic lineages, Chlorophyta and Streptophyta, and a residuum of green algae, which have been referred to prasinophytes or micromonadophytes. Nuclear DNA content estimates have been published for less than 1% of the described green algal members of Chlorophyta, which includes multicellular green marine algae and freshwater flagellates (e.g. Chlamydomonas and Volvox). The present investigation summarizes the state of our knowledge and adds substantially to our database of C-values, especially for the streptophyte charophycean lineage which is the sister group of the land plants. A recent list of 2C nuclear DNA contents for isolates and species of green algae is expanded by 72 to 157. Methods The DNA-localizing fluorochrome DAPI (4′,6-diamidino-2-phenylindole) and red blood cell (chicken erythrocytes) standard were used to estimate 2C values with static microspectrophotometry. Key Results In Chlorophyta, including Chlorophyceae, Prasinophyceae, Trebouxiophyceae and Ulvophyceae, 2C DNA estimates range from 0·01 to 5·8 pg. Nuclear DNA content variation trends are noted and discussed for specific problematic taxon pairs, including Ulotrichales–Ulvales, and Cladophorales–Siphonocladales. For Streptophyta, 2C nuclear DNA contents range from 0·2 to 6·4 pg, excluding the highly polyploid Charales and Desmidiales, which have genome sizes of up to 14·8 and 46·8 pg, respectively. Nuclear DNA content data for Streptophyta superimposed on a contemporary molecular phylogeny indicate that early diverging lineages, including some members of Chlorokybales, Coleochaetales and Klebsormidiales, have genomes as small as 0·1–0·5 pg. It is proposed that the streptophyte ancestral nuclear genome common to both the charophyte and the embryophyte lineages can be characterized as 1C = 0·2 pg and 1n = 6

  20. Tracking Chemotactic Migration of a Genetically Engineered Bacterium in the Presence of Constructed Nutrient Gradients Within a Sandy Aquifer in Cape Cod, Massachusetts

    NASA Astrophysics Data System (ADS)

    Harvey, R. W.; Ford, R. M.; Metge, D. W.; Wang, M.; Toepfer, A. A.; McGowan, S. B.

    2008-05-01

    injection. The study demonstrated substantive vertical migration of P. stutzeri toward higher acetate and nitrate concentrations. In contrast, the bacterial controls (P. stutzeri that had been chemotactically impaired by a priori exposure to the fluorochrome DAPI, 4,6'-diamidino-2-phenylindole, and co-injected with the test bacteria) were advected horizontally with the groundwater and did not migrate vertically toward higher acetate and nitrate concentrations. The study further demonstrated that chemotactic migration can be considerably faster in the aquifer than would be predicted from laboratory studies and may have an important role in aquifer remediation.

  1. Theranostic nanoshells: from probe design to imaging and treatment of cancer.

    PubMed

    Bardhan, Rizia; Lal, Surbhi; Joshi, Amit; Halas, Naomi J

    2011-10-18

    therapy approaches. We describe the fabrication of DNA-conjugated nanoshell complexes and compare the efficiency of light-induced and thermally-induced release of DNA. Double-stranded DNA nanoshells also provide a way to deliver small molecules into cells: we describe the delivery and light-triggered release of DAPI (4',6-diamidino-2-phenylindole), a dye molecule used to stain DNA in the nuclei of cells.

  2. Control of Vertebrate Skeletal Mineralization by Polyphosphates

    PubMed Central

    Omelon, Sidney; Georgiou, John; Henneman, Zachary J.; Wise, Lisa M.; Sukhu, Balram; Hunt, Tanya; Wynnyckyj, Chrystia; Holmyard, Douglas; Bielecki, Ryszard; Grynpas, Marc D.

    2009-01-01

    calcium lowers the relative apatite saturation, preventing formation of apatite crystals. Identified in situ within resorbing bone and mineralizing cartilage by the fluorescent reporter DAPI (4′,6-diamidino-2-phenylindole), polyphosphate formation prevents apatite crystal precipitation while accumulating high local concentrations of total calcium and phosphate. When mineralization is required, tissue non-specific alkaline phosphatase, an enzyme associated with skeletal and cartilage mineralization, cleaves orthophosphates from polyphosphates. The hydrolytic degradation of polyphosphates in the calcium-polyphosphate complex increases orthophosphate and calcium concentrations and thereby favors apatite mineral formation. The correlation of alkaline phosphatase with this process may be explained by the destruction of polyphosphates in calcifying cartilage and areas of bone formation. Conclusions/Significance We hypothesize that polyphosphate formation and hydrolytic degradation constitute a simple mechanism for phosphate accumulation and enzymatic control of biological apatite saturation. This enzymatic control of calcified tissue mineralization may have permitted the development of a phosphate-based, mineralized endoskeleton that can be continually remodeled. PMID:19492083

  3. Microbial structures in an Alpine Thermal Spring - Microscopic techniques for the examination of Biofilms in a Subsurface Environment

    NASA Astrophysics Data System (ADS)

    Dornmayr-Pfaffenhuemer, Marion; Pierson, Elisabeth; Janssen, Geert-Jan; Stan-Lotter, Helga

    2010-05-01

    The research into extreme environments hast important implications for biology and other sciences. Many of the organisms found there provide insights into the history of Earth. Life exists in all niches where water is present in liquid form. Isolated environments such as caves and other subsurface locations are of interest for geomicrobiological studies. And because of their "extra-terrestrial" conditions such as darkness and mostly extreme physicochemical state they are also of astrobiological interest. The slightly radioactive thermal spring at Bad Gastein (Austria) was therefore examined for the occurrence of subsurface microbial communities. The surfaces of the submerged rocks in this warm spring were overgrown by microbial mats. Scanning electron microscopy (SEM) performed by the late Dr. Wolfgang Heinen revealed an interesting morphological diversity in biofilms found in this environment (1, 2). Molecular analysis of the community structure of the radioactive subsurface thermal spring was performed by Weidler et al. (3). The growth of these mats was simulated using sterile glass slides which were exposed to the water stream of the spring. Those mats were analysed microscopically. Staining, using fluorescent dyes such as 4',6-Diamidino-2-phenylindol (DAPI), gave an overview of the microbial diversity of these biofilms. Additional SEM samples were prepared using different fixation protocols. Scanning confocal laser microscopy (SCLM) allowed a three dimensional view of the analysed biofilms. This work presents some electron micrographs of Dr. Heinen and additionally new microscopic studies of the biofilms formed on the glass slides. The appearances of the new SEM micrographs were compared to those of Dr. Heinen that were done several years ago. The morphology and small-scale distribution in the microbial mat was analyzed by fluorescence microscopy. The examination of natural biomats and biofilms grown on glass slides using several microscopical techniques

  4. Quantitative Expression and Co-Localization of Wnt Signalling Related Proteins in Feline Squamous Cell Carcinoma

    PubMed Central

    Marote, Georgina; Abramo, Francesca; McKay, Jenny; Thomson, Calum; Beltran, Mariana; Millar, Michael; Priestnall, Simon; Dobson, Jane; Costantino-Casas, Fernando; Petrou, Terry; McGonnell, Imelda M.; Davies, Anthony J.; Weetman, Malcolm; Garden, Oliver A.; Masters, John R.; Thrasivoulou, Christopher; Ahmed, Aamir

    2016-01-01

    Feline oral squamous cell carcinoma (FOSCC) is an aggressive neoplasm in cats. Little is known about the possible molecular mechanisms that may be involved in the initiation, maintenance and progression of FOSCC. Wnt signalling is critical in development and disease, including many mammalian cancers. In this study, we have investigated the expression of Wnt signalling related proteins using quantitative immunohistochemical techniques on tissue arrays. We constructed tissue arrays with 58 individual replicate tissue samples. We tested for the expression of four key Wnt/ß-catenin transcription targets, namely Cyclin D1 (CCND1 or CD1), FRA1, c-Myc and MMP7. All antibodies showed cross reactivity in feline tissue except MMP7. Quantitative immunohistochemical analysis of single proteins (expressed as area fraction / amount of tissue for normal vs tumor, mean ± SE) showed that the expression of CD1 (3.9 ± 0.5 vs 12.2 ± 0.9), FRA1 (5.5 ± 0.6 vs 16.8 ± 1.1) and c-Myc (5.4 ± 0.5 vs 12.5 ± 0.9) was increased in FOSCC tissue by 2.3 to 3 fold compared to normal controls (p<0.0001). By using a multilabel, quantitative fluorophore technique we further investigated if the co-localization of these proteins (all transcription factors) with each other and in the nucleus (stained with 4',6-diamidino-2-phenylindole, DAPI) was altered in FOSCC compared to normal tissue. The global intersection coefficients, a measure of the proximity of two fluorophore labeled entities, showed that there was a significant change (p < 0.01) in the co-localization for all permutations (e.g. CD1/FRA1 etc), except for the nuclear localization of CD1. Our results show that putative targets of Wnt signalling transcription are up-regulated in FOSCC with alterations in the co-localization of these proteins and could serve as a useful marker for the disease. PMID:27559731

  5. Unique polarized light-matter interaction in single one-dimensional semiconducting oxide nanomaterials and applications of indium tin oxide nanorod networks as surface enhanced raman spectroscopy and photodetection platforms

    NASA Astrophysics Data System (ADS)

    Choi, Daniel So Ri

    rhodamine 6G (R6G) and 4',6-diamidino-2-phenylindole (DAPI) and comparing the Raman signals to that of Si and AuNRs substrates. Additionally, the presented 1D SCOs as detector of visible light is realized as photoresponse is observed from networks of all four materials types upon introduction of sub-bandgap, visible wavelength light yielding comparable performance to commercially available devices for ITO NR network based device.

  6. Mortality associated with melarsomine dihydrochloride administration in two North American river otters (Lontra canadensis) and a red panda (Ailurus fulgens fulgens).

    PubMed

    Neiffer, Donald L; Klein, Edwin C; Calle, Paul P; Linn, Michael; Terrell, Scott P; Walker, Rodney L; Todd, Donna; Vice, Carol C; Marks, Steven K

    2002-09-01

    Two adult North American river otters (Lontra canadensis) and an adult red panda (Ailurus fulgens fulgens) at three separate institutions died within 22 hr after receiving single 2.5- to 2.7-mg/kg doses of melarsomine dihydrochloride administered in the epaxial musculature as a treatment for filarid nematodes. One otter had a suspected Dirofilaria immitis infection, the other had a confirmed D. lutrae infection, and the red panda had a confirmed Dirofilaria sp. infection, presumably with D. immitis. Postmortem examinations revealed similar gross lesions, although they were less severe in the red panda. The trachea and primary bronchi contained abundant foamy fluid, the lungs were mottled with areas of consolidation, and the pulmonary parenchyma exuded abundant fluid at the cut section. Histologic evaluation revealed acute pulmonary edema, which resulted in respiratory failure and death. There may have been direct pulmonary cellular toxicity of melarsomine dihydrochloride or a severe systemic anaphylactic reaction to antigens released after parasite death. An idiosyncratic drug reaction or a low therapeutic index of melarsomine probably caused the death of the three individuals. Melarsomine dihydrochloride use should be avoided in North American river otters and red pandas.

  7. In vitro antimicrobial activity of sodium hypochlorite, chlorhexidine gluconate and octenidine dihydrochloride in elimination of microorganisms within dentinal tubules of primary and permanent teeth.

    PubMed

    Tirali, Resmiye-Ebru; Bodur, Haluk; Ece, Gülden

    2012-05-01

    The aim of this study was to evaluate the effectiveness of different irrigation solutions at different time intervals for the elimination of E. faecalis and C. albicans penetrated into the dentine tubules of primary and permanent teeth in vitro. The 4 mm primary and permanent teeth sections were sterilized and contaminated with a mixture of E. faecalis and C. albicans strains. After the application of different irrigation solutions (Sodium hypochlorite, Chlorhexidine gluconate, Octenidine Dihydrochloride, saline) to the contaminated tooth sections according to study groups, neutralizers were applied for inactivation of the solutions after 30 sec, 1 min and 5 min. Dentine shavings were placed into TSB and 10 µL from each tube was inoculated on agar plates, followed by an incubation period of 24 h at 37°C. The colonies were counted macroscopically. The results were compared by using Kruskal-Wallis and Mann Whitney U tests, with a significance level at p<0.05. Among the irrigation solutions that were tested against E. faecalis on primary and permanent teeth, the most effective one was found as 5-minute application of 0.1% Octenidine Dihydrochloride. The antibacterial effects of the tested solutions on the same time periods against C. albicans revealed no significant difference. There were no statistically significant differences between primary and permanent teeth with respect to the antimicrobial activity of the tested solutions. Moreover, Octenidine Dihydrochloride may be used as an alternative endodontic irrigant.

  8. UV-activated conversion of Hoechst 33258, DAPI, and Vybrant DyeCycle fluorescent dyes into blue-excited, green-emitting protonated forms.

    PubMed

    Zurek-Biesiada, Dominika; Kędracka-Krok, Sylwia; Dobrucki, Jurek W

    2013-05-01

    Hoechst 33258, DAPI and Vybrant DyeCycle are commonly known DNA fluorescent dyes that are excited by UV and emit in the blue region of the spectrum of visible light. Conveniently, they leave the reminder of the spectrum for microscopy detection of other cellular targets labeled with probes emitting in green, yellow or red. However, an exposure of these dyes to UV induces their photoconversion and results in production of the forms of these dyes that are excited by blue light and show fluoresce maxima in green and a detectable fluorescence in yellow and orange regions of the spectrum. Photoconversion of Hoechst 33258 and DAPI is reversible and independent of the dye concentration or the presence of DNA. Spectrofluorimetry and mass spectrometry analyses indicate that exposure to UV induces protonation of Hoechst 33258 and DAPI.

  9. Cetirizine dihydrochloride loaded microparticles design using ionotropic cross-linked chitosan nanoparticles by spray-drying method.

    PubMed

    Li, Feng-Qian; Ji, Rui-Rui; Chen, Xu; You, Ben-Ming; Pan, Yong-Hua; Su, Jia-Can

    2010-12-01

    To control the release rate and mask the bitter taste, cetirizine dihydrochloride (CedH) was entrapped within chitosan nanoparticles (CS-NPs) using an ionotropic gelation process, followed by microencapsulation to produce CS matrix microparticles using a spray-drying method. The aqueous colloidal CS-NPs dispersions with a drug encapsulation efficiency (EE) of <15%, were then spray dried to produce a powdered nanoparticles-in-microparticles system with an EE of >70%. The resultant spherical CS microparticles had a smooth surface, were free of organic solvent residue and showed a diameter range of 0.5~5 μm. The in vitro drug release properties of CedH encapsulated microparticles showed an initial burst effect during the first 2 h. Drug release from the matrix CS microparticles could be retarded by the crosslinking agent pentasodium tripolyphosphate or the wall material. The technique of 'ionotropic gelation' combined with 'spray-drying' could be applicable for preparation of CS nanoparticlesin-microparticles drug delivery systems. CS-NPs based microparticles might provide a potential micro-carrier for oral administration of the freely water-soluble drug--CedH.

  10. Ultrasound-induced killing of monocytic U937 cells enhanced by 2,2'-azobis(2-amidinopropane) dihydrochloride.

    PubMed

    Feril, Loreto B; Tsuda, Yuko; Kondo, Takashi; Zhao, Qing-Li; Ogawa, Ryohei; Cui, Zheng-Guo; Tsukada, Kazuhiro; Riesz, Peter

    2004-02-01

    To determine the effect of 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) on ultrasound (US)-induced cell killing, human monocytic leukemia cells (U937) were incubated at various temperatures (25.0, 37.0 and 40.0 degrees C) for 1 min in air-saturated phosphate-buffered solution (PBS) containing 50 mM AAPH before exposure to nonthermal 1 MHz US for 1 min at an intensity of 2.0 W/cm(2). Cell viability was determined by means of the Trypan blue dye exclusion test immediately after sonication. Apoptosis was measured after 6-h incubation post-sonication by flow cytometry. Free radicals generated by AAPH, a temperature-dependent free radical generator, or US or both were also investigated using electron paramagnetic resonance (EPR) spin trapping. The results showed that US-induced cell lysis and apoptosis were enhanced in the presence of AAPH regardless of the temperature at the time of sonication. At 40.0 degrees C, US alone induced increased cell killing, while AAPH alone is capable of inducing significant but minimal apoptosis at this temperature. Although free radicals were increased in the combined treatment, this increase did not correlate well with cell killing. The mechanism of enhancement points to the increased uptake of the agent during sonication rather than potentiation by AAPH. These findings suggest the clinical potential of temperature-dependent free radical generators in cancer therapy with therapeutic US.

  11. Oxidative modification and inactivation of Cu,Zn-superoxide dismutase by 2,2'-azobis(2-amidinopropane) dihydrochloride.

    PubMed

    Kwon, H Y; Choi, S Y; Won, M H; Kang, T; Kang, J H

    2000-11-30

    We have investigated oxidative modification of human Cu, Zn-superoxide dismutase (SOD) by alkylperoxyl radicals and alkylperoxides. To generate free radicals, we used the hydrophilic azocompound, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). When Cu,Zn-SOD was incubated with AAPH, the enzyme activity was decreased gradually in a time-dependent manner. The oxidative damage to Cu,Zn-SOD by AAPH-derived radicals led to protein fragmentation which is associated with the inactivation of enzyme. Incubation with AAPH resulted in the release of copper ions from Cu,Zn-SOD and the generation of protein carbonyl derivatives. Catalase did not protect the fragmentation of Cu,Zn-SOD whereas azide, glutathione and a metal chelator, diethylenetriamine pentaacetic acid inhibited the protein fragmentation. When Cu,Zn-SOD that has been exposed to AAPH was subsequently analyzed by amino acid analysis, lysine, histidine, proline, and valine residues were particularly sensitive. It is suggested that oxidative damage of Cu,Zn-SOD by AAPH-derived radicals may induce the perturbation of cellular antioxidant defense systems and subsequently lead to the deleterious condition in cells.

  12. A water extract of Artemisia capillaris prevents 2,2'-azobis(2-amidinopropane) dihydrochloride-induced liver damage in rats.

    PubMed

    Han, Kyu-Ho; Jeon, You-Jin; Athukorala, Yasantha; Choi, Kang-Duk; Kim, Cheon-Jei; Cho, Jin-Kook; Sekikawa, Mitsuo; Fukushima, Michiro; Lee, Chi-Ho

    2006-01-01

    A water extract of Artemisia capillaris Thunberg (Compositae) was investigated for protective effects against oxidative stress induced by 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) in Sprague-Dawley male rats. Rats were orally administered A. capillaris water extract (ACWE; 7.5 g/kg) for 7 days before AAPH treatment (60 mg/kg). AAPH intoxication significantly elevated enzyme markers of liver injury (glutamic oxaloacetic transaminase and glutamic pyruvic transaminase). The pre-administration of ACWE significantly reduced the liver-damaging effects of AAPH as indicated by the low levels of these enzymes. Moreover, the ACWE administration significantly attenuated the accumulation of thiobarbituric acid-reactive substances in both plasma and liver tissues compared with those of rats administered AAPH alone. Furthermore, ACWE administration slightly improved the liver reduced glutathione levels and enhanced the production of antioxidant enzymes like catalase. A. capillaris contained 10.1 mg of catechin in 100 g of dried sample; the high-performance liquid chromatography results showed catechin composition in the ACWE to be 28% (-)-epigallocatechin gallate, 49% (-)- epigallocatechin, and 23% other catechins. These observations clearly indicate that ACWE contains antioxidant catechins capable of ameliorating the AAPH-induced hepatic injury by virtue of its antioxidant activity.

  13. Protective effects of beta-blockers against 2,2'-azobis(2-amidinopropane)-dihydrochloride-induced damage.

    PubMed

    Miura, T; Muraoka, S; Ogiso, T

    1995-06-30

    The protective effects of beta-blockers against 2,2'-azobis(2- amidinopropane)-dihydrochloride (AAPH)-induced damage were investigated. With the exception of pindolol, none of the beta-blockers tested inhibited arachidonate peroxidation induced by AAPH in the absence of iron. In contrast, ADP-Fe(3+)- and NADPH-dependent microsomal lipid peroxidation was inhibited by all the beta-blockers tested, although the inhibitory effects of atenolol and metoprolol were very slight. Oxidation of tryptophan residues in bovine serum albumin (BSA) induced by AAPH was strongly inhibited by pindolol and propranolol but not by atenolol or metoprolol. All the beta-blockers tested, however, inhibited AAPH-induced carbonyl formation of BSA. Furthermore, all the beta-blockers tested also strongly inhibited the deoxyribose degradation induced by AAPH, suggesting that these agents act as hydroxyl radical scavengers to inhibit carbonyl formation. DNA strand scission was induced by AAPH in the absence or presence of O2. Only pindolol strongly inhibited the DNA damage in the absence of O2. In the presence of O2, however, all the beta-blockers tested effectively prevented the DNA damage. These results suggested that the hydroxyl radicals produced from AAPH damaged DNA and, that beta-blockers might act as hydroxyl radical scavengers to protect DNA against the AAPH-induced oxidative damage.

  14. Antioxidant activity of sugarcane molasses against 2,2'-azobis(2-amidinopropane) dihydrochloride-induced peroxyl radicals.

    PubMed

    Asikin, Yonathan; Takahashi, Makoto; Mishima, Takashi; Mizu, Masami; Takara, Kensaku; Wada, Koji

    2013-11-01

    Sugarcane molasses is a rich source of antioxidant materials with peroxyl radical scavenging effects. To explore the potent antioxidant activity of sugarcane molasses against 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced peroxyl radicals, 7 methanolic fractions of sugarcane molasses (F1-F7) were separated via bioassay-guided fractionation and evaluated by oxygen radical absorbance capacity (ORAC), cellular antioxidant activity (CAA), and oxidative DNA damage protective activity assays. The ORAC values of sugarcane molasses fractions ranged from 4399 to 6,266 μmol TE/g, whilst the EC50 values for CAA ranged from 3.7 to 5.9 μg/ml. Moreover, it was found that sugarcane molasses fractions, particularly F6 and F7, could protect against oxidative DNA damage caused by peroxyl radicals at an effective concentration of 100 μg/ml. Ten phenolic constituents were identified in the fractions, including known antioxidative compounds, viz., schaftoside, isoschaftoside, ferulic acid, p-coumaric acid, and p-hydroxybenzaldehyde.

  15. Effect of treatment with betahistine dihydrochloride on the postural stability in patients with different duration of benign paroxysmal positional vertigo.

    PubMed

    Stambolieva, Katerina; Angov, Georgi

    2010-01-01

    The effect of betahistine dihydrochloride on the postural stability after repositioning Epley's maneuver (EM) in patients with BPPV was evaluated by static posturography in open and closed eyes conditions. Ninety patients were divided into four groups by duration (less and above 60 days of BPPV) and by treatment (with and without treatment with betahistine). The investigation was made one hour after the positive Dix-Hallpike test, 10 and 20 days after the treatment with EM. "Sway velocity" (SV) was calculated to evaluate postural stability. The results show dependence between efficacy of treatment with betahistine applied after EM and duration of BPPV. Betahistine normalized postural stability of patients with duration of BPPV less than 60 days after 10 days of treatment and had less effect on patients with duration of BPPV above 60 days. We assume that after removing the otoconia betahistine plays an important role for improving blood flow in the inner ear. The short presence of otoconia didn't damage sensory receptor, and restoring the normal function of motion-sensitive hairs cells and stabilizing the posture was observed.

  16. Development of novel sustained release matrix pellets of betahistine dihydrochloride: effect of lipophilic surfactants and co-surfactants.

    PubMed

    Shamma, Rehab Nabil; Basalious, Emad B; Shoukri, Raguia

    2012-01-01

    Sustained release matrix pellets of the freely water soluble drug, betahistine dihydrochloride (BH), were prepared using freeze pelletization technique. Different waxes and lipids (cetyl alcohol, beeswax, glyceryl tripalmitate (GTP) and glyceryl tristearate) were evaluated for the preparation of matrix pellets. A D-optimal design was employed for the optimization and to explore the effect of drug loading (X(1)), concentration of lipophilic surfactant (X(2)), concentration of co-surfactant (X(3)) and wax type (X(4)) on the release extent of the drug from matrix pellets. The entrapment efficiency (Y(1)), pellet diameter (Y(2)), and the percentage drug released at given times were selected as dependent variables. Results revealed a significant impact of all independent variables on drug release from the formulated pellets. The lipophilic surfactant significantly increased both the entrapment efficiency and the in vitro drug release and significantly decreased the pellet size. The optimized BH-loaded pellets were composed of 19.95% drug loading, 9.95% Span(®) 80 (surfactant), 0.25% Capmul(®) (co-surfactant) using glyceryl tripalmitate as a matrix former. The release profiles of the drug from hard gelatin capsule containing optimized pellets equivalent to 32 mg BH was similar to that of target release model for once-daily administration based on similarity factor. It could be concluded that a promising once-daily capsule containing sustained release pellets of BH was successfully designed.

  17. Cytogenetic analysis of Otiorhynchus bisulcatus (Fabricius, 1781) and O.(Zadrehus) atroapterus (De Geer, 1775) (Coleoptera, Curculionidae, Entiminae) using C bands, NORs, and DAPI/CMA3 staining.

    PubMed

    Holecová, Milada; Maryańska-Nadachowska, Anna; Rozek, Maria

    2013-01-01

    The structure of the karyotypes of two Otiorhynchus species belonging to separate subgenera, viz. Otiorhynchus s.str. bisulcatus and O. (Zadrehus) atroapterus, is compared and described for the first time. Both species have the same chromosome number (2n = 22), sex chromosome system of an achiasmate parachute type (Xy(p)), symmetric karyotype with the prevalence of metacentrics, similar meiotic behaviour, localization of NORs and positive DAPI signals. The main differences involve the morphology of autosomes and the X chromosome in the C-banding pattern and DAPI/CMA3 signals as well as in the presence of additional B chromosomes.

  18. A novel family of diarylpyrimidines (DAPYs) featuring a diatomic linker: Design, synthesis and anti-HIV activities.

    PubMed

    Gu, Shuang-Xi; Qiao, Heng; Zhu, Yuan-Yuan; Shu, Qi-Chao; Liu, Hui; Ju, Xiu-Lian; De Clercq, Erik; Balzarini, Jan; Pannecouque, Christophe

    2015-10-15

    To improve the conformational flexibility and positional adaptability of the traditional diarylpyrimidines (DAPYs), a family of diarylpyrimidines featuring a C-N diatomic linker between the left wing benzene ring and the central pyrimidine was firstly designed, synthesized, and evaluated for in vitro anti-HIV activity. Most of target molecules showed excellent activities against wild-type (WT) HIV-1. Among them the most potent two compounds 12h and 12r displayed extremely potent WT HIV-1 inhibitory activities with an EC50 of 2.6 nM and 3.0 nM, respectively, while their selective index (CC50/EC50) values were both over 1000. Another compound 12b (EC50 14.9 nM) was also noteworthy due to its high SI of 18,614. Moreover, all of compounds were evaluated for their WT HIV-1 reverse transcriptase activities, which shown that the newly synthesized CH2NH-DAPYs bind to HIV-1 RT and belong to the genuine NNRTIs. However, the synthesized compounds lack the activities against HIV-1 double mutant (RES056) and HIV-2 (ROD). Thus it is an upcoming objective to improve the activities against HIV-1 double mutants.

  19. Fluorescence lifetime imaging of DAPI-stained nuclei as a novel diagnostic tool for the detection and classification of B-cell chronic lymphocytic leukemia.

    PubMed

    Yahav, Gilad; Hirshberg, Abraham; Salomon, Ophira; Amariglio, Ninette; Trakhtenbrot, Luba; Fixler, Dror

    2016-07-01

    B-cell chronic lymphocytic leukaemia (B-CLL) and B-cell precursor acute lymphoblastic leukaemia (B-ALL) are the most common type of leukaemia in adults and children, respectively. Today, fluorescence in situ hybridization (FISH) is the standard for detecting chromosomal aberrations that reflect adverse and favorable outcome. This study revealed a new, simple, and fast diagnostic tool to detect pathological cells by measuring and imaging the fluorescence lifetime (FLT) using FLT imaging microscopy (FLIM) of the peripheral blood (PB) cells of B-CLL samples that were labeled with the DNA binder, DAPI. The FLT of DAPI in healthy individuals was found to be 2.66 ± 0.12 ns. In contrast, PB cells of B-CLL and BM cells of B-ALL patients were characterized by a specific group distribution of the FLT values. The FLT of DAPI was divided into four subgroups, relative to 2.66 ns: short+, normal, prolonged, and prolonged+. These alterations could be related to different chromatin arrangements of B-CLL and B-ALL interphase nuclei. Notably, extremely long FLT of nuclear DAPI correlate with the presence of extra chromosome 12, while moderate increases compared to normal characterize the deletion of p53. Such correlations potentially enable a FLT-based rapid automatic diagnosis and classification of B-CLL even when the frequency of genetic and chromosomal abnormalities is low. © 2016 International Society for Advancement of Cytometry.

  20. A new oxidative stress model, 2,2-azobis(2-amidinopropane) dihydrochloride induces cardiovascular damages in chicken embryo.

    PubMed

    He, Rong-Rong; Li, Yan; Li, Xiao-Di; Yi, Ruo-Nan; Wang, Xiao-Yu; Tsoi, Bun; Lee, Kenneth Ka Ho; Abe, Keiichi; Yang, Xuesong; Kurihara, Hiroshi

    2013-01-01

    It is now well established that the developing embryo is very sensitive to oxidative stress, which is a contributing factor to pregnancy-related disorders. However, little is known about the effects of reactive oxygen species (ROS) on the embryonic cardiovascular system due to a lack of appropriate ROS control method in the placenta. In this study, a small molecule called 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH), a free radicals generator, was used to study the effects of oxidative stress on the cardiovascular system during chick embryo development. When nine-day-old (stage HH 35) chick embryos were treated with different concentrations of AAPH inside the air chamber, it was established that the LD50 value for AAPH was 10 µmol/egg. At this concentration, AAPH was found to significantly reduce the density of blood vessel plexus that was developed in the chorioallantoic membrane (CAM) of HH 35 chick embryos. Impacts of AAPH on younger embryos were also examined and discovered that it inhibited the development of vascular plexus on yolk sac in HH 18 embryos. AAPH also dramatically repressed the development of blood islands in HH 3+ embryos. These results implied that AAPH-induced oxidative stress could impair the whole developmental processes associated with vasculogenesis and angiogenesis. Furthermore, we observed heart enlargement in the HH 40 embryo following AAPH treatment, where the left ventricle and interventricular septum were found to be thickened in a dose-dependent manner due to myocardiac cell hypertrophy. In conclusion, oxidative stress, induced by AAPH, could lead to damage of the cardiovascular system in the developing chick embryo. The current study also provided a new developmental model, as an alternative for animal and cell models, for testing small molecules and drugs that have anti-oxidative activities.

  1. Carcinogenicity of ortho-phenylenediamine dihydrochloride in rats and mice by two-year drinking water treatment.

    PubMed

    Matsumoto, Michiharu; Suzuki, Masaaki; Kano, Hirokazu; Aiso, Shigetoshi; Yamazaki, Kazunori; Fukushima, Shoji

    2012-05-01

    The carcinogenicity of ortho-phenylenediamine (o-PD) was examined by administrating o-phenylenediamine dihydrochloride (o-PD2HCl) in dinking water to groups of 50 F344/DuCrj rats and 50 Crj:BDF₁ mice of both sexes for 2 years. The drinking water concentration of o-PD2HCl was 0, 500, 1,000 or 2,000 ppm (wt/wt) for male rats; 0, 250, 500 or 1,000 ppm for female rats; 0, 500, 1,000 or 2,000 ppm for male mice; and 0, 1,000, 2,000 or 4,000 ppm for female mice. Two-year administration of o-PD2HCl produced a dose-dependent increase in the incidences of hepatocellular adenomas and carcinomas in rats of both sexes and in female mice, and hepatocellular adenomas in male mice. In mice, the incidences of hepatocellular adenomas were increased at the lowest dose used in both males and females. Metastasis from hepatocellular carcinomas of rats occurred predominantly in the lung. Incidences of transitional cell papillomas and carcinomas in the urinary bladder were noted in male rats administered 2,000 ppm, together with an increased incidence of papillary and/or nodular hyperplasia of transitional epithelium. In mice, the incidence of papillary adenomas in the gall bladder, which is rare in mice, was increased in both males and females administered 2,000 ppm. Thus, o-PD is carcinogenic in two species, i.e., rats and mice of both sexes.

  2. Inactivation of Listeria monocytogenes, Salmonella spp. and Escherichia coli O157:H7 on cantaloupes by octenidine dihydrochloride.

    PubMed

    Upadhyay, Abhinav; Chen, Chi-Hung; Yin, Hsinbai; Upadhyaya, Indu; Fancher, Samantha; Liu, Yanyan; Nair, Meera Surendran; Jankelunas, Leanne; Patel, Jitendra R; Venkitanarayanan, Kumar

    2016-09-01

    The efficacy of a new generation disinfectant, octenidine dihydrochloride (OH), as wash and coating treatments for reducing Listeria monocytogenes (LM), Salmonella spp. (SAL), and Escherichia coli O157:H7 (EC) on cantaloupe was investigated. Cantaloupe rind plugs inoculated separately with the three bacterial species (∼8 log CFU/cm(2)) were washed for 1, 3, 5 min at 25 °C in water, or chlorine (200 ppm), ethanol (1%), OH (0.01, 0.05, 0.1%) and surviving populations were measured after treatment. Additionally, inoculated cantaloupe rind plugs were coated with 2% chitosan or chitosan containing OH (0.01, 0.05, 0.1%) and sampled for surviving pathogens. Subsequently, the antimicrobial efficacy of OH wash and coating (0.1, 0.2%) on whole cantaloupes was determined. All OH wash reduced LM, SAL, and EC on cantaloupe rinds by > 5 log CFU/cm(2) by 2 min, and reduced populations to undetectable levels (below 2 log CFU/cm(2)) by 5 min (P < 0.05). Similarly, OH coating on cantaloupe rinds reduced the pathogens by 3-5 log /cm(2) (P < 0.05). Washing and coating whole cantaloupes with OH reduced the three pathogens by at least 5 log and 2 log CFU/cm(2), respectively (P < 0.05). Results suggest that OH could be used as antimicrobial wash and coating to reduce LM, SAL, and EC on cantaloupes.

  3. Development of novel elastic vesicle-based topical formulation of cetirizine dihydrochloride for treatment of atopic dermatitis.

    PubMed

    Goindi, Shishu; Kumar, Gautam; Kumar, Neeraj; Kaur, Amanpreet

    2013-12-01

    Cetirizine is a piperazine-derived second-generation antihistaminic drug recommended for treatment of pruritus associated with atopic dermatitis. The present investigation encompasses development of a nanosized novel elastic vesicle-based topical formulation of cetirizine dihydrochloride using combination of Phospholipon® 90G and edge activators with an aim to have targeted peripheral H1 antihistaminic activity. The formulation was optimized with respect to phospholipid/drug/charge inducer ratio along with type and concentration of edge activator. The optimized formulation was found to be satisfactory with respect to stability, drug content, entrapment efficiency, pH, viscosity, vesicular size, spreadability, and morphological characteristics. The ex vivo permeation studies through mice skin were performed using Franz diffusion cell assembly. It was found that the mean cumulative percentage amount permeated in 8 h was almost twice (60.001 ± 0.332) as compared to conventional cream (33.268 ± 0.795) and aqueous solution of drug (32.616 ± 0.969), suggesting better penetration and permeation of cetirizine from the novel vesicular delivery system. Further, therapeutic efficacy of optimized formulation was assessed against oxazolone-induced atopic dermatitis in mice. It was observed that the developed formulation was highly efficacious in reducing the itching score (4.75 itches per 20 min) compared to conventional cream (9.75 itches per 20 min) with profound reduction in dermal eosinophil count and erythema score. To conclude, a novel vesicular, dermally safe, and nontoxic topical formulation of cetirizine was successfully developed and may be used to treat atopic dermatitis after clinical investigation.

  4. INVESTIGATION AND OPTIMIZATION OF TITRIMETRIC AND SPECTROPHOTOMETRIC METHODS FOR THE ASSAY OF FLUNARIZINE DIHYDROCHLORIDE USING IN SITU BROMINE.

    PubMed

    Prashanth, Kudige Nagaraj; Swamy, Nagaraju; Basavaiah, Kanakapura

    2016-01-01

    Three indirect methods for the assay of flunarizine dihydrochloride (FNH) in bulk drug and commercial formulation based on titrimetric and spectrophotometric techniques using bromate-bromide mixture are described. In titrimetry, a measured excess of bromate-bromide mixture is added to an acidified solution of FNH and the unreacted bromine is determined iodometrically (method A). Spectrophotometry involves the addition of a known excess of bromate-bromide mixture to FNH in acid medium followed by estimation of unreacted bromine by its reaction with excess iodide and the liberated iodine (I₃⁻) is either measured at 370 nm (method B) or liberated iodine reacted with starch followed by the measurement of the blue colored starch-iodide complex at 575 run (method C). Titrimetric method is applicable over the range 4.5-30.0 mg FNH (method A), and the reaction stoichiometry is found to be 1:2 (FNH:KBrO₃). The spectrophotometric methods are applicable over the concentration ranges 0.8-16.0 µg/mL and 0.4-8.0 µg/mL FNH for method B and method C, respectively. The molar absorptivities are calculated to be 2.83 x 10⁴ and 4.96 x 10⁴ L mol⁻¹cm⁻¹ for method B and method C, respectively, and the corresponding Sandell sensitivity values are 0.0168 and 0.0096 µg cm⁻². The proposed methods have been applied successfully for the determination of FNH in pure form and in its dosage form and the results were compared with those of a literature method by applying the Student's t-test and F-test.

  5. Role of potassium channels in rabbit intestinal motility disorders induced by 2, 2'-azobis (2-amidinopropane) dihydrochloride (AAPH).

    PubMed

    Hernandez, L; Grasa, L; Fagundes, D S; Gonzalo, S; Arruebo, M P; Plaza, M A; Murillo, M D

    2010-06-01

    Oxidative stress appears to play a role in the pathogenesis of several inflammatory gastrointestinal diseases. Changes in intestinal motility have been reported in different models of intestinal inflammation. The initiating factor of altered motility could be an alteration of gut redox status. The aim of this study was to investigate the effect of oxidative stress evoked by 2, 2'-azobis (2-amidinopropane) dihydrochloride (AAPH) on the intestinal motility of rabbit duodenum and the possible contribution of different K(+) channels in mediating this response. Whole thickness segments of rabbit duodenum were suspended in the direction of the longitudinal or circular smooth muscle fibres in an organ bath to study the effects of AAPH alone, or in the presence of different K(+) channel blockers on the amplitude, frequency and tone of spontaneous contractions. In circular muscle, AAPH 20 mM induced a reduction of the amplitude, the frequency and tone of the spontaneous contractions. In longitudinal muscle, AAPH 10 mM induced a reduction of the amplitude and tone of the spontaneous contractions. The reduction of the amplitude and tone induced by AAPH was reverted by BaCl2 (1 mM) and TEA (5 mM). Charybdotoxin (100 nM) and iberiotoxin (100 nM) only reverted the reduction of the tone induced by AAPH. In conclusion, our results show that the peroxyl radicals released by AAPH reduced the amplitude and the tone of the spontaneous contractions of the longitudinal smooth muscle from rabbit small intestine. Inward rectifier and intermediate and large-conductance Ca(2+)-activated K(+) channels could be involved in these effects.

  6. Hyperthermic sensitization by the radical initiator 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH). I. In vitro studies.

    PubMed

    Krishna, M C; Dewhirst, M W; Friedman, H S; Cook, J A; DeGraff, W; Samuni, A; Russo, A; Mitchell, J B

    1994-01-01

    AAPH (2,2'-azobis-(2-amidinopropane dihydrochloride)) is a water-soluble, heat-labile azo compound which undergoes thermal decomposition to produce carbon-centred free radicals. These carbon-centred radicals might be directly cytotoxic or may react with oxygen to produce potentially cytotoxic alkoxyl and peroxyl radicals. The rate of free radical production as a result of AAPH thermal decomposition increases with increasing temperature. We have evaluated the efficacy of AAPH as a heat sensitizer for Chinese hamster V79 cells by the clonogenic assay. AAPH (50 mM) was not cytotoxic to V79 cells at 37 degrees C for exposures up to 3 h. In contrast, AAPH (50 mM) was found to markedly sensitize cells exposed to 42, 43 and 45 degrees C. For a 75 min exposure to 42 degrees C alone, cell survival was reduced to 9 x 10(-1); however, a 75 min exposure at 42 degrees C+AAPH resulted in survival of 5.5 x 10(-4). For 43 and 45.5 degrees C heating, cell survival was potentiated by AAPH at the 1% survival level by 4.1 and 1.4-fold, respectively. AAPH was also found to sensitize both hypoxic cells and thermotolerant cells. These findings would encourage in vivo evaluation of AAPH (or analogues) as a temperature-dependent heat sensitizer. AAPH represents a new class of heat sensitizers which may have use in unravelling the mechanism(s) of heat killing and may have utility in local hyperthermia treatment.

  7. 2,2'-Azobis (2-Amidinopropane) Dihydrochloride Is a Useful Tool to Impair Lung Function in Rats.

    PubMed

    Moreira Gomes, Maria D; Carvalho, Giovanna M C; Casquilho, Natalia V; Araújo, Andressa C P; Valença, Samuel S; Leal-Cardoso, Jose H; Zin, Walter A

    2016-01-01

    Recently, several studies have reported that respiratory disease may be associated with an increased production of free radicals. In this context, 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) is a free radical-generating compound widely used to mimic the oxidative stress state. We aimed to investigate whether AAPH can generate lung functional, inflammatory, histological and biochemical impairments in the lung. Wistar rats were divided into five groups and instilled with saline solution (714 μL/kg, CTRL group) or different amounts of AAPH (25, 50, 100, and 200 mg/kg, 714 μL/kg, AAPH groups). Seventy-two hours later the animals were anesthetized, paralyzed, intubated and static elastance (Est), viscoelastic component of elastance (ΔE), resistive (ΔP1) and viscoelastic (ΔP2) pressures were measured. Oxidative damage, inflammatory markers and lung morphometry were analyzed. ΔP1 and Est were significantly higher in AAPH100 and AAPH200 than in the other groups. The bronchoconstriction indexes were larger in AAPH groups than in CTRL. The area occupied by collagen and elastic fibers, polymorpho- and mononuclear cells, malondialdehyde and carbonyl groups levels were significantly higher in AAPH200 than in CTRL. In comparison to CTRL, AAPH200 showed significant decrease and increase in the activities of superoxide dismutase and catalase, respectively. AAPH augmented the release of pro-inflammatory cytokines IL-1β, IL-6 e TNF-α. Hence, exposure to AAPH caused significant inflammatory alterations and redox imbalance accompanied by altered lung mechanics and histology. Furthermore, we disclosed that exposure to AAPH may represent a useful in vivo tool to trigger lung lesions.

  8. Impact of antiseptics on radical metabolism, antioxidant status and genotoxic stress in blood cells: povidone-iodine versus octenidine dihydrochloride.

    PubMed

    Wagner, Karl-Heinz; Jürss, Alice; Zarembach, Beate; Elmadfa, Ibrahim

    2004-08-01

    No sufficient data are available of the of antiseptics' influence on human blood cells. Effects of two antiseptics, povidone-iodine (PVD-I) versus octenidine dihydrochloride (OD), were tested on antioxidant status, radical formation, antioxidant defence enzymes and genotoxic stress in blood cells, in vitro. Human blood was taken by venipuncture, enriched with PVD-I or OD (0.0001-20% final concentration) and incubated at 37 degrees C between 30 and 120 min. alpha-Tocopherol was assessed in erythrocytes and granulocytes. Superoxide-dismutase (SOD) and glutathione (GSH) were determined in erythrocytes, the total anti-oxidative capacity (TAC) and malondialdehyde (MDA) in their ghosts. In granulocytes status of hydrogen peroxide (H(2)O(2)), superoxide anions and MDA was observed. Genotoxic stress was determined by counting sister chromatide exchanges (SCE) in lymphocytes after enrichment within 0.05-0.4% of antiseptics. Based on all biomarker tested, concentrations up to 0.05% incubated for 30 min did not affect cell metabolism. 1% and 10% PVD-I reduced the activity of SOD (-40%), GSH (-62%) and the content of alpha-tocopherol more than OD (p<0.05). No significant differences between the antiseptics were observed for TAC and MDA. H(2)O(2) and superoxide anions were significantly reduced after the 10% addition for both substances independent on the exposure. Without having changes in lipid oxidation, the reduction of antioxidative defence mechanisms must be due to the oxidation caused by the antiseptics, mainly PVD-I. An increased SCE rate was neither observed with PVD-I nor with OD within an enrichment with 0.05-0.4%. Higher concentrations (1% and more) could not be tested on SCE formation because they caused cell bursts. The results presented indicate that concentrations up to 0.05% incubated for 30 min are safe for exposing blood cells of healthy subjects.

  9. 2,2′-Azobis (2-Amidinopropane) Dihydrochloride Is a Useful Tool to Impair Lung Function in Rats

    PubMed Central

    Moreira Gomes, Maria D.; Carvalho, Giovanna M. C.; Casquilho, Natalia V.; Araújo, Andressa C. P.; Valença, Samuel S.; Leal-Cardoso, Jose H.; Zin, Walter A.

    2016-01-01

    Recently, several studies have reported that respiratory disease may be associated with an increased production of free radicals. In this context, 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH) is a free radical-generating compound widely used to mimic the oxidative stress state. We aimed to investigate whether AAPH can generate lung functional, inflammatory, histological and biochemical impairments in the lung. Wistar rats were divided into five groups and instilled with saline solution (714 μL/kg, CTRL group) or different amounts of AAPH (25, 50, 100, and 200 mg/kg, 714 μL/kg, AAPH groups). Seventy-two hours later the animals were anesthetized, paralyzed, intubated and static elastance (Est), viscoelastic component of elastance (ΔE), resistive (ΔP1) and viscoelastic (ΔP2) pressures were measured. Oxidative damage, inflammatory markers and lung morphometry were analyzed. ΔP1 and Est were significantly higher in AAPH100 and AAPH200 than in the other groups. The bronchoconstriction indexes were larger in AAPH groups than in CTRL. The area occupied by collagen and elastic fibers, polymorpho- and mononuclear cells, malondialdehyde and carbonyl groups levels were significantly higher in AAPH200 than in CTRL. In comparison to CTRL, AAPH200 showed significant decrease and increase in the activities of superoxide dismutase and catalase, respectively. AAPH augmented the release of pro-inflammatory cytokines IL-1β, IL-6 e TNF-α. Hence, exposure to AAPH caused significant inflammatory alterations and redox imbalance accompanied by altered lung mechanics and histology. Furthermore, we disclosed that exposure to AAPH may represent a useful in vivo tool to trigger lung lesions. PMID:27812337

  10. Measuring antioxidant efficiency of wort, malt, and hops against the 2,2'-azobis(2-amidinopropane) dihydrochloride-induced oxidation of an aqueous dispersion of linoleic acid.

    PubMed

    Liégeois, C; Lermusieau, G; Collin, S

    2000-04-01

    This paper presents a simple, convenient method for determining the efficiency of antioxidants in aqueous systems. Production of conjugated diene hydroperoxide by oxidation of linoleic acid in an aqueous dispersion is monitored at 234 nm. 2, 2'-Azobis(2-amidinopropane) dihydrochloride is used as a free radical initiator. Among 12 antioxidants tested, phenolic compounds proved to be the most efficient, both kinetically and in terms of the inhibition time (T(inh)). Applied to wort, malt, and hops, the method confirmed a significant antioxidant activity in such products, especially hops. This assay can be used to follow oxidative changes throughout the brewing process and to understand the contribution of each raw material.

  11. Molecular characterization of constitutive heterochromatin in three species of Trypoxylon (Hymenoptera: Crabronidae: Trypoxylini) by CMA3/DAPI staining

    PubMed Central

    Menezes, Rodolpho Santos Telles; Carvalho, Antonio Freire; Silva, Janisete Gomes; Costa, Marco Antonio

    2011-01-01

    Abstract Previous cytogenetic analyses in Trypoxylon Latreille, 1796 have been basically restricted to C-banding. In the present study, base-specific CMA3 and DAPI fluorochrome staining were used to characterize the constitutive heterochromatin in three Trypoxylon species. The heterochromatin was GC-rich in all the species studied; however, in Trypoxylon nitidum F. Smith, 1856the molecular composition of the heterochromatinwasdifferent among chromosome pairs. Conversely, the euchromatin was AT-rich in the three species. These results suggest high conservatism in the euchromatic regions as opposed to the heterochromatic regions that have a high rate of changes. In this study, we report the karyotype of Trypoxylon rugifrons F. Smith, 1873which has the lowest chromosome number in the genus and other characteristics of the likely ancestral Trypoxylon karyotype. PMID:24260620

  12. Molecular characterization of constitutive heterochromatin in three species of Trypoxylon (Hymenoptera: Crabronidae: Trypoxylini) by CMA3/DAPI staining.

    PubMed

    Menezes, Rodolpho Santos Telles; Carvalho, Antonio Freire; Silva, Janisete Gomes; Costa, Marco Antonio

    2011-01-01

    Previous cytogenetic analyses in Trypoxylon Latreille, 1796 have been basically restricted to C-banding. In the present study, base-specific CMA3 and DAPI fluorochrome staining were used to characterize the constitutive heterochromatin in three Trypoxylon species. The heterochromatin was GC-rich in all the species studied; however, in Trypoxylon nitidum F. Smith, 1856the molecular composition of the heterochromatinwasdifferent among chromosome pairs. Conversely, the euchromatin was AT-rich in the three species. These results suggest high conservatism in the euchromatic regions as opposed to the heterochromatic regions that have a high rate of changes. In this study, we report the karyotype of Trypoxylon rugifrons F. Smith, 1873which has the lowest chromosome number in the genus and other characteristics of the likely ancestral Trypoxylon karyotype.

  13. Extraction-Free Ion-Pair Methods for the Assay of Trifluoperazine Dihydrochloride in Bulk Drug, Tablets, and Spiked Human Urine Using Three Sulfonphthalein Dyes

    NASA Astrophysics Data System (ADS)

    Prashanth, K. N.; Swamy, N.; Basavaiah, K.

    2014-11-01

    Three simple and sensitive extraction-free spectrophotometric methods are described for the determination of trifluoperazine dihydrochloride (TFH). The methods are based on ion pair complex formation between the nitrogenous compound trifluoperazine (TFP) converted from trifluoperazine dihydrochloride and sulfonphthalein dyes, namely, bromocresol green (BCG), bromothymol blue (BTB), and bromophenol blue (BPB) in dichloromethane medium in which all the above experimental variables were circumvented. The colored products are measured at 425 nm in the BCG method, 415 nm in the BTB method, and 420 nm in the BPB method. The stoichiometry of the ion-pair complexes formed between the drug and dye (1:1) was determined by Job's continuous variations method, and the stability constants of the complexes were also calculated. These methods quantify TFP over the concentration ranges of 1.25-20.0 μg/ml in the BCG method, 1.5-21.0 μg/ml in the BTB method, and 1.5-18.0 μg/ml in the BPB method. The molar absorptivity (l·mol-1·cm-1) and Sandell sensitivity (ng/cm2) were calculated to be 2.06·104 and 0.0197; 1.82·104 and 0.0224; and 2.22·104 and 0.0183 for the BCG, BTB, and BPB methods, respectively. The methods were successfully applied to the determination of TFP in pure drug, pharmaceuticals, and in spiked human urine with good accuracy and precision.

  14. Comparison of the Antimicrobial Efficacy of Octenidine Dihydrochloride and Chlorhexidine with and Without Passive Ultrasonic Irrigation - An Invitro Study

    PubMed Central

    Cherian, Bastin; Manjunath, Mysore Krishnaswamy

    2016-01-01

    Introduction Elimination of microorganisms from infected root canals is a complicated task. Numerous measures have been described to reduce the microbial load in the root canal system, including the use of various instrumentation techniques, irrigation regimens and intracanal medicaments. The drawbacks of few commonly used irrigants include toxic and harmful side effects, microbial resistance to antimicrobial agents and staining. Hence there is a need for alternative agents which are nontoxic, effective and safe. Aim To compare and evaluate antimicrobial effects of 2% Chlorhexidine (CHX) versus 0.1% Octenidine Dihydrochloride (OCT) as root canal irrigant with and without passive ultrasonic irrigation against Enterococcus faecalis (E. faecalis) in vitro and to evaluate the depth of penetration of irrigant solution into the dentinal tubules at the junction of middle and apical third. Materials and Methods Forty eight freshly extracted, single rooted human mandibular premolars were decoronated and root specimen standardized to 14mm. Biofilm of E. faecalis (strain ATCC 29212) was grown for seven days and the specimens were divided into four groups (n=12) based on irrigation protocol : Group I- Conventional Syringe Irrigation (CSI) with 2% CHX, Group II- CSI + 0.1% OCT, Group III-Passive Ultrasonic Irrigation (PUI) + 2% CHX and Group IV- PUI+ 0.1% OCT. Dentin shavings were collected at two depths (200μm and 400μm) and total number of colony forming units were determined. The data were statistically analyzed using ANOVA, Scheffes multiple comparison of means and paired t-test (p<0.05). Results Group III and IV (PUI) showed significant difference compared to Group I and II (CSI) both at 200μm and 400μm (p=0.000). For Group III and Group IV no significant differences were found at 200μm and 400μm (p=1.000 and 0.363 respectively), however significant difference was found between data at 200μm and 400μm for all the four groups (p=0.000). Conclusion Octenidine (0

  15. Karyotype analysis of four jewel-beetle species (Coleoptera, Buprestidae) detected by standard staining, C-banding, AgNOR-banding and CMA3/DAPI staining

    PubMed Central

    Karagyan, Gayane; Lachowska, Dorota; Kalashian, Mark

    2012-01-01

    Abstract The male karyotypes of Acmaeodera pilosellae persica Mannerheim, 1837 with 2n=20 (18+neoXY), Sphenoptera scovitzii Faldermann, 1835 (2n=38–46), Dicerca aenea validiuscula Semenov, 1895 – 2n=20 (18+Xyp) and Sphaerobothris aghababiani Volkovitsh et Kalashian, 1998 – 2n=16 (14+Xyp) were studied using conventional staining and different chromosome banding techniques: C-banding, AgNOR-banding, as well as fluorochrome Chromomycin A3 (CMA3) and DAPI. It is shown that C-positive segments are weakly visible in all four species which indicates a small amount of constitutive heterochromatin (CH). There were no signals after DAPI staining and some positive signals were discovered using CMA3 staining demonstrating absence of AT-rich DNA and presence of GC-rich clusters of CH. Nucleolus organizing regions (NORs) were revealed using Ag-NOR technique; argentophilic material mostly coincides with positive signals obtained using CMA3 staining. PMID:24260661

  16. Karyotype analysis of four jewel-beetle species (Coleoptera, Buprestidae) detected by standard staining, C-banding, AgNOR-banding and CMA3/DAPI staining.

    PubMed

    Karagyan, Gayane; Lachowska, Dorota; Kalashian, Mark

    2012-01-01

    The male karyotypes of Acmaeodera pilosellae persica Mannerheim, 1837 with 2n=20 (18+neoXY), Sphenoptera scovitzii Faldermann, 1835 (2n=38-46), Dicerca aenea validiuscula Semenov, 1895 - 2n=20 (18+Xyp) and Sphaerobothris aghababiani Volkovitsh et Kalashian, 1998 - 2n=16 (14+Xyp) were studied using conventional staining and different chromosome banding techniques: C-banding, AgNOR-banding, as well as fluorochrome Chromomycin A3 (CMA3) and DAPI. It is shown that C-positive segments are weakly visible in all four species which indicates a small amount of constitutive heterochromatin (CH). There were no signals after DAPI staining and some positive signals were discovered using CMA3 staining demonstrating absence of AT-rich DNA and presence of GC-rich clusters of CH. Nucleolus organizing regions (NORs) were revealed using Ag-NOR technique; argentophilic material mostly coincides with positive signals obtained using CMA3 staining.

  17. FISH and DAPI staining of the synaptonemal complex of the Nile tilapia (Oreochromis niloticus) allow orientation of the unpaired region of bivalent 1 observed during early pachytene.

    PubMed

    Ocalewicz, Konrad; Mota-Velasco, Jose C; Campos-Ramos, Rafael; Penman, David J

    2009-01-01

    Bivalent 1 of the synaptonemal complex (SC) in XY male Oreochromis niloticus shows an unpaired terminal region in early pachytene. This appears to be related to recombination suppression around a sex determination locus. To allow more detailed analysis of this, and unpaired regions in the karyotype of other Oreochromis species, we developed techniques for FISH on SC preparations, combined with DAPI staining. DAPI staining identified presumptive centromeres in SC bivalents, which appeared to correspond to the positions observed in the mitotic karyotype (the kinetochores could be identified only sporadically in silver-stained EM SC images). Furthermore, two BAC clones containing Dmo (dmrt4) and OniY227 markers that hybridize to known positions in chromosome pair 1 in mitotic spreads (near the centromere, Flpter 0.25, and the putative sex-determination locus, Flpter 0.57, respectively) were used as FISH probes on SCs to verify that the presumptive centromere identified by DAPI staining was located in the expected position. Visualization of both the centromere and FISH signals on bivalent 1 allowed the unpaired region to be positioned at Flpter 0.80 to 1.00, demonstrating that the unpaired region is located in the distal part of the long arm(s). Finally, differences between mitotic and meiotic measurements are discussed.

  18. A study of embryonic development in eriophyoid mites (Acariformes, Eriophyoidea) with the use of the fluorochrome DAPI and confocal microscopy.

    PubMed

    Chetverikov, Philipp E; Desnitskiy, Alexey G

    2016-01-01

    The embryonic development of four eriophyoid mite species, Cecidophyopsis ribis, Phytoptus avellanae, Oziella liroi and Loboquintus subsquamatus, has been studied with the use of fluorochrome DAPI and confocal microscopy. The first three nuclear divisions occur on the egg periphery (the groups of 2, 4, and 6 nuclei have been recorded), while the biggest part of yolk remains undivided. After four or five nuclear divisions all nuclei are situated only in one sector of the embryo, while other sectors contain only yolk suggesting possible meroblastic cleavage. Later, the formation of superficial blastoderm takes place. A few large yolk cells are situated inside the embryo. Germ band formation initiates as funnel-like cell invagination and leads to formation of a typical stage with four paired prosomal buds (chelicerae, palps, legs I and II). Each palp contains two lobes (anterior and posterior), the adult subcapitulum is presumably a fusion product of the anterior pair of the lobes. Neither rudiments of legs III and IV, traces of opisthosomal segments nor remnants of the prelarval exuvium under the egg shell were detected. Overall, the pattern of embryonic development in eriophyoids re-emphasizes the peculiarity of this ancient group of miniaturized phytoparasitic animals, and invites researches to pursue a deeper investigation of various fundamental aspects of this aberrant group of Acari. Further studies using various fluorescent dyes and transmission electron microscopy are needed to visualize plasma membranes and clarify the pattern of early cleavage of eriophyoids.

  19. Cytogenetic characterization of complex karyotypes in seven established melanoma cell lines by multiplex fluorescence in situ hybridization and DAPI banding.

    PubMed

    Schulten, Hans Jürgen; Gunawan, Bastian; Otto, Friedrich; Hassmann, René; Hallermann, Christian; Noebel, Albrecht; Füzesi, László

    2002-03-01

    We report the use of multiplex fluorescence in situ hybridization (M-FISH) to resolve chromosomal aberrations in seven established melanoma cell lines with hypotriploid to hypertetraploid complex karyotypes. By simultaneous identification of all human chromosomes in single FISH experiments using a set of 52 directly labeled, whole chromosome painting probes, cryptic chromosomal translocations and the origin of unclear chromosomal material in structural rearranged and marker chromosomes could be identified, refining the tumor karyotypes in all seven cell lines. The number of structural aberrations in each cell line assigned with combined M-FISH and DAPI banding analysis ranged from 15 to 45. Altogether, 275 breakpoints could be assigned to defined chromosomal regions or bands. The chromosome arms 1p, 6q, 7p, 9p, and 11q which are known to be nonrandomly associated with melanoma tumorigenesis, were frequently involved in chromosomal breaks and/or copy number changes. This study also demonstrated the practical usefulness of combining M-FISH with conventional cytogenetic banding techniques for the characterization of complex tumor karyotypes with massive genomic alterations.

  20. Utility of positron annihilation lifetime technique for the assessment of spectroscopic data of some charge-transfer complexes derived from N-(1-Naphthyl)ethylenediamine dihydrochloride.

    PubMed

    Refat, Moamen S; Adam, Abdel Majid A; Sharshar, T; Saad, Hosam A; Eldaroti, Hala H

    2014-03-25

    In this work, structural, thermal, morphological, pharmacological screening and positron annihilation lifetime measurements were performed on the interactions between a N-(1-Naphthyl)ethylenediamine dihydrochloride (NEDA·2HCl) donor and three types of acceptors to characterize these CT complexes. The three types of acceptors include π-acceptors (quinol and picric acid), σ-acceptors (iodine) and vacant orbital acceptors (tin(IV) tetrachloride and zinc chloride). The positron annihilation lifetime parameters were found to be dependent on the structure, electronic configuration, the power of acceptors and molecular weight of the CT complexes. The positron annihilation lifetime spectroscopy can be used as a probe for the formation of charge-transfer (CT) complexes.

  1. Utility of positron annihilation lifetime technique for the assessment of spectroscopic data of some charge-transfer complexes derived from N-(1-Naphthyl)ethylenediamine dihydrochloride

    NASA Astrophysics Data System (ADS)

    Refat, Moamen S.; Adam, Abdel Majid A.; Sharshar, T.; Saad, Hosam A.; Eldaroti, Hala H.

    2014-03-01

    In this work, structural, thermal, morphological, pharmacological screening and positron annihilation lifetime measurements were performed on the interactions between a N-(1-Naphthyl)ethylenediamine dihydrochloride (NEDA·2HCl) donor and three types of acceptors to characterize these CT complexes. The three types of acceptors include π-acceptors (quinol and picric acid), σ-acceptors (iodine) and vacant orbital acceptors (tin(IV) tetrachloride and zinc chloride). The positron annihilation lifetime parameters were found to be dependent on the structure, electronic configuration, the power of acceptors and molecular weight of the CT complexes. The positron annihilation lifetime spectroscopy can be used as a probe for the formation of charge-transfer (CT) complexes.

  2. Carcinogenesis studies of 4,4'-methylenedianiline dihydrochloride given in drinking water to F344/N rats and B6C3F1 mice.

    PubMed

    Lamb, J C; Huff, J E; Haseman, J K; Murthy, A S; Lilja, H

    1986-01-01

    Carcinogenesis studies of 4,4'-methylenedianiline dihydrochloride (98.6% pure) were conducted by administering this chemical in the drinking water of F344/N rats and B6C3F1 mice. Groups of 50 rats and 50 mice of each sex received drinking water containing 150 or 300 ppm 4,4'-methylenedianiline dihydrochloride (dosage expressed as the free base) for 103 wk. Groups of 50 rats and 50 mice of each sex, given drinking water adjusted with 0.1 N HCl to the pH (3.7) of the 300-ppm formulation, served as controls. Survival was comparable among groups except for male mice receiving the 300-ppm dose of 4,4'-methylenedianiline dihydrochloride; survival in that group was lower than that in controls. Mean body weight was reduced in 300-ppm-dose female rats and 300-ppm-dose male and female mice compared to controls. Water consumption was reduced in a dose-related manner in both sexes of rats. No compound-related clinical effects were observed. Under the conditions of these studies, there was clear evidence of carcinogenicity for F344/N rats and for B6C3F1 mice in that 4,4'-methylenedianiline dihydrochloride caused increased incidences of (1) follicular-cell carcinomas of the thyroid gland (controls, 0/49; low dose, 0/47; high dose, 7/48, 15%; p less than or equal to 0.012) and neoplastic nodules of the liver (controls, 1/50, 2%; low dose, 12/50, 24%; high dose, 25/50, 50%; p less than or equal to 0.001) in male rats, (2) follicular-cell adenomas (controls, 0/47; low dose, 2/47, 4%; high dose, 17/48, 35%; p less than or equal to 0.001) and C-cell adenomas (controls, 0/47; low dose, 3/47, 6%; high dose, 6/48, 13% p less than or equal to 0.029) of the thyroid gland in female rats, (3) follicular-cell adenomas of the thyroid gland (controls, 0/47; low dose, 3/49, 6%; high dose, 16/49, 33%; p less than or equal to 0.001), carcinomas of the liver (controls, 10/49, 20%; low dose, 33/50, 66%; high dose, 29/50, 58%; p less than or equal to 0.001), and pheochromocytomas of the adrenal

  3. Electro-oxidation and determination of antihistamine drug, cetirizine dihydrochloride at glassy carbon electrode modified with multi-walled carbon nanotubes.

    PubMed

    Patil, Roopa H; Hegde, Rajesh N; Nandibewoor, Sharanappa T

    2011-03-01

    A multi-walled carbon nanotube (MWCNT) film-modified glassy carbon electrode (GCE) was constructed for the determination of an antihistamine drug, cetirizine dihydrochloride (CTZH) using cyclic voltammetry (CV). Owing to the unique structure and extraordinary properties of MWCNT, the MWCNT film has shown an obvious electrocatalytic activity towards oxidation of CTZH, since it facilitates the electron transfer and significantly enhances the oxidation peak current of CTZH. All experimental parameters have been optimized. Under the optimum conditions, the oxidation peak current was linearly proportional to the concentration of CTZH in the range from 5.0×10(-7) to 1.0×10(-5)M. The detection limit was 7.07×10(-8)M with 180s accumulation. Finally, the proposed sensitive and simple electrochemical method was successfully applied to CTZH determination in pharmaceutical and urine samples.

  4. Changes in ascorbic acid content in primary cultured rat hepatocytes exposed to 2,2'-azobis (2-amidinopropane) dihydrochloride, a radical initiator.

    PubMed

    Sasakii, K; Kimura, K; Kitaguchi, Y; Onoue, T; Ogura, H; Aoyagi, Y

    2001-08-01

    Changes in ascorbate content in primary cultured rat hepatocytes exposed to oxidative stress derived from water soluble radical initiator 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) were examined. Cells were exposed to 0.05 and 5 mg/ml of AAPH as 'mild' and severe' oxidative stresses, respectively. Lipid peroxidation in hepatocytes was induced by 'severe' oxidative stress, but not by 'mild' oxidative stress. Ascorbate decreased at 6 hr after administration of both mild' and severe' oxidative stresses, and recovered to the control level after a further 6 hr. In cells treated with 'severe oxidative stress, however, total ascorbate (reduced form plus oxidized form) had increased 24 hr after administration. These results indicated that consumption alone did not account for the increase of ascorbate in hepatocytes under oxidative stress.

  5. Damage to biological tissues induced by radical initiator 2,2'-azobis(2-amidinopropane) dihydrochloride and its inhibition by chain-breaking antioxidants.

    PubMed

    Terao, K; Niki, E

    1986-01-01

    A water-soluble azo compound, 2,2'-azobis(2-amidinopropane) dihydrochloride, a well-known free radical initiator, was administered intraperitoneally to mice to study the toxicological effects on biological tissues in vivo and their inhibition by chain-breaking antioxidants. It caused damage to biological tissues without biotransformation. No specific target organ was observed. The most striking fine structural changes were the degeneration, swelling, and disruption of the endothelium lining cells of the capillaries in various organs. Furthermore, the death of lymphocytes in the lymphoid tissues and the fatty degeneration of the liver and kidneys have also been observed. Water-soluble chain-breaking antioxidants, such as 2-carboxy-2,5,7,8-tetramethyl-6-chromanol (a vitamin E analogue), uric acid, cysteine, and glutathione suppressed the above damage, whereas vitamin C was ineffective.

  6. Protective effect of ferulic acid against 2,2'-azobis(2-amidinopropane) dihydrochloride-induced oxidative stress in PC12 cells.

    PubMed

    Shen, Y; Zhang, H; Wang, L; Qian, H; Qi, Y; Miao, X; Cheng, L; Qi, X

    2016-01-31

    Oxidative stress is closely related to the pathogenesis of neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease. However, the underlying antioxidant mechanisms of ferulic acid (FA) aganist oxidantive stress are poorly understood. We evaluated the potential protective effects of FA against 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced damage in PC12 cells. Our results indicated that pretreatment with FA prior to AAPH exposure significantly increased PC12 cell survival, and also increased catalase and superoxide dismutase activity. Furthermore, FA treatment reduced cellular lactate dehydrogenase release and malondialdehyde levels. It attenuated AAPH-induced apoptosis in PC12 cells, as determined by flow cytometric detection of annexin V. Reductions in mitochondrial membrane potential and accumulation of intracellular Ca2+ were also inhibited by FA treatment. These findings suggested that FA protected PC12 cells against AAPH-induced oxidative stress, and may be a neuroprotective agent.

  7. Development of a Bladder Bioreactor for Tissue Engineering in Urology.

    PubMed

    Davis, Niall F; Callanan, Anthony

    2016-01-01

    A urinary bladder bioreactor was constructed to replicate physiological bladder dynamics. A cyclical low-delivery pressure regulator mimicked filling pressures of the human bladder. Cell growth was evaluated by culturing human urothelial cells (UCs) on porcine extracellular matrix scaffolds (ECMs) in the bioreactor and in static growth conditions for 5 consecutive days. UC proliferation was compared with quantitative viability indicators and by fluorescent markers for intracellular esterase activity and plasma membrane integrity. Scaffold integrity was characterized with scanning electron microscopy and 4,6-diamidino-2-phenylindole staining.

  8. Dielectric barrier discharge plasma in Ar/O{sub 2} promoting apoptosis behavior in A549 cancer cells

    SciTech Connect

    Huang Jun; Li Hui; Chen Wei; Lv Guohua; Wang Xingquan; Zhang Guoping; Wang Pengye; Ostrikov, Kostya; Yang Size

    2011-12-19

    The Ar/O{sub 2} plasma needle in the induction of A549 cancer cells apoptosis process is studied by means of real-time observation. The entire process of programmed cell death is observed. The typical morphological changes of A549 apoptosis are detected by 4', 6-diamidino-2-phenylindole staining, for example, chromatin condensation and nuclear fragmentation. Cell viability is determined and quantified by neutral red uptake assay, and the survival rate of A549 from Ar/O{sub 2} plasmas is presented. Further spectral analysis indicates the reactive species, including O and OH play crucial roles in the cell inactivation.

  9. Dielectric barrier discharge plasma in Ar/O2 promoting apoptosis behavior in A549 cancer cells

    NASA Astrophysics Data System (ADS)

    Huang, Jun; Li, Hui; Chen, Wei; Lv, Guo-Hua; Wang, Xing-Quan; Zhang, Guo-Ping; Ostrikov, Kostya; Wang, Peng-Ye; Yang, Si-Ze

    2011-12-01

    The Ar/O2 plasma needle in the induction of A549 cancer cells apoptosis process is studied by means of real-time observation. The entire process of programmed cell death is observed. The typical morphological changes of A549 apoptosis are detected by 4', 6-diamidino-2-phenylindole staining, for example, chromatin condensation and nuclear fragmentation. Cell viability is determined and quantified by neutral red uptake assay, and the survival rate of A549 from Ar/O2 plasmas is presented. Further spectral analysis indicates the reactive species, including O and OH play crucial roles in the cell inactivation.

  10. Combined FISH, anti-γ-Hb and DAPI for detection of fetal nucleated RBCs in maternal blood

    NASA Astrophysics Data System (ADS)

    Farhad, Mona; Price, Jeffrey H.

    2002-05-01

    Since the 1970s, extensive research has been devoted to the development of a standard procedure for the isolation of fetal nucleated red cells (fnRBCs) from maternal blood. Since these cells are sources of fetal DNA, cytogenetic analysis would lead to a minimally-invasive method for the prenatal diagnosis of chromosomal and genetic disorders early in gestation. FnRBCs constitute a significant portion of the fetal blood, have a short and finite life span, and are rare in peripheral adult blood. They have been reported to exist in the maternal circulation at frequencies as low as 1:105 - 1:109 maternal nucleated cells. Due to these ultra-rare frequencies, isolation with minimal loss has been a time and labor-intensive process. To overcome this problem, a fully automated scanning cytometer that incorporates high-performance autofocus and image segmentation has been built and shown higher rate, quantity, sensitivity (true positive rate) and specificity (true negative rate) in a model cell preparation. For detecting fnRBCs, two discriminating characteristics may suffice: (1) the presence of fetal hemoglobin, which is the major intracytoplasmic protein found in fetal red cells from 5 to 35 weeks gestation, and (2) the presence of a nucleus. In clinical trials, the fetal origin of the isolated cells will be confirmed by fluorescence in situ hybridization (FISH) on the X and Y chromosomes in male pregnancies. The aim of the present study was to develop a reliable and reproducible staining method for combined immunofluorescence and FISH analysis for these clinical trials. This staining technique was developed using fnRBCs extracted from fetal liver blood and a human erythroleukemia cell line (HEL) that expresses fetal hemoglobin. The resulting method for four-color X- and Y-FISH , anti-(gamma) -Hb fluorescence and DAPI staining was consistent and bright.

  11. New DAPI and FISH findings on egg maturation processes in related hybridogenetic and parthenogenetic Bacillus hybrids (Insecta, Phasmatodea).

    PubMed

    Marescalchi, O; Scali, V

    2001-10-01

    Bacillus stick insects have proved adequate for studying a wide array of reproductive modes: sexual, parthenogenetic, hybridogenetic, androgenetic. Hybridogenetic strains (B. rossius-grandii) were thought to discard the paternal "grandii" haploset during first meiotic division and keep the "rossius" hemiclone, whereas the clonal B. whitei (=rossius/grandii) would maintain its hybrid structure by fusing back two nonsister nuclei-each derived from previously segregated heterospecific complements-by the end of the 2(nd) meiotic division. New investigations on laid eggs and ovariole squashes, either DAPI stained or FISH labeled, revealed that in hybridogens the "grandii" set is excluded from the germ line prior to meiosis and that a DNA extra-synthesis should occur to produce hemiclonal eggs after two cytologically normal meiotic divisions. On the other hand, in B. whitei eggs no genome segregation appears to occur and an intrameiotic DNA extra-synthesis must take place to produce 2n tetrachromatidic oocytes I; these divide twice and give unreduced clonal eggs. The new findings bring hybridogenetic oogenesis of Bacillus to be coincident with that of the known hemiclonal organisms and point to an independent onset of B. whitei from hemiclonal strains. In addition, B. whitei gains a closer resemblance to B. lynceorum owing to the sharing of a cytologically identical egg maturation mechanism, of the same maternal ancestor and of peculiar chromosomal features. It is here suggested that B. lynceorum originated from the incorporation of an "atticus" genome into a B. whitei egg, according to a pathway of repeated hybridization often occurred with other polyploid hybrids.

  12. Genome exclusion and gametic DAPI-DNA content in the hybridogenetic Bacillus rossius-grandii benazzii complex (Insecta Phasmatodea).

    PubMed

    Tinti, F; Scali, V

    1992-11-01

    Among Sicilian stick insects, two hybridogenetic complexes have been discovered: Bacillus rossius-grandii benazzii and B. rossius-grandii grandii, which also produce androgenetic offspring. The egg maturation of the former is analyzed here through DAPI fluorometry, which, besides the assessment of the meiotic stages, also allows their DNA measurements and the analysis of sperm-head evolution into male pronuclei in these polyspermic eggs. Hybridogenetic eggs undergo an extrasynthesis of chromosomes, because two groups of n autobivalents (4C each) are segregated at metaphase 1st; the two groups must correspond to the pure parental species haplosets. Then the grandii chromosomes degenerate (1st polar body), while the rossius chromosomes divide further to produce two groups of n autodiads (2C each); one of them degenerates (2nd polar body), and the other is ready to perform syngamy (female pronucleus). Meanwhile, several B. grandii sperm evolve into male pronuclei by doubling their DNA (from 1C to 2C content) and assuming an interphase nucleus appearance. If regular mixis occurs, the F1 hybrid constitution is restored but, if it fails, a fusion between two sperms may occur, originating fully paternal descendants (natural androgenesis). The genome exclusion mechanism of stick-insect hybridogens appears to be more primitive than those observed in the already known hybridogenetic complexes of Poeciliopsis and Rana esculenta. Unfertilized eggs of hybridogens are capable of self-activation, but the cytology of the related clonally reproducing B. whitei indicates that its parthenogenetic mechanism stems from the hybridization event (hybrid theory) rather than from tychoparthenogenetic potentialities (spontaneous theory).

  13. Assessment of a dye permeability assay for determination of inactivation rates of Cryptosporidium parvum oocysts.

    PubMed Central

    Jenkins, M B; Anguish, L J; Bowman, D D; Walker, M J; Ghiorse, W C

    1997-01-01

    The ability to determine inactivation rates of Cryptosporidium parvum oocysts in environmental samples is critical for assessing the public health hazard of this gastrointestinal parasite in watersheds. We compared a dye permeability assay, which tests the differential uptake of the fluorochromes 4'-6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) by the oocysts (A. T. Campbell, L. J. Robertson, and H. V. Smith, Appl. Environ. Microbiol. 58:3488-3493, 1992), with an in vitro excystation assay, which tests their ability to excyst and, thus, their metabolic potential and potential for infectivity (J.B. Rose, H. Darbin, and C.P. Gerba, Water Sci. Technol. 20:271-276, 1988). Formaldehyde-fixed (killed) oocysts and untreated oocysts were permeabilized with sodium hypochlorite and subjected to both assays. The results of the dye permeability assays were the same, while the excystation assay showed that no excystation occurred in formaldehyde-fixed oocysts. This confirmed that oocyst wall permeability, rather than metabolic activity potential, was the basis of the dye permeability viability assessment. A previously developed protocol (L. J. Anguish and W. C. Ghiorse, Appl. Environ. Microbiol. 63:724-733, 1997) for determining viability of oocysts in soil and sediment was used to examine further the use of oocyst permeability status as an indicator of oocyst viability in fecal material stored at 4 degrees C and in water at various temperatures. Most of the oocysts in fresh calf feces were found to be impermeable to the fluorochromes. They were also capable of excystation, as indicated by the in vitro excystation assay, and were infective, as indicated by a standard mouse infectivity assay. The dye permeability assay further showed that an increase in the intermediate population of oocysts permeable to DAPI but not to PI occurred over time. There was also a steady population of oocysts permeable to both dyes. Further experiments with purified oocysts suspended in

  14. X-Ray induced cataract is preceded by LEC loss, and coincident with accumulation of cortical DNA, and ROS; similarities with age-related cataracts

    PubMed Central

    Zitnik, Galynn; Tsai, Ryan; Wolf, Norman

    2010-01-01

    Purpose To compare age-related cataractous (ARC) changes in unirradiated mice lenses to those induced by head-only X-irradiation of 3 month-old mice. Methods lens epithelial cells (LECs) as well as partially degraded cortical DNA were visualized in fixed sections using 4',6-diamidino-2-phenylindole (DAPI) staining, and in fresh lenses using the vital stain Hoechst 33342. reactive oxygen species (ROS) activity was also visualized directly in fresh lenses using the vital dye Dihydrorhodamine (DHR). In fixed lenses an antibody specific for 8-OH Guanosine (8-OH-G) lesions was used to visualize DNA oxidative adducts from ROS damage. Alpha smooth muscle actin was visualized using specific antibodies to determine if myofibroblasts were present. Fluorescence was quantified using Laser Scanning Confocal Microscopy (LSCM). The degree of lens opacity and cataract formation was determined by slit lamp, or from digitalized images of light reflections taken with a low magnification light microscope. Results Using DNA- and ROS-specific vital fluorescent dyes, and laser scanning confocal microscopy we have previously described 4 changes in the aging rodent lenses: 1) a significantly decreased density of surface LECs in lenses from old compared to younger mice and rats; 2) a very large increase in retained cortical nuclei and DNA fragments in the secondary lens fibers of old rodent lenses; 3) increased cortical ROS in old rodent lenses; 4) increased cataract concomitantly with the cortical DNA and ROS increases. In the current study we report that these same 4 changes also occur in an accelerated fashion in mice given head-only X-irradiation at 3 months of age. In addition to vital staining of fresh lenses, we also examined sections from fixed eyes stained with DAPI or hematoxylin and eosin (H&E) and found the same loss of surface LECs and accumulation of undigested nuclei and debris in secondary lens fibers occur with age or following X-irradiation. In addition sections from fixed

  15. [Investigation of chromosomes in varieties and translocation lines of pea Pisum sativum L. by FISH, Ag-NOR, and differential DAPI staining].

    PubMed

    Samatadze, T E; Muravenko, O M; Bol'sheva, N L; Amosova, A B; Gostimsckiĭ, S A; Zelenin, A V

    2005-12-01

    The DNA intercalator 9-aminoachridine was used for obtaining high-resolution DAPI patterns of chromosomes of Pisum sativum L. with more than 300 bands per haploid chromosome set. The karyotypes of three pea varieties, Viola, Capital, and Rosa Crown, and two translocation lines, L-108 (T(2-4s)) and M-10 (T(2-7s)), were examined. Based on the results of DAPI staining, we have identified chromosomes, constructed idiograms, and established breakpoints of chromosome translocations. Lines L-108 (T(2-4s)) and M-10 (T(2-7s)) were shown to appear as a result of respectively one translocation between chromosomes 2 and 4 and two translocations between chromosomes 2 and 7. All varieties and translocation lines of pea were examined using fluorescence in situ hybridization (FISH) with telomere repetition probes, 5S and 45S wheat DNA probes. Transcriptional activity of 45S rRNA was detected by Ag-NOR staining. Telomere repetitions were shown to be located only in telomeric chromosome regions. Using high-resolution DAPI staining allowed us to verify localization of 5S genes on pea chromosomes 1, 3, and 5. 45S rDNAs were localized in the secondary constriction regions on the satellite and the satellite thread of chromosome and on the satellite thread and in more proximal satellite heterochromatic region of chromosome 7. The size of 45S rDNA signal on chromosome 7 was larger and its transcriptional activity, higher than the corresponding parameters on chromosome 4 in most of the forms studied. A visual comparison of the results of FISH and Ag-NOR staining of normal and translocated pea chromosomes did not reveal any significant differences between them. The translocations of the satellite chromosomes apparently did not cause significant changes either in the amount of the ribosomal genes or in their transcriptional activity.

  16. DNA staining with the fluorochromes EtBr, DAPI and YOYO-1 in the comet assay with tobacco plants after treatment with ethyl methanesulphonate, hyperthermia and DNase-I.

    PubMed

    Gichner, Tomás; Mukherjee, Anita; Velemínský, Jirí

    2006-06-16

    We applied the alkaline version of the single-cell gel electrophoresis (comet) assay to roots and leaves of tobacco (Nicotiana tabacum var. xanthi) seedlings or isolated leaf nuclei treated with: (1) the alkylating agent ethyl methanesulphonate, (2) necrotic heat treatments at 50 degrees C, and (3) DNase-I. All three treatments induced a dose-dependent increase in DNA migration, expressed as percentage of tail DNA. A comparison of the fluorochrome DNA dyes ethidium bromide, DAPI and YOYO-1 demonstrated that for the alkaline version of the comet assay in plants, the commonly used fluorescent dye ethidium bromide can be used with the same efficiency as DAPI or YOYO-1.

  17. Karyotype analysis of Panax ginseng C.A.Meyer, 1843 (Araliaceae) based on rDNA loci and DAPI band distribution.

    PubMed

    Waminal, Nomar Espinosa; Park, Hye Mi; Ryu, Kwang Bok; Kim, Joo Hyung; Yang, Tae-Jin; Kim, Hyun Hee

    2012-01-01

    Ginseng has long been considered a valuable plant owing to its medicinal properties; however, genomic information based on chromosome characterization and physical mapping of cytogenetic markers has been very limited. Dual-color FISH karyotype and DAPI banding analyses of Panax ginseng C. A. Meyer, 1843 were conducted using 5S and 45S rDNA probes. The somatic chromosome complement was 2n=48 with lengths from 3.3 μm to 6.3 μm. The karyotype was composed of 12 metacentric, 9 submetacentric, and 3 subtelocentric pairs. The 5S rDNA probe localized to the intercalary region of the short arm of pair 11, while the 45S rDNA was located at the secondary constriction of the subtelocentric satellited chromosome 14. DAPI bands were clearly observed for most chromosomes, with various signal intensities and chromosomal distributions that consequently improved chromosome identification. As a result, all 24 chromosomes could be distinguished and numbers were assigned to each chromosome for the first time. The results presented here will be useful for the on-going ginseng genome sequencing and further molecular-cytogenetic studies and breeding programs of ginseng.

  18. Karyotype analysis of Panax ginseng C.A.Meyer, 1843 (Araliaceae) based on rDNA loci and DAPI band distribution

    PubMed Central

    Waminal, Nomar Espinosa; Park, Hye Mi; Ryu, Kwang Bok; Kim, Joo Hyung; Yang, Tae-Jin; Kim, Hyun Hee

    2012-01-01

    Abstract Ginseng has long been considered a valuable plant owing to its medicinal properties; however, genomic information based on chromosome characterization and physical mapping of cytogenetic markers has been very limited. Dual-color FISH karyotype and DAPI banding analyses of Panax ginseng C. A. Meyer, 1843 were conducted using 5S and 45S rDNA probes. The somatic chromosome complement was 2n=48 with lengths from 3.3 μm to 6.3 μm. The karyotype was composed of 12 metacentric, 9 submetacentric, and 3 subtelocentric pairs. The 5S rDNA probe localized to the intercalary region of the short arm of pair 11, while the 45S rDNA was located at the secondary constriction of the subtelocentric satellited chromosome 14. DAPI bands were clearly observed for most chromosomes, with various signal intensities and chromosomal distributions that consequently improved chromosome identification. As a result, all 24 chromosomes could be distinguished and numbers were assigned to each chromosome for the first time. The results presented here will be useful for the on-going ginseng genome sequencing and further molecular-cytogenetic studies and breeding programs of ginseng. PMID:24260682

  19. Fluorescent complexes of DNA with DAPI 4′,6-diamidine-2-phenyl indole.2HCl or DCI 4′,6-dicarboxyamide-2-phenyl indole

    PubMed Central

    Kapuściński, Jan; Skoczylas, Bogna

    1978-01-01

    4′,6-Dioarboxyamide-2-phenyl indole (DCI), a non-ionic structural analogue of 4′,6-diamidine-2-phenyl indole·2HCl (DAPI), was synthesized in order to verify the hypothesis of intercalation of both dyes into the DNA double helix. The influence of pH, viscosity, and different concentrations of SDS (sodium dodecylsulphate) or NaCl on the optical and fluorescent properties and the changes in thermal transition of both dye complexes with DNA confirm the affinity of the dyes to the double helix as well as their stabilizing influence on the secondary DNA structure. The results of binding studies, carried out by fluorescent methods have shown that the dyes are strongly bound to DNA, though the number of binding sites is small. According to the experimental data, the fluorescent properties of DAPI and DCI complexes with DNA are connected with the intercalating binding mechanism of these dyes. On the other hand, the eventual ionic or hydrogen bonds of dyes outside the DNA helix do not change noticeably their fluorescent properties. PMID:31603

  20. Heterochromatin in the chromosomes of the gorilla: characterization with distamycin A/DAPI, D287/170, chromomycin A3, quinacrine, and 5-azacytidine.

    PubMed

    Schmid, M; Haaf, T; Ott, G; Scheres, J M; Wensing, J A

    1986-01-01

    The chromosomes of the gorilla were extensively studied with various staining techniques labeling the different classes of heterochromatin. The chromosomal distribution of distamycin A/DAPI-, D287/170-, quinacrine-, and chromomycin A3-positive heterochromatic regions, as well as the nucleolus organizer regions, is described and compared with the karyotypes of other hominoid species. Lymphocyte cultures were treated with low doses of 5-azacytidine during the last hours of culture. This cytidine analog induces distinct undercondensation in 37 heterochromatic regions in the 24 gorilla chromosomes. The 5-azacytidine-induced undercondensations are localized not only in most of the distamycin A/DAPI-bright heterochromatic regions but also in many telomeric C-bands of the chromosomes. Furthermore, 5-azacytidine preserves the somatic pairing between heterochromatic regions from the interphase nuclei into the metaphase stage. The homeologies and differences in the chromosomal localization of the various classes of heterochromatin, 5-azacytidine-sensitive regions, 5-methylcytosine-rich DNA sequences, and satellite DNAs in the gorilla, chimpanzee, orangutan, and man are discussed.

  1. Embryonation and infectivity of Ascaris suum eggs isolated from worms expelled by pigs treated with albendazole , pyrantel pamoate, ivermectin or piperazine dihydrochloride.

    PubMed

    Boes, J; Eriksen, L; Nansen, P

    1998-02-28

    The effect of anthelmintic treatment of pigs on the embryonation and infectivity of Ascaris suum eggs isolated from expelled worms was investigated. Four groups of two naturally infected pigs were dosed with albendazole, pyrantel pamoate, ivermectin or piperazine dihydrochloride, respectively. Following worm expulsion, the eggs were removed from the uteri of female worms and embryonated in sulphuric acid. The infectivity of the embryonated eggs was tested through mouse inoculation. Egg development appeared normal in cultures from worms of the piperazine. pyrantel and ivermectin treated groups. In the albendazole cultures, egg development was largely arrested at the one-cell stage (81%). Where development occurred, irregular cell division was observed and only 7% of the eggs in the culture developed into fullgrown larvae. Following mouse inoculation with 2500 embryonated eggs, significantly lower lung larval counts on day 8 post inoculation (p.i.) were observed for mice in the piperazine and pyrantel treated groups (P < 0.01) compared to untreated controls. The larvae that developed in the eggs from ivermectin and albendazole treated groups appeared fully infective for mice. It was concluded that ovicidal activity of albendazole in vivo inhibits subsequent A. suum egg development in vitro; albendazole is, therefore, not suitable to obtain worms for egg embryonation to produce experimental inoculums. The anthelmintic treatment of pigs with ivermectin had only a limited effect on both embryonation and infectivity of A. suum eggs isolated from expelled worms.

  2. Validated derivative and ratio derivative spectrophotometric methods for the simultaneous determination of levocetirizine dihydrochloride and ambroxol hydrochloride in pharmaceutical dosage form

    NASA Astrophysics Data System (ADS)

    Ali, Omnia I. M.; Ismail, Nahla S.; Elgohary, Rasha M.

    2016-01-01

    Three simple, precise, accurate and validated derivative spectrophotometric methods have been developed for the simultaneous determination of levocetirizine dihydrochloride (LCD) and ambroxol hydrochloride (ABH) in bulk powder and in pharmaceutical formulations. The first method is a first derivative spectrophotometric method (1D) using a zero-crossing technique of measurement at 210.4 nm for LCD and at 220.0 nm for ABH. The second method employs a second derivative spectrophotometry (2D) where the measurements were carried out at 242.0 and 224.4 nm for LCD and ABH, respectively. In the third method, the first derivative of the ratio spectra was calculated and the first derivative of the ratio amplitudes at 222.8 and 247.2 nm was selected for the determination of LCD and ABH, respectively. Calibration graphs were established in the ranges of 1.0-20.0 μg mL- 1 for LCD and 4.0-20.0 μg mL- 1 for ABH using derivative and ratio first derivative spectrophotometric methods with good correlation coefficients. The developed methods have been successfully applied to the simultaneous determination of both drugs in commercial tablet dosage form.

  3. Phototoxic process after rapid photosensitive membrane damage of 5,5"-bis(aminomethyl)-2,2':5',2"-terthiophene dihydrochloride.

    PubMed

    Saito, T K; Takahashi, M; Muguruma, H; Niki, E; Mabuchi, K

    2001-08-30

    We report a new aspect of rapid (<30 s) light-induced cell membrane damage photosensitized by 5,5"-bis(aminomethyl)-2,2':5',2"-terthiophene dihydrochloride (BAT), which is a water-soluble alpha-terthienyl analogue, using a high-power laser (light intensity 1.6 W cm(-2)). In this paper, we will discuss the relationship between the exposure time of the cells to the photosensitizer and the phototoxic process. Three toxic processes can be identified: first, a non-light-mediated toxicity dependent on BAT-cell incubation; second, a phototoxicity independent of BAT exposure time when the BAT concentration is in the 2-10-microM range; third, a phototoxicity dependent on BAT exposure time when BAT concentration becomes 20 microM. The cytotoxicity decreases when alpha-tocopherol, an antioxidant, is added to a cell membrane. This pattern of phototoxicity is the typical of a phospholipid peroxidation chain reaction and oxidative damage of membrane proteins triggered by a reactive oxygen species generated by a triplet state of BAT. The BAT exposure time is clearly correlated with the partition of the photosensitizer in the cell membrane and inside the cell.

  4. Anti-proliferative and apoptotic effects of etoricoxib, a selective COX-2 inhibitor, on 1,2-dimethylhydrazine dihydrochloride-induced colon carcinogenesis.

    PubMed

    Tanwar, Lalita; Piplani, Honit; Sanyal, Sn

    2010-01-01

    In the present study, we assessed effects of etoricoxib, a non-steroidal anti-inflammatory drug, on proliferation and apoptosis in 1,2-dimethylhydrazine dihydrochloride (DMH) induced colon lesion development. Male SD rats were divided into four groups: Group 1 controls receiving the vehicle treatment; Group 2 administered DMH weekly (30 mg/kg body weight, subcutaneously) alone; Group 3, DMH weekly plus etoricoxib (0.64 mg/kg body weight, orally) daily; and Group 4, etoricoxib alone. After six weeks of treatment, animals were sacrificed and colons were analysed for morphological and histopathological features. Well characterized pre-neoplastic aberrations such as multiple plaque lesions, hyperplasia and dysplasia were found in the DMH treated group whereas these features were reduced with co-administration of etoricoxib. To study apoptosis, colonocytes were isolated by metal chelation from colonic sacs and studied by fluorescent staining and further confirmed by DNA fragmentation. The DMH treated animals had fewer apoptotic nuclei as compared to the controls, but numbers were higher with DMH+etoricoxib as well as etoricoxib alone. Expression of proliferative cell nuclear antigen (PCNA), assessed by Western blot analysis and immunohistochemistry, was found to be elevated by DMH treatment group and again reduced by etoricoxib. Results for bromodeoxyuridine incorporation (BrdU) were in agreement. It may be concluded that the drug, etoricoxib, has the potential to act as an anti-apoptotic and anti- proliferative agent in the colon.

  5. Validated derivative and ratio derivative spectrophotometric methods for the simultaneous determination of levocetirizine dihydrochloride and ambroxol hydrochloride in pharmaceutical dosage form.

    PubMed

    Ali, Omnia I M; Ismail, Nahla S; Elgohary, Rasha M

    2016-01-15

    Three simple, precise, accurate and validated derivative spectrophotometric methods have been developed for the simultaneous determination of levocetirizine dihydrochloride (LCD) and ambroxol hydrochloride (ABH) in bulk powder and in pharmaceutical formulations. The first method is a first derivative spectrophotometric method ((1)D) using a zero-crossing technique of measurement at 210.4 nm for LCD and at 220.0 nm for ABH. The second method employs a second derivative spectrophotometry ((2)D) where the measurements were carried out at 242.0 and 224.4 nm for LCD and ABH, respectively. In the third method, the first derivative of the ratio spectra was calculated and the first derivative of the ratio amplitudes at 222.8 and 247.2 nm was selected for the determination of LCD and ABH, respectively. Calibration graphs were established in the ranges of 1.0-20.0 μg mL(-1) for LCD and 4.0-20.0 μg mL(-1) for ABH using derivative and ratio first derivative spectrophotometric methods with good correlation coefficients. The developed methods have been successfully applied to the simultaneous determination of both drugs in commercial tablet dosage form.

  6. Original research paper. Characterization and taste masking evaluation of microparticles with cetirizine dihydrochloride and methacrylate-based copolymer obtained by spray drying.

    PubMed

    Amelian, Aleksandra; Szekalska, Marta; Ciosek, Patrycja; Basa, Anna; Winnicka, Katarzyna

    2017-03-01

    Taste of a pharmaceutical formulation is an important parameter for the effectiveness of pharmacotherapy. Cetirizine dihydrochloride (CET) is a second-generation antihistamine that is commonly administered in allergy treatment. CET is characterized by extremely bitter taste and it is a great challenge to successfully mask its taste; therefore the goal of this work was to formulate and characterize the microparticles obtained by the spray drying method with CET and poly(butyl methacrylate-co-(2-dimethylaminoethyl) methacrylate-co-methyl methacrylate 1:2:1 copolymer (Eudragit E PO) as a barrier coating. Assessment of taste masking by the electronic tongue has revealed that designed formulations created an effective taste masking barrier. Taste masking effect was also confirmed by the in vivo model and the in vitro release profile of CET. Obtained data have shown that microparticles with a drug/polymer ratio (0.5:1) are promising CET carriers with efficient taste masking potential and might be further used in designing orodispersible dosage forms with CET.

  7. Efficient synthesis of α-fluoromethylhistidine di-hydrochloride and demonstration of its efficacy as a glutathione S-transferase inhibitor.

    PubMed

    Considine, Kelly L; Stefanidis, Lazaros; Grozinger, Karl G; Audie, Joseph; Alper, Benjamin J

    2017-03-15

    Histidine decarboxylase (HDC) is an enzyme that converts histidine to histamine. Inhibition of HDC has several medical applications, and HDC inhibitors are of considerable interest for the study of histidine metabolism. (S)-α-Fluoromethylhistidine di-hydrochloride (α-FMH) is a potent HDC inhibitor that is commercially available at high cost in small amounts only. Here we report a novel, inexpensive, and efficient method for synthesis of α-FMH using methyl 2-aziridinyl-3-(N-triphenylmethyl-4-imidazolyl) propionate and HF/pyridine, with experimental yield of 57%. To identify novel targets for α-FMH, we developed a three step in silico work-flow for identifying physically plausible protein targets. The work-flow resulted in 21 protein target hits, including several enzymes involved in glutathione metabolism, and notably, two isozymes of the glutathione S-transferase (GST) superfamily, which plays a central role in drug metabolism. In view of this predictive data, the efficacy of α-FMH as a GST inhibitor was investigated in vitro. α-FMH was demonstrated to be an effective inhibitor of GST at micromolar concentration, suggesting that off-target effects of α-FMH may limit physiological drug metabolism and elimination by GST-dependent mechanisms. The present study therefore provides new avenues for obtaining α-FMH and for studying its biochemical effects, with potential implications for drug development.

  8. The effect of methyl linoleate hydroperoxides and radical initiator 2,2'-azobis (2-amidinopropane) dihydrochloride on antibody production by mouse spleen cells.

    PubMed

    Oarada, M; Kurita, N; Miyaji, M

    1997-06-01

    Methyl linoleate hydroperoxides (MLHPO) and 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH), a widely used free radical initiator, were examined for effects on antibody production by spleen cells using plaque-forming cell response against sheep red blood cells (SRBC). The in vitro primary antibody response was enhanced in the presence of MLHPO at a concentration range of 2-20 microM or AAPH at 0.1 microM, but was suppressed with higher concentrations of these compounds. In the in vitro secondary antibody responses, both MLHPO and AAPH failed to increase plaque-forming cell response above that of the control culture. Following the oral administration of MLHPO (2.29 g/kg) four times to mice, in vivo primary plaque-forming cell response was significantly suppressed. After, a single intraperitoneal injection of AAPH (60 mg/kg) to mice, in vitro primary plaque-forming cell response was also suppressed. These findings suggest that primary antibody response can be affected by lipid hydroperoxides and oxygen radicals in vivo.

  9. Dysfunction of mouse liver mitochondria induced by 2,2'-azobis-(2-amidinopropane) dihydrochloride, a radical initiator, in vitro and in vivo.

    PubMed

    Kanno, T; Utsumi, T; Ide, A; Takehara, Y; Saibara, T; Akiyama, J; Yoshioka, T; Utsumi, K

    1994-09-01

    Mouse liver mitochondria were uncoupled in a time dependent by intraperitoneal injection of a radical initiator, 2,2'-azobis-(2-amidinopropane) dihydrochloride (AAPH) (100 mg/kg). State 3 respiration, ADP/O ratio and respiratory control ratio (RCR) were decreased 30 min after injection but there was no effect on state 4 respiration. Lipid peroxidation was increased and oxidative phosphorylation was uncoupled at one hr after drug injection but gradually recovered to normal levels after 14 hr in vivo. State 3 respiration, RCR and ADP/O ratio but not state 4 respiration of isolated mouse mitochondria were inhibited by short term incubation with AAPH in vitro. This inhibitory action was concentration dependent (ID50 = 5 mM) but was not prevented by alpha-tocopherol. AAPH had no effect on electron transport or the membrane potential of these isolated mitochondria. However, mitochondria were uncoupled via lipid peroxidation and swelling by long term incubation with AAPH. These inhibitory effects of AAPH were reduced by its spontaneous degradation not only in vitro but also in vivo. Thus AAPH induces mitochondrial dysfunction by direct action in the early period of treatment and free radicals produced from AAPH mediate mitochondrial swelling via lipid peroxidation in the late period. From these findings, it is concluded that mitochondrial phosphorylation plays an important role in the pathogenesis of liver injury induced by AAPH and that radicals generated by AAPH might be a source of liver injury and mitochondrial dysfunction in vivo.

  10. F1F0-ATPase, early target of the radical initiator 2,2'-azobis-(2-amidinopropane) dihydrochloride in rat liver mitochondria in vitro.

    PubMed

    Beauseigneur, F; Goubern, M; Chapey, M F; Gresti, J; Vergely, C; Tsoko, M; Demarquoy, J; Rochette, L; Clouet, P

    1996-12-01

    This study was designed to determine which enzyme activities were first impaired in mitochondria exposed to 2,2'-azobis-(2-amidinopropane) dihydrochloride (AAPH), a known radical initiator. EPR spin-trapping revealed generation of reactive oxygen species although malondialdehyde formation remained very low. With increasing AAPH concentrations, State-3 respiration was progressively depressed with unaltered ADP/O ratios. A top-down approach demonstrated that alterations were located at the phosphorylation level. As shown by inhibitor titrations, ATP/ADP translocase activity was unaffected in the range of AAPH concentrations used. In contrast, AAPH appeared to exert a deleterious effect at the level of F1F0-ATPase, comparable with dicyclohexylcarbodi-imide, which alters Fo proton channel. A comparison of ATP hydrolase activity in uncoupled and broken mitochondria reinforced this finding. In spite of its pro-oxidant properties, AAPH was shown to act as a dose-dependent inhibitor of cyclosporin-sensitive permeability transition initiated by Ca2+, probably as a consequence of its effect on F1F0-ATPase. Resveratrol, a potent antiperoxidant, completely failed to prevent the decrease in State-3 respiration caused by AAPH. The data suggest that AAPH, when used under mild conditions, acted as a radical initiator and was capable of damaging F1F0-ATPase, thereby slowing respiratory chain activity and reducing mitochondrial antioxidant defences.

  11. Primary aminophospholipids in the external layer of liposomes protect their component polyunsaturated fatty acids from 2,2'-azobis(2-amidinopropane)- dihydrochloride-mediated lipid peroxidation.

    PubMed

    Kubo, Kazuhiro; Sekine, Seiji; Saito, Morio

    2005-02-09

    We showed in our previous study that docosahexaenoic acid-rich phosphatidylethanolamine in the external layer of small-size liposomes, as a model for biomembranes, protected its docosahexaenoic acid from 2,2'-azobis(2-amidinopropane)dihydrochloride- (AAPH-) mediated lipid peroxidation in vitro. Besides phosphatidylethanolamine, both phosphatidylserine and an alkenyl-acyl analogue of phosphatidylethanolamine, phosphatidylethanolamine plasmalogen, are reported to possess characteristic antioxidant activities. However, there are few reports about the relationship between the protective activity of phosphatidylethanolamine plasmalogen and/or phosphatidylserine against lipid peroxidation and their distribution in a phospholipid bilayer. Furthermore, it is unclear whether phosphatidylethanolamine plasmalogen and/or phosphatidylserine protect their component polyunsaturated fatty acids (PUFAs) from lipid peroxidation. In the present study, we examined the relationship between the transbilayer distribution of aminophospholipids, such as phosphatidylethanolamine rich in arachidonic acid, phosphatidylethanolamine plasmalogen, and phosphatidylserine, and the oxidative stability of their component PUFAs. The transbilayer distribution of these aminophospholipids in liposomes was modulated by coexisting phosphatidylcholine bearing two types of acyl chain: dipalmitoyl or dioleoyl. The amounts of these primary aminophospholipids in the external layer became significantly higher in liposomes containing dioleoylphosphatidylcholine than in those containing dipalmitoylphosphatidylcholine. Phosphatidylethanolamine rich in arachidonic acid, phosphatidylethanolamine plasmalogen or phosphatidylserine in the external layer of liposomes, as well as external docosahexaenoic acid-rich phosphatidylethanolamine, were able to protect their component PUFAs from AAPH-mediated lipid peroxidation.

  12. Anticancer Effect of Ursodeoxycholic Acid in Human Oral Squamous Carcinoma HSC-3 Cells through the Caspases.

    PubMed

    Pang, Liang; Zhao, Xin; Liu, Weiwei; Deng, Jiang; Tan, Xiaotong; Qiu, Lihua

    2015-05-05

    Bear bile was used as a traditional medicine or tonic in East Asia, and ursodeoxycholic acid (UDCA) is the most important compound in bear bile. Further, synthetic UDCA is also used in modern medicine and nutrition; therefore, its further functional effects warrant research, in vitro methods could be used for the fundamental research of its anticancer effects. In this study, the apoptotic effects of UDCA in human oral squamous carcinoma HSC-3 cells through the activation of caspases were observed by the experimental methods of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay, DAPI (4',6-diamidino-2-phenylindole) staining, flow cytometry analysis, RT-PCR (reverse transcription-polymerase chain reaction) assay and Western blot assay after HSC-3 cells were treated by different concentrations of UDCA. With 0 to 400 μg/mL UDCA treatment, UDCA had strong growth inhibitory effects in HSC-3 cells, but had almost no effect in HOK normal oral cells. At concentrations of 100, 200 and 400 μg/mL, UDCA could induce apoptosis compared to untreated control HSC-3 cells. Treatment of 400 μg/mL UDCA could induce more apoptotic cancer cells than 100 and 200 μg/mL treatment; the sub-G1 DNA content of 400 μg/mL UDCA treated cancer cells was 41.3% versus 10.6% (100 μg/mL) and 22.4% (200 μg/mL). After different concentrations of UDCA treatment, the mRNA and protein expressions of caspase-3, caspase-8, caspase-9, Bax, Fas/FasL (Fas ligand), TRAIL (TNF-related apoptosis-inducing ligand), DR4 (death receptor 4) and DR5 (death receptor 5) were increased in HSC-3 cells, and mRNA and protein expressions of Bcl-2 (B-cell lymphoma 2), Bcl-xL (B-cell lymphoma-extra large), XIAP (X-linked inhibitor of apoptosis protein), cIAP-1 (cellular inhibitor of apoptosis 1), cIAP-2 (cellular inhibitor of apoptosis 2) and survival were decreased. Meanwhile, at the highest concentration of 400 μg/mL, caspase-3, caspase-8, caspase-9, Bax, Fas/FasL, TRAIL, DR4, DR5, and Iκ

  13. Variability in cytogenetic adaptive response of cultured human lymphocytes to mitomycin C, bleomycin, quinacrine dihydrochloride, Co60 gamma-rays and hyperthermia.

    PubMed

    Krishnaja, A P; Sharma, N K

    2008-03-01

    Adaptive response (AR) is a well-documented phenomenon by which cells or organisms exposed to low dose of a genotoxicant become less sensitive to subsequent high-dose exposure to the same or another genotoxicant. AR, if induced can modify the efficacy leading to drug or radio-resistance, during anti-neoplastic drug or radiation treatment. Contradictions exist in AR induction by different genotoxicants with respect to the biomarkers, time schedules, and inter-individual variability, reflecting the complexity of AR in eukaryotic cells. In order to further ascertain these factors, AR induced by anti-neoplastic agents mitomycin C (MMC), bleomycin (BLM) and chemosterilant quinacrine dihydrochloride was examined in different donors and time schedules using cytogenetic biomarkers chromosome aberrations, sister chromatid exchanges and micronuclei (MN). BLM- and hyperthermia (HT)-induced cross-resistance to gamma rays and MMC/BLM, respectively, was also studied. Difference between MMC- and BLM-induced protective effects in biomarkers examined in the same donors was noticed. Adaptation to BLM and HT showed cross-resistance to chromosome damage induction by gamma rays and BLM/MMC, respectively. Cell cycle analysis indicated that adaptation is not caused by change in the rate of cell proliferation after challenge dose. MN as a chromosomal biomarker in large-scale population studies on AR is advocated, based on similar AR induced in all donors by MMC/BLM and rapid assessment in binucleated cells. Influence of certain genotypes on chromosomal biomarkers used in AR studies and role of AR in radiation and chemotherapy need to be further deciphered.

  14. Individualized long-term outcomes in blood phenylalanine concentrations and dietary phenylalanine tolerance in 11 patients with primary phenylalanine hydroxylase (PAH) deficiency treated with Sapropterin-dihydrochloride.

    PubMed

    Stockler-Ipsiroglu, Sylvia; Yuskiv, Nataliya; Salvarinova, Ramona; Apatean, Delia; Ho, Gloria; Cheng, Barbara; Giezen, Alette; Lillquist, Yolanda; Ueda, Keiko

    2015-03-01

    We analyzed long-term sustainability of improved blood Phenylalanine (Phe) control and changes to dietary Phe tolerance in 11 patients (1 month to 16 years), with various forms of primary PAH deficiency (classic, moderate, severe phenylketonuria [PKU], mild hyperphenylalaninemia [HPA]), who were treated with 15-20mg/kg/d Sapropterin-dihydrochloride during a period of 13-44 months. 7/11 patients had a sustainable, significant reduction of baseline blood Phe concentrations and 6 of them also had an increase in mg/kg/day Phe tolerance. In 2 patients with mild HPA, blood Phe concentrations remained in the physiologic range even after a 22 and 36% increase in mg/kg/day Phe tolerance and an achieved Phe intake at 105% and 268% of the dietary reference intake (DRI) for protein. 2 of these responders had classic PKU. 1 patient with mild HPA who started treatment at 2 months of life, had a significant and sustainable reduction in pretreatment blood Phe concentrations, but no increase in the mg/kg/day Phe tolerance. An increase in Phe tolerance could only be demonstrated when expressing the patient's daily Phe tolerance with the DRI for protein showing an increase from 58% at baseline to 78% of normal DRI at the end of the observation. Long-term follow-up of patients with an initial response to treatment with Sapropterin is essential to determine clinically meaningful outcomes. Phenylalanine tolerance should be expressed in mg/kg/day and/or % of normal DRI to differentiate medical therapy related from physiologic growth related increase in daily Phe intake.

  15. Transbuccal delivery of betahistine dihydrochloride from mucoadhesive tablets with a unidirectional drug flow: in vitro, ex vivo and in vivo evaluation

    PubMed Central

    El-Nabarawi, Mohamed A; Ali, Adel A; Aboud, Heba M; Hassan, Amira H; Godah, Amany H

    2016-01-01

    Objective Betahistine dihydrochloride (BH.2HCl), an anti-vertigo histamine analog used in the treatment of Ménière’s disease, undergoes extensive first-pass metabolism and suffers from short biological half-life. The aim of the present work was to develop and estimate controlled release mucoadhesive buccal tablets of BH.2HCl with a unidirectional drug flow to overcome this encumbrance. Methods A direct compression method was adopted for preparation of the tablets using mucoadhesive polymers like guar gum, hydroxypropyl methyl cellulose K4M, sodium carboxymethyl cellulose and their combinations. The tablets were coated from all surfaces except one surface with a solution of 5% (w/v) cellulose acetate and 1% (w/v) dibutyl phthalate. Different permeation enhancers like 2% sodium deoxycholate, 2% sodium cholate hydrate (SCH) and 5% menthol were tested. Swelling index, ex vivo residence time, mucoadhesion strength, in vivo testing of mucoadhesion time, in vitro dissolution and ex vivo permeation were carried out. Furthermore, compatibility and accelerated stability studies were performed for the drug excipients. Finally, drug bioavailability of the BH.2HCl-optimized buccal mucoadhesive formulation was compared with that of the orally administered Betaserc® 24 mg tablet in six healthy male volunteers. Results Formulation F10, which contained a combination of 35% guar gum and 5% sodium carboxymethyl cellulose, exhibited long adhesion time, high adhesion strength and diminished irritation to volunteers and showed zero-order release kinetics. SCH produced a significant enhancement in permeation of BH.2HCl across buccal mucosa. BH.2HCl-optimized buccal mucoadhesive formulation showed percentage relative bioavailability of 177%. Conclusion The developed mucoadhesive tablets represent a promising alternative for the buccal delivery of BH.2HCl. PMID:28008227

  16. Histological and immunohistochemical observations of mucin-depleted foci (MDF) stained with Alcian blue, in rat colon carcinogenesis induced with 1,2-dimethylhydrazine dihydrochloride.

    PubMed

    Yoshimi, Naoki; Morioka, Takamitsu; Kinjo, Tatsuya; Inamine, Morihiko; Kaneshiro, Tatsuya; Shimizu, Takahiro; Suzui, Masumi; Yamada, Yasuhiro; Mori, Hideki

    2004-10-01

    The usefulness of mucin-depleted foci (MDF), which have recently been proposed as a new preneoplastic biomarker in rat colon carcinogenesis, was histologically investigated in rat colonic tissues treated with 1,2-dimethylhydrazine dihydrochloride (DMH). The relationship among aberrant crypt foci (ACF), MDF and beta-catenin accumulated crypts (BCAC) was examined by comparing the corresponding computer-captured images. Twelve male F344 rats were given DMH s.c. at a dose of 40 mg/kg body weight, once a week for 2 weeks, and randomly divided into two groups. Rats in group 1 were given normal drinking water, while those in group 2 were given drinking water containing indomethacin (IND) at 16 ppm for 6 weeks. All animals were sacrificed 8 weeks after the first DMH treatment. The resected colons were fixed in 10% formalin, and stained with Alcian blue for observation of ACF and MDF. Histological and immunohistochemical analysis revealed that the numbers of ACF, MDF and overlapping lesions in group 2 (treated with IND) were significantly decreased, compared with those in group 1. The number of BCAC in group 2 was also significantly lower than that in group 1. The reduction (61.5%) of MDF by IND was much greater than that (29.3%) of ACF. Analyses of the computer-captured images indicated that MDF had more frequent dysplastic changes and overexpression of beta-catenin than did ACF. MDF having over 4 crypts or MDF with the appearance of ACF corresponded well to BCAC. These results suggest that MDF may be useful as an early biomarker in colon carcinogenesis.

  17. Enhancement of hyperthermia-induced apoptosis by a free radical initiator, 2,2'-azobis (2-amidinopropane) dihydrochloride, in human histiocytic lymphoma U937 cells.

    PubMed

    Li, F J; Kondo, T; Zhao, Q L; Tanabe, K; Ogawa, R; Li, M; Arai, Y

    2001-09-01

    To elucidate the mechanism how a free radical initiator, 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH), induces cell death at hyperthermic temperatures, apoptosis in a human histiocytic lymphoma cell line, U937, was investigated. Free radical formation deriving from the thermal decomposition of AAPH was examined by spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). An assay for DNA fragmentation, observation of nuclear morphological changes, and flow cytometry for phosphatidylserine (PS) externalization were used to detect apoptosis and revealed enhancement of 44.0 degrees C hyperthermia-induced apoptosis by free radicals due to AAPH. However, free radicals alone derived from AAPH did not induce apoptosis. Hyperthermia induced the production of lipid peroxidation (LPO), an increase in intracellular Ca2+ concentration ([Ca2+]i) and enhanced expression of the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1). The effects of hyperthermia on LPO and [Ca2+]i were enhanced markedly by the combination with AAPH. A significant decrease in Bcl-2 expression, increase in Bax expression, a loss of mitochondrial membrane potential (delta psi m) and a marked increase in cytochrome c expression were found only in cells treated with hyperthermia and AAPH. Although an intracellular Ca2+ ion chelator, BAPTA-AM, completely inhibited DNA fragmentation, water-soluble vitamin E, Trolox, only partially suppressed DNA fragmentation and the increase in [Ca2+]i. In contrast, LPO was inhibited completely by Trolox, but no inhibition by BAPTA-AM was found. These results suggest that apoptosis induced by hyperthermia alone is due to the increase in [Ca2+]i arising from increased expression of IP3R1 and LPO. Additional increase in [Ca2+]i due to increased LPO and the activation of mitochondria-caspase dependent pathway play a major role in the enhancement of apoptosis by the combination with hyperthermia and AAPH.

  18. Kinetics of peroxidation of linoleic acid incorporated into DPPC vesicles initiated by the thermal decomposition of 2,2'-azobis(2-amidinopropane) dihydrochloride.

    PubMed

    Cubillos, M A; Lissi, E A; Abuin, E B

    2001-07-01

    In a previous work [Chem. Phys. Lipids 2000 104, 49], we have derived the following rate law for the oxidation of lipids in compartmentalized systems: R(T)=(k(1)/k(t))(0.5) k(p) [In](0.5) c(0.5) [LH], where, R(T) is the total rate of oxidation, k(1) is the rate constant for the production of free radicals, k(t) and k(p) are the intra-particle rate constants for the termination and propagation sets, respectively, [In] is the concentration of a water-soluble initiator, c is the concentration of particles, and [LH] is the intra-particle concentration of oxidable lipid. In the present work, we have investigated on the applicability of the proposed kinetic rate law for a system where it takes place the oxidation of a reactive lipid incorporated into an inert matrix. With this purpose, we have measured the rate of oxidation of linoleic acid incorporated into dipalmitoylphosphatidylcholine vesicles initiated by the thermal decomposition of 2,2'-azobis(2-amidinopropane) dihydrochloride as a function of the initiator, particles, and intra-particle LH concentrations. The experimentally determined kinetic orders obtained were 0.54+/-0.02, 0.48+/-0.05 and 0.83+/-0.04 for the dependence of the oxidation rate with initiator, particles, and LH intra-particle concentrations, respectively, in agreement with those theoretically predicted. The lower value obtained for the kinetic order in LH is attributed to a change in k(t) with the increase in oxidable lipid intra-particle concentration. The main point to be emphazised from the results here obtained is that the kinetic rate law for the oxidation of lipids in compartmentalized systems can be significantly different than that observed when to the oxidation takes place in homogeneous solution.

  19. Activation of caspase-3 by lysosomal cysteine proteases and its role in 2,2'-azobis-(2-amidinopropane)dihydrochloride (AAPH)-induced apoptosis in HL-60 cells.

    PubMed

    Ishisaka, R; Kanno, T; Akiyama, J; Yoshioka, T; Utsumi, K; Utsumi, T

    2001-01-01

    We previously reported that in addition to mitochondrial cytochrome c dependent activation, lysosomal cysteine proteases were also involved in the activation of caspase-3. In this study, we have separately obtained the lysosomal and mitochondrial caspase-3 activating factors in a crude mitochondrial fraction and characterized their ability to activate pro-caspase-3 in the in vitro assay system. When a rat liver crude mitochondrial fraction containing lysosomes (ML) was treated with a low concentration of digitonin, lysosomal factors were selectively released without the release of a mitochondrial factor (cytochrome c, Cyt.c). Treatment of ML with Ca(2+) in the presence of inorganic phosphate (P(i)), in contrast, released mitochondrial Cyt.c without the release of lysosomal factors. The obtained lysosomal and mitochondrial factors activated caspase-3 in different manners; caspase-3 activation by lysosomal and mitochondrial factors was specifically suppressed by E-64, a cysteine protease inhibitor, and caspase-9 inhibitor, respectively. Thus, the activation of caspase-3 by lysosomal factors was found to be distinct from the activation by mitochondrial Cyt.c dependent formation of the Apaf-1/caspase-9 complex. To further determine whether or not the activation of caspase-3 by lysosomal cysteine proteases is involved in cellular apoptosis, the effect of E-64-d, a cell-permeable inhibitor of cysteine protease, on 2,2'-azobis-(2-amidinopropane)dihydrochloride (AAPH)-induced apoptosis in HL-60 cells was investigated. As a result, DNA fragmentation induced by AAPH was found to be remarkably (up to 50%) reduced by pretreatment with E-64-d, indicating the participation of lysosomal cysteine proteases in AAPH-induced apoptosis in HL-60 cells.

  20. Holocentric chromosomes of psocids (Insecta, Psocoptera) analysed by C-banding, silver impregnation and sequence specific fluorochromes CMA3 and DAPI.

    PubMed

    Golub, Natalia V; Nokkala, Seppo; Kuznetsova, Valentina G

    2004-01-01

    The pattern of nucleolus attachment and C-heterochromatin distribution and molecular composition in the karyotypes of psocid species Psococerastis gibbosa (2n = 16+X), Blaste conspurcata (2n = 16+X) and Amphipsocus japonicus (2n = 14+neo-XY) were studied by C-banding, silver impregnation and sequence specific fluorochromes CMA3 and DAPI. Every species was found to have a single nucleolus in male meiosis. In P. gibbosa the nucleolus is attached to an autosomal bivalent; in B. conspurcata to the X-chromosome; in A. japonicus to the neo-XY bivalent. The species show a rather small amount of constitutive heterochromatin, C-blocks demonstrating telomeric localization with rare exceptions. P. gibbosa is characterized by a polymorphism for C-blocks occurrence and distribution. In the autosomes of this species, C-heterochromatin consists of AT-rich DNA except for the nucleolus organizing region, which is also GC-rich; the X-chromosome shows both AT- and GC-rich clusters. In A. japonicus and B. conspurcata, C-heterochromatin of the autosomes and sex chromosomes consists of both GC-rich and AT-rich DNA clusters, which are largely co-localized.

  1. Simultaneous detection and semiquantification of DNA damage in normal and apoptotic cells: triple-immunofluorescent labeling using DAPI, antibodies, and TUNEL.

    PubMed

    Agrawal, Anant; Godar, Dianne E

    2012-07-01

    We developed a triple-labeling immunofluorescence technique that simultaneously identifies total DNA (DAPI), DNA damage (antibodies), and dead cells [terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive cells] and a method that semiquantifies DNA damage in paraffin-embedded tissues. Using this technique in combination with our analysis method, scientists can now simultaneously detect and compare the relative amounts of DNA damage of almost any kind (except single-strand and double-strand breaks), using indirect fluorescent antibody labeling, in both normal and dying cells of different tissues. Simultaneous labeling of DNA damage and dead or TUNEL-positive cells can reduce processing costs and analysis time, and can lead to discoveries concerning how cells die from different DNA damages. We used increasing doses of UV (290 to 400 nm) radiation to create DNA damage in the form of cyclobutane pyrimidine dimers and 6-4 photoproducts that kill some of the cells in 3-dimensional tissue-engineered skin and vaginal samples. We describe a protocol that reliably detects and semiquantifies DNA damage in both normal and apoptotic cells. We show this triple-labeling immunofluorescence technique and analysis method yields linear UV dose response curves for damage to DNA bases that allows semiquantification of cyclobutane pyrimidine dimers and calculation of its repair rate (T=1 and 24 h), whereas TUNEL allows quantification of the number of apoptotic cells. Scientists can now create beautiful fluorescent pictures that simultaneously detect DNA damage in both normal and apoptotic cells to assess and semiquantify the damage to understand better how different insults lead to the cell's demise.

  2. Synbiotic (Lactobacillus rhamnosus+Lactobacillus acidophilus+inulin) attenuates oxidative stress and colonic damage in 1,2 dimethylhydrazine dihydrochloride-induced colon carcinogenesis in Sprague-Dawley rats: a long-term study.

    PubMed

    Verma, Angela; Shukla, Geeta

    2014-11-01

    The inexorable increase in the incidence of colorectal cancer has led to growing interest in its prevention by natural interventions. Thus, the present study was designed with the aim of delineating the antioxidative and antitumorigenic effects of synbiotics in experimental colon carcinogenesis. It was observed that administration of a synbiotic, before 1,2 dimethylhydrazine dihydrochloride (DMH)-induced colon carcinogenesis in Sprague-Dawley rats, led to increased body weight and growth rate, and decreased tumor incidence compared with the DMH-only-treated group of animals. Most notably, the level of malondialdehyde, a measure of lipid peroxidation, decreased, and levels of the antioxidants, glutathione reductase, superoxide dismutase, and glutathione peroxidase increased in animals in the Lactobacillus acidophilus+DMH, inulin+DMH, and synbiotic+DMH groups compared with DMH-only-treated animals. Histopathological observations of the colon also documented fewer dysplastic changes and increased the number of goblet cells in the probiotic-treated, prebiotic-treated, and synbiotic-treated animals compared with DMH-only-treated animals. Taken together, the present study shows that the use of synbiotics is a better prophylactic strategy than the use of probiotic and prebiotic alone because of the greater increase in antioxidants associated with the higher degree of attenuation of DMH-induced tumorigenesis.

  3. Synthesis of new N,N'-bis[1-aryl-3-(piperidine-1-yl)propylidene]hydrazine dihydrochlorides and evaluation of their cytotoxicity against human hepatoma and breast cancer cells.

    PubMed

    Kucukoglu, Kaan; Gul, H Inci; Cetin-Atalay, Rengul; Baratli, Yosra; Charles, Anne-Laure; Sukuroglu, Murat; Gul, Mustafa; Geny, Bernard

    2014-06-01

    N,N'-Bis[1-aryl-3-(piperidine-1-yl)propylidene]hydrazine dihydrochlorides were synthesized by the reaction of 2 mols of 1-aryl-3-(piperidine-1-yl)-1-propanone hydrochlorides with 1 mol of hydrazine hydrate. Aryl part was C₆H₅ (P1), 4-CH₃C₆H₄ (P2), 4-CH₃OC₆H₄ (P3), 4-HOC₆H₄ (P4), 4-ClC₆H₄ (P5), 3-CH₃OC₆H₄ (P6), 4-FC₆H₄ (P7) and 4-BrC₆H₄ (P8). Except P1, all compounds were reported for the first time. The chemical structures were confirmed by UV, (1)H NMR, (13)C NMR and HRMS spectra. P1, P2, P7 and P8 against human hepatoma (Huh7) cells and P1, P2, P4, P5, P6, P7 and P8 against breast cancer (T47D) cells have shown cytotoxicity. P1, P2 and P7 had more potent cytotoxicity against Huh7 cells than the reference compound 5-FU, whereas only P2 was more potent than the 5-FU against T47D cells. Representative compound P7 inhibited the mitochondrial respiration at 144, 264 and 424 µM concentrations dose-dependantly in liver homogenates. The results suggest that P1, P2, P7 and P8 may serve as model compounds for further synthetic studies.

  4. Single-strand breaks in DNA of various organs of mice induced by methyl methanesulfonate and dimethylsulfoxide determined by the alkaline unwinding technique

    SciTech Connect

    Solveig Walles, S.A.; Erixon, K.

    1984-03-01

    The method for determination of single-strand breaks (SSB) in DNA by the technique of alkaline unwinding and hydroxylapatite chromatography has been applied for cell nuclei from organs of mice. Male mice were given methyl methane-sulfonate (MMS) and dimethylsulfoxide (DMSO) by i.p. administration. Cell nuclei were prepared from various organs and then lysed in alkali. The amount of DNA was determined by fluorometry using 4',6-diamidino-2-phenylindole.2HCl. The relative level of SSB in DNA was determined in various organs (liver, kidney, lung, spleen, testis and brain) 1-24 h after administration of the agent. After MMS-treatment the number of SSB in DNA increased to about the same extent in all organs 1 h post-treatment but then decreased by time. The SSB persisted for the longest time in brain- and lung-DNA. DMSO induced SSB only in DNA of kidney.

  5. Multiphoton microscopy with clearing for three dimensional histology of kidney biopsies

    PubMed Central

    Olson, Eben; Levene, Michael J.; Torres, Richard

    2016-01-01

    We present a multiphoton microscopy approach with clearing optimized for pathology evaluation producing image quality comparable to traditional histology. Use of benzyl alcohol/benzyl benzoate with 4',6-diamidino-2-phenylindole and eosin in an optimized imaging setup results in optical sections nearly indistinguishable from traditionally-cut sections. Application to human renal tissue demonstrates diagnostic-level image quality can be maintained through 1 millimeter of tissue. Three dimensional perspectives reveal changes of glomerular capsule cells not evident on single sections. Collagen-derived second harmonic generation can be visualized through entire biopsies. Multiphoton microscopy with clearing has potential for increasing the yield of histologic evaluation of biopsy specimens. PMID:27570700

  6. Rule governing the division pattern in Escherichia coli minB and wild-type filaments.

    PubMed Central

    Jaffé, A; Boye, E; D'Ari, R

    1990-01-01

    Escherichia coli minB mutants form anucleate minicells and multinucleate filaments. We show here that the overwhelming majority of nucleate cells contain 2n (n = 0, 1, 2, ...) nucleoids, as determined by 4',6-diamidino-2-phenylindole staining, and 2n (n = 1, 2, 3, ...) copies of the replication origin, as determined by flow cytometry. This shows that division sites are not chosen randomly among the available sites in minB filaments. Similarly, wild-type cells contain 2n nucleoids, both during cell division inhibition and when furazlocillin-induced filaments are allowed to divide. We conclude that the min+ function is only to prevent septation only at polar sites; the placement of internal cell division sites must obey strict rules, which are the same in minB and wild-type cells. PMID:2188963

  7. Fluorescent nanoparticles for observing primo vascular system along sciatic nerve.

    PubMed

    Jia, Zhao-Feng; Lee, Byung-Cheon; Eom, Ki-Hoon; Cha, Jin-Myung; Lee, Jin-Kyu; Su, Zhen-Dong; Yu, Wen-Hui; Ryu, Pan Dong; Soh, Kwang-Sup

    2010-09-01

    The primo vascular system was found in the epineurium along the rat sciatic nerve following subcutaneous injection of fluorescent nanoparticles at the Zusanli acupoint (ST-36). Nanoparticles were injected into the primo-vessel near ST-36 and flowed along the sciatic nerve. Fluorescence revealed a structure in the epineurium that was hardly detectable. Images of the isolated sample stained with 4',6-diamidino-2-phenylindole were captured using confocal microscopy. These images showed the distinctive nuclei distribution and multi-lumen structure of primo-vessels that differentiate them from lymphatic vessels, blood capillaries and nerves. This study demonstrates a new use for nanoparticles in fluorescence reflectance imaging techniques during in vivo imaging of primo-vessels.

  8. Base-sequence specificity of Hoechst 33258 and DAPI binding to five (A/T)4 DNA sites with kinetic evidence for more than one high-affinity Hoechst 33258-AATT complex.

    PubMed

    Breusegem, Sophia Y; Clegg, Robert M; Loontiens, Frank G

    2002-02-01

    The binding of Hoechst 33258 and DAPI to five different (A/T)4 sequences in a stable DNA hairpin was studied exploiting the substantial increase in dye fluorescence upon binding. The two dyes have comparable affinities for the AATT site (e.g. association constant K(a)=5.5 x 10(8) M(-1) for DAPI), and their affinities decrease in the series AATT > TAAT approximately equal to ATAT > TATA approximately equal to TTAA. The extreme values of K(a) differ by a factor of 200 for Hoechst 33258 but only 30 for DAPI. The binding kinetics of Hoechst 33258 were measured by stopped-flow under pseudo-first order conditions with an (A/T)4 site in excess. The lower-resolution experiments can be well represented by single exponential processes, corresponding to a single-step binding mechanism. The calculated association-rate parameters for the five (A/T)4 sites are similar (2.46 x 10(8) M(-1) s(-1) to 0.86 x 10(8) M(-1) s(-1)) and nearly diffusion-controlled, while the dissociation-rate parameters vary from 0.42 s(-1) to 96 s(-1). Thus the association constants are kinetically controlled and are close to their equilibrium-determined values. However, when obtained with increased signal-to-noise ratio, the kinetic traces for Hoechst 33258 binding at the AATT site reveal two components. The concentration dependencies of the two time constants and amplitudes are consistent with two different kinetically equivalent two-step models. In the first model, fast bimolecular binding is followed by an isomerization of the initial complex. In the second model, two single-step associations form two complexes that mutually exclude each other. For both models the four reaction-rate parameters are calculated. Finally, specific dissociation kinetics, using poly[d(A-5BrU)], show that the kinetics are even more complex than either two-step model. We correlate our results with the different binding orientations and locations of Hoechst 33258 in the DNA minor groove found in several structural studies in

  9. Use of 5-cyano-2,3-ditolyl tetrazolium chloride for quantifying planktonic and sessile respiring bacteria in drinking water.

    PubMed

    Schaule, G; Flemming, H C; Ridgway, H F

    1993-11-01

    Direct microscopic quantification of respiring (i.e., viable) bacteria was performed for drinking water samples and biofilms grown on different opaque substrata. Water samples or biofilms developed in flowing drinking water were incubated with the vital redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and R2A medium. One hour of incubation with 0.5 mM CTC was sufficient to obtain intracellular reduction of CTC to the insoluble fluorescent formazan (CTF) product, which was indicative of cellular respiratory (i.e., electron transport) activity. This result was obtained with both planktonic and biofilm-associated cells. Planktonic bacteria were captured on 0.2-microns-pore-size polycarbonate membrane filters and examined by epifluorescence microscopy. Respiring cells containing CTF deposits were readily detected and quantified as red-fluorescing objects on a dark background. The number of CTC-reducing bacteria was consistently greater than the number of aerobic CFU determined on R2A medium. Approximately 1 to 10% of the total planktonic population (determined by counterstaining with 4,6-diamidino-2-phenylindole) were respirometrically active. The proportion of respiring bacteria in biofilms composed of drinking water microflora was greater, ranging from about 5 to 35%, depending on the substratum. Respiring cells were distributed more or less evenly in biofilms, as demonstrated by counterstaining with 4,6-diamidino-2-phenylindole. The amount of CTF deposited in single cells of Pseudomonas putida that formed monospecies biofilms was quantified by digital image analysis and used to indicate cumulative respiratory activity. These data indicated significant cell-to-cell variation in respiratory activity and reduced electron transport following a brief period of nutrient starvation.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Development of a Decellularized Lung Bioreactor System for Bioengineering the Lung: The Matrix Reloaded

    PubMed Central

    Price, Andrew P.; England, Kristen A.; Matson, Amy M.; Blazar, Bruce R.

    2010-01-01

    We developed a decellularized murine lung matrix bioreactor system that could be used to evaluate the potential of stem cells to regenerate lung tissue. Lungs from 2–3-month-old C57BL/6 female mice were excised en bloc with the trachea and heart, and decellularized with sequential solutions of distilled water, detergents, NaCl, and porcine pancreatic DNase. The remaining matrix was cannulated and suspended in small airway growth medium, attached to a ventilator to simulate normal, murine breathing-induced stretch. After 7 days in an incubator, lung matrices were analyzed histologically. Scanning electron microscopy and histochemical staining demonstrated that the pulmonary matrix was intact and that the geographic placement of the proximal and distal airways, alveoli and vessels, and the basement membrane of these structures all remained intact. Decellularization was confirmed by the absence of nuclear 4′,6-diamidino-2-phenylindole staining and negative polymerase chain reaction for genomic DNA. Collagen content was maintained at normal levels. Elastin, laminin, and glycosaminglycans were also present, although at lower levels compared to nondecellularized lungs. The decellularized lung matrix bioreactor was capable of supporting growth of fetal alveolar type II cells. Analysis of day 7 cryosections of fetal-cell-injected lung matrices showed pro-Sp-C, cytokeratin 18, and 4′,6-diamidino-2-phenylindole-positive cells lining alveolar areas that appeared to be attached to the matrix. These data illustrate the potential of using decellularized lungs as a natural three-dimensional bioengineering matrix as well as provide a model for the study of lung regeneration from pulmonary stem cells. PMID:20297903

  11. Role of cytokines and Jak3/Stat3 signaling in the 1,2-dimethylhydrazine dihydrochloride-induced rat model of colon carcinogenesis: early target in the anticancer strategy.

    PubMed

    Saini, Manpreet Kaur; Vaish, Vivek; Sanyal, Sankar Nath

    2013-05-01

    The molecular mechanisms by which colon cancer cells regulate the expression of various proinflammatory and anti-inflammatory cytokines and transcription factors resulting in tumor progression have not been well clarified. The present study thus explores the effect of cancer cell-derived cytokines and transcription factors on the chemoprevention of a rat model of early colon carcinogenesis. Elevated expression of proinflammatory cytokines [interleukin-1β (IL-1β), IL-2, interferon γ, and tumor necrosis factor-α] and the transcription factors [Janus kinase 3 (Jak3) and signal transducer and activator of transcription 3 (Stat3)] was found in the 1,2-dimethylhydrazine dihydrochloride (DMH) group; however, this elevated expression was reversed by the individual and combination treatment with piroxicam, a traditional nonsteroidal anti-inflammatory drug [inhibiting both cyclooxygenase-1 (COX-1) and COX-2] and c-phycocyanin, a cyanobacterium-derived biliprotein from Spirulina platensis (selective COX-2 inhibitor). In the DMH group, low expression of IL-4, an anti-inflammatory cytokine, was further observed with respect to the other groups. Expression of inducible nitric oxide synthase and nitric oxide/citrulline levels was also analyzed and was found to be elevated with DMH treatment. Increased apoptotic index and stimulated levels of Bcl-2-associated death promoter (Bad), a proapoptotic protein, were observed in piroxicam-treated and c-phycocyanin-treated rats. In-silico molecular docking of piroxicam as a ligand with several regulatory proteins was performed, indicating that, except inducible nitric oxide synthase, it effectively binds with COX-1, COX-2, Jak3, and Stat3. Piroxicam and c-phycocyanin perhaps showed chemopreventive properties by inhibiting proinflammatory cytokines and Jak3/Stat3 signaling while promoting apoptosis. In addition, a combination regimen was found to be more beneficial than monotherapy.

  12. Comparison of the antibacterial effect of silver sulfadiazine 1%, mupirocin 2%, Acticoat and octenidine dihydrochloride in a full-thickness rat burn model contaminated with multi drug resistant Acinetobacter baumannii.

    PubMed

    Selçuk, Caferi Tayyar; Durgun, Mustafa; Ozalp, Burhan; Tekin, Alicem; Tekin, Recep; Akçay, Cemal; Alabalık, Ulaş

    2012-12-01

    In this study, our aim is to compare the efficacy of different topical antibacterial agents in a rat model contaminated with a multi drug resistant (MDR) standard Acinetobacter baumannii strain. The study was carried out on 40 Sprague-Dawley rats of 250-300 g each. For the purposes of this study, the rats were divided into 5 groups, with 8 rats in each group: Group 1 control; Group 2 silver sulfadiazine; Group 3 mupirocin; Group 4 Acticoat group; and Group 5 octenidine dihydrochloride group. Following to the formation of the full-thickness burn areas in rats, the MDR A. baumannii standard strain was inoculated into the burned area. The rats in all the groups were sacrificed at the end of the 10th day and subjected to histopathological and microbiological evaluation. In the histopathological evaluation, the lowest inflammatory cell response and bacterial density in the eschar and muscle tissues were observed in the Acticoat group. While these results were found to be statistically significant compared to the silver sulfadiazine group, only the bacterial density in the muscle tissue was found as significant in comparison to the mupirocin and octenidine groups. In the microbiological evaluation, the lowest growth in the muscle tissue culture among all the groups was observed in the Acticoat group. The growth in the eschar tissue culture was significantly lower in the Acticoat and octenidine groups in comparison to the silver sulfadiazine group. At the end of the study, it has been observed that Acticoat was effective both in eschar and muscle, while octenidine was effective in eschar tissues in a rat burn model contaminated with MDR A. baumannii.

  13. Food restriction alters N'-propyl-4,5,6,7-tetrahydrobenzothiazole-2,6-diamine dihydrochloride (pramipexole)-induced yawning, hypothermia, and locomotor activity in rats: evidence for sensitization of dopamine D2 receptor-mediated effects.

    PubMed

    Collins, Gregory T; Calinski, Diane M; Newman, Amy Hauck; Grundt, Peter; Woods, James H

    2008-05-01

    Food restriction enhances sensitivity to the reinforcing effects of a variety of drugs of abuse including opiates, nicotine, and psychostimulants. Food restriction has also been shown to alter a variety of behavioral and pharmacological responses to dopaminergic agonists, including an increased sensitivity to the locomotor stimulatory effects of direct- and indirect-dopamine agonists, elevated extracellular dopamine levels in responses to psychostimulants, as well as suppression of agonist-induced yawning. Behavioral and molecular studies suggest that augmented dopaminergic responses observed in food-restricted animals result from a sensitization of the dopamine D2 receptor; however, little is known about how food restriction affects dopamine D3 receptor function. The current studies were aimed at better defining the effects of food restriction on D2 and D3 receptor function by assessing the capacity of N'-propyl-4,5,6,7-tetrahydrobenzothiazole-2,6-diamine dihydrochloride (pramipexole) to induce yawning, penile erection (PE), hypothermia, and locomotor activity in free-fed and food-restricted rats. Food restriction resulted in a suppression of pramipexole-induced yawning, a sensitized hypothermic response, and an enhanced locomotor response to pramipexole, effects that are suggestive of an enhanced D2 receptor activity; no effect on pramipexole-induced PE was observed. Antagonist studies further supported a food restriction-induced enhancement of the D2 receptor activity because the D2 antagonist 3-[4-(4-chlorophenyl)-4-hydroxypiperidin-l-yl]methyl-1H-indole (L741,626) recovered pramipexole-induced yawning to free-fed levels, whereas yawning and PE were suppressed following pretreatment with the D3 antagonist N-{4-[4-(2,3-dichlorophenyl)-piperazin-1-yl]-trans-but-2-enyl}-4-pyridine-2-yl-benzamide hydrochloride (PG01037). The results of the current studies suggest that food restriction sensitized rats to the D2-mediated effects of pramipexole while having no effect

  14. Use of a standardized JaCVAM in vivo rat comet assay protocol to assess the genotoxicity of three coded test compounds; ampicillin trihydrate, 1,2-dimethylhydrazine dihydrochloride, and N-nitrosodimethylamine.

    PubMed

    McNamee, J P; Bellier, P V

    2015-07-01

    As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo rat alkaline comet assay (comet assay), our laboratory examined ampicillin trihydrate (AMP), 1,2-dimethylhydrazine dihydrochloride (DMH), and N-nitrosodimethylamine (NDA) using a standard comet assay validation protocol (v14.2) developed by the JaCVAM validation management team (VMT). Coded samples were received by our laboratory along with basic MSDS information. Solubility analysis and range-finding experiments of the coded test compounds were conducted for dose selection. Animal dosing schedules, the comet assay processing and analysis, and statistical analysis were conducted in accordance with the standard protocol. Based upon our blinded evaluation, AMP was not found to exhibit evidence of genotoxicity in either the rat liver or stomach. However, both NDA and DMH were observed to cause a significant increase in % tail DNA in the rat liver at all dose levels tested. While acute hepatoxicity was observed for these compounds in the high dose group, in the investigators opinion there were a sufficient number of consistently damaged/measurable cells at the medium and low dose groups to judge these compounds as genotoxic. There was no evidence of genotoxicity from either NDA or DMH in the rat stomach. In conclusion, our laboratory observed increased DNA damage from two blinded test compounds in rat liver (later identified as genotoxic carcinogens), while no evidence of genotoxicity was observed for the third blinded test compound (later identified as a non-genotoxic, non-carcinogen). This data supports the use of a standardized protocol of the in vivo comet assay as a cost-effective alternative genotoxicity assay for regulatory testing purposes.

  15. Evolutionary chromosomal differentiation among four species of Conoderus Eschscholtz, 1829 (Coleoptera, Elateridae, Agrypninae, Conoderini) detected by standard staining, C-banding, silver nitrate impregnation, and CMA3/DA/DAPI staining.

    PubMed

    Schneider, Marielle Cristina; Almeida, Mara Cristina; Rosa, Simone Policena; Costa, Cleide; Cella, Doralice Maria

    2006-01-01

    The speciose Brazilian Elateridae fauna is characterized by high karyotypic diversity, including one species (Chalcolepidius zonatus Eschscholtz, 1829) with the lowest diploid number within any Coleoptera order. Cytogenetic analysis of Conoderus dimidiatus Germar, 1839, C. scalaris (Germar, 1824,) C. ternarius Germar, 1839, and C. stigmosus Germar, 1839 by standard and differential staining was performed with the aim of establishing mechanisms of karyotypic differentiation in these species. Conoderus dimidiatus, C. scalaris, and C. ternarius have diploid numbers of 2n(male) = 17 and 2n(female) = 18, and a X0/XX sex determination system, similar to that encountered in the majority of Conoderini species. The karyotype of C. stigmosus was characterized by a diploid number of 2n = 16 and a neoXY/neoXX sex determination system that was highly differentiated from other species of the genus. Some features of the mitotic and meiotic chromosomes suggest an autosome/ancestral X chromosome fusion as the cause of the neoXY system origin in C. stigmosus. C-banding and silver impregnation techniques showed that the four Conoderus species possess similar chromosomal characteristics to those registered in most Polyphaga species, including pericentromeric C band and autosomal NORs. Triple staining techniques including CMA3/DA/DAPI also provided useful information for differentiating these Conoderus species. These techniques revealed unique GC-rich heterochromatin associated with NORs in C. scalaris and C. stigmosus and CMA3-heteromorphism in C. scalaris and C. ternarius.

  16. Chromosome studies of Astyanax jacuhiensis Cope, 1894 (Characidae) from the Tramandai River Basin, Brazil, using in situ hybridization with the 18S rDNA probe, DAPI and CMA3 staining.

    PubMed

    da Silva, Laura Lahr Lourenço; Giuliano-Caetano, Lucia; Dias, Ana Lúcia

    2012-01-01

    The genus Astyanax comprises 86 species of fish distributed in Brazilian river basins and is considered of the Incertae sedis group within the family Characidae. This study presents an analysis of 12 specimens of Astyanax jacuhiensis from the Tramandai River Basin, RS Brazil: 6 from the Maquiné River and 6 from the Quadros Lagoon. All specimens showed a diploid number equal to 50 chromosomes with different karyotypic formula between the two localities. The population from the Maquiné River showed 10m+26sm+6st+8a (FN=92). Fish from the Quadros Lagoon showed 12m+20sm+6st+12a (FN=88). AgNORs were evidenced in the short arm of one acrocentric chromosome pair in both populations, confirmed by FISH with the 18S rDNA probe. CMA3 fluorochrome corresponded with the AgNOR sites, while DAPI staining was negative in these regions. C banding revealed that heterochromatin was weakly distributed, mainly in the pericentromeric and terminal regions in most chromosomes. Analyses of male gonadal tissue were conducted with the objective of characterizing the meiotic chromosome behavior in A. jacuhiensis. The following stages were evidenced: spermatogonial with 50 chromosomes, pachytene and metaphase I with 25 bivalents, and metaphase II with 25 chromosomes, thus confirming the diploid number of the species. Chromosomal abnormalities were not observed. This study shows preliminary data on A. jacuhiensis from the Tramandai River Basin, contributing with more chromosomal information for this group of fish.

  17. A New Class of Molecular Targeted Radioprotectors: GSK-3beta Inhibitors

    SciTech Connect

    Thotala, Dinesh K.; Geng Ling; Dickey, Amy K.; Hallahan, Dennis E.; Yazlovitskaya, Eugenia M.

    2010-02-01

    Purpose: Development of new treatments is critical to effective protection against radiation-induced injury. We investigate the potential of developing small-molecule inhibitors of glycogen synthase kinase 3beta (GSK-3beta)-SB216763 or SB415286-as radioprotective agents to attenuate intestinal injury. Methods and Materials: A survival study was done by use of C57BL/6J mice to evaluate the radioprotective effect of GSK-3beta inhibitors. Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay and immunohistochemical staining for Bax and Bcl-2 were used to assess apoptosis in the small intestines of the treated mice. A clonogenic survival study, apoptosis assays (staining with annexin V or 4',6-diamidino-2-phenylindole), and immunoblot analysis of beta-catenin, Bcl-2, Bax, and caspase 3 were done by use of Rat intestinal epithelial cell line IEC-6 cells. Results: Pretreatment with SB415286 significantly improved survival of mice irradiated with 8 and 12 Gy. Mice pretreated with SB216763 or SB415286 showed a significant reduction in TUNEL- and Bax-positive cells and an increase in Bcl-2-positive cells in intestinal crypts at 4 and/or 12 h after radiation with 4 and/or 8 Gy compared with radiation alone. Pretreatment of irradiated IEC-6 cells with GSK-3beta inhibitors significantly increased clonogenic survival compared with cells treated with radiation alone. This increase was due to the attenuation of radiation-induced apoptosis, as shown by annexin V and 4',6-diamidino-2-phenylindole assays, as well as immunoblot analysis of Bcl-2, Bax, and caspase 3. Conclusions: Glycogen synthase kinase 3beta small-molecule inhibitors protect mouse intestine from radiation-induced damage in cell culture and in vivo and improve survival of mice. Molecular mechanisms of this protection involve attenuated radiation-induced apoptosis regulated by Bcl-2, Bax, and caspase 3. Therefore GSK-3beta inhibitors reduce deleterious consequences of intestinal irradiation and

  18. Kinetin induces cell death in root cortex cells of Vicia faba ssp. minor seedlings.

    PubMed

    Kunikowska, Anita; Byczkowska, Anna; Kaźmierczak, Andrzej

    2013-08-01

    The double fluorescence staining with acridine orange and ethidium bromide (AO/EB) revealed that treatment of Vicia faba ssp. minor seedlings with kinetin-induced programmed cell death (PCD) in root cortex cells. Kinetin-induced cell death reflected by the morphological changes of nuclei including their invagination, volume increase, chromatin condensation and degradation as well as formation of micronuclei showed by AO/EB and 4,6-diamidino-2-phenylindol staining was accompanied by changes including increase in conductivity of cell electrolytes secreted to culture media, decrease in the number of the G1- and G2-phase cells and appearance of fraction of hypoploid cells as the effect of DNA degradation without ladder formation. Decrease in the number of mitochondria and in the activity of cellular dehydrogenases, production of reactive oxygen species (ROS), appearance of small and then large lytic vacuoles and increase in the amount of cytosolic calcium ions were also observed. The PCD was also manifested by increased width and weight of apical fragments of roots as well as decreased length of cortex cells which led to shortening of the whole roots. The kinetin-induced PCD process was almost completely inhibited by adenine, an inhibitor of phosphoribosyl transferase, and mannitol, an inhibitor of ROS production. These cell-death hallmarks and pathway of this process suggested that the induction of kinetin-specific vacuolar type of death, expressed itself with similar intensity on both morphological and metabolic levels, was a transient protecting whole roots and whole seedlings against elimination.

  19. Fermented Chinese Formula Shuan-Tong-Ling Protects Brain Microvascular Endothelial Cells against Oxidative Stress Injury

    PubMed Central

    Tan, Lingjing; Zhang, Xiang; Wang, Jinfeng; Li, Xiaoli; Huang, Weifeng; Yang, Songbai

    2016-01-01

    Fermented Chinese formula Shuan-Tong-Ling (STL), composed of fourteen medicinal herbs, was an experiential formula by Dr. Zhigang Mei for treating vascular encephalopathy, but the underlying mechanisms remained unknown. In this study, we aimed to investigate the protective effects of fermented STL on hydrogen peroxide- (H2O2-) induced injury in rat brain microvascular endothelial cells (BMECs) and the possible mechanisms. Cultured BMECs were treated with H2O2, STL, or nicotinamide (NAM, a SIRT1 inhibitor). Then, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was employed to detect cell proliferation and senescence-associated beta-galactosidase (SA-β-gal) was used to examine cell senescence. Cell nuclei were observed by 4′,6-diamidino-2-phenylindole. Additionally, changes in reactive oxygen species (ROS), superoxide dismutase (SOD), and glutathione (GSH) levels were measured. Expression of SIRT1, p21, and PGC-1α was determined by western blot. Cell proliferation significantly increased with STL treatment in a dose-dependent manner. H2O2 treatment could intensify cell senescence and nuclei splitting or pyknosis. With STL treatment, the reduced ROS level was accompanied by increased SOD and GSH activity. Further assays showed upregulation of SIRT1 and PGC-1α and downregulation of p21 after STL treatment. The results revealed that STL could protect BMECs against oxidative stress injury at least partially through the SIRT1 pathway. PMID:28096886

  20. Spatiotemporal organization of AT- and GC-rich DNA and their association with transition proteins TP1 and TP2 in rat condensing spermatids.

    PubMed

    Kolthur-Seetharam, Ullas; Pradeepa, Madapura M; Gupta, Nikhil; Narayanaswamy, Rammohan; Rao, Manchanahalli R Satyanarayana

    2009-10-01

    Transition protein 1 (TP1) and TP2 replace histones during midspermiogenesis (stages 12-15) and are finally replaced by protamines. TPs play a predominant role in DNA condensation and chromatin remodeling during mammalian spermiogenesis. TP2 is a zinc metalloprotein with two novel zinc finger modules that condenses DNA in vitro in a GC-preference manner. TP2 also localizes to the nucleolus in transfected HeLa and Cos-7 cells, suggesting a GC-rich preference, even in vivo. We have now studied the localization pattern of TP2 in the rat spermatid nucleus. Colocalization studies using GC-selective DNA-binding dyes chromomycin A3 and 7-amino actinomycin D and an AT-selective dye, 4',6-diamidino-2-phenylindole, indicate that TP2 is preferentially localized to GC-rich sequences. Interestingly, as spermatids mature, TP2 and GC-rich DNA moves toward the nuclear periphery, and in the late stages of spermatid maturation, TP2 is predominantly localized at the nuclear periphery. Another interesting observation is the mutually exclusive localization of GC- and AT-rich DNA in the elongating and elongated spermatids. A combined immunofluorescence experiment with anti-TP2 and anti-TP1 antibodies revealed several foci of overlapping localization, indicating that TP1 and TP2 may have concerted functional roles during chromatin remodeling in mammalian spermiogenesis.

  1. Dynamics of Nuclear DNA Quantities during Zygote Development in Barley.

    PubMed Central

    Mogensen, H. L.; Holm, P. B.

    1995-01-01

    Quantities of DNA were estimated in the nuclei of mechanically isolated egg and zygote protoplasts in two cultivars of barley using 4[prime],6-diamidino-2-phenylindole staining and microfluorometry. Unlike many previous studies on DNA amounts within the sex cells of flowering plants, we obtained consistent and unambiguous results indicating that the egg and sperm nuclei are at the 1C DNA level (basic haploid amount) at the time of karyogamy. Karyogamy was initiated within 60 min postpollination, and the male chromatin became completely integrated into the egg nucleus within 6 to 7 hr postpollination (hpp). Zygotic nuclear DNA levels began to increase at ~9 to 12 hpp in cultivar Alexis and at 12 to 15 hpp in cultivar Igri. The 4C DNA complement was reached in most zygotes by 22 to 26 hpp in cultivar Alexis and by 23 to 29 hpp in cultivar Igri. These data are fundamental to a better understanding of fertilization and zygote maturation in flowering plants. They are also relevant to studies in which the timing of zygotic DNA replication is of interest, such as ongoing investigations on genetic transformations in barley using the microinjection technique. PMID:12242375

  2. Bioactive chemicals from carrot (Daucus carota) juice extracts for the treatment of leukemia.

    PubMed

    Zaini, Rana; Clench, Malcolm R; Le Maitre, Christine L

    2011-11-01

    Overwhelming evidence indicates that consumption of fruits and vegetables with antioxidant properties correlates with reduced risk for cancers, including leukemia. Carrots contain beneficial agents, such as β-carotene and polyacetylenes, which could be effective in the treatment of leukemia. This study investigated the effect of carrot juice extracts on myeloid and lymphoid leukemia cell lines together with normal hematopoietic stem cells. Leukemia cell lines and nontumor control cells were treated with carrot juice extracts for up to 72 hours in vitro. Induction of apoptosis was investigated by using annexin V/propidium iodide staining followed by flow cytometric analysis, and results were confirmed by using 4'-6-diamidino-2-phenylindole morphology. Effects on cellular proliferation were investigated via cell cycle analysis and cell counts. Treatment of leukemia cell lines with carrot juice extract induced apoptosis and inhibited progression through the cell cycle. Lymphoid cell lines were affected to a greater extent than were myeloid cell lines, and normal hematopoietic stem cells were less sensitive than most cell lines. This study has shown that extracts from carrots can induce apoptosis and cause cell cycle arrest in leukemia cell lines. The findings suggest that carrots may be an excellent source of bioactive chemicals for the treatment of leukemia.

  3. A potent antimicrobial peptide derived from the protein LsGRP1 of Lilium.

    PubMed

    Lin, Chia-Hua; Chang, Min-Wei; Chen, Chao-Ying

    2014-04-01

    LsGRP1 is a defense-related gene differentially expressed in lily leaves in response to pathogen attack. The difficulty in the expression of LsGRP1 in Escherichia coli suggested the presence of antimicrobial activity in LsGRP1. To evaluate the antimicrobial trait of LsGRP1, three LsGRP1-derived peptides were chemically synthesized; namely LsGRP1(N) (N-terminal region without the signal peptide), LsGRP1(G) (glycine-rich region), and LsGRP1(C) (C-terminal cysteine-rich region). LsGRP1(C) was proposed to be a potential antimicrobial agent according to its broad-spectrum and effective antimicrobial activity. LsGRP1(C) displayed inhibition effects on bacterial and fungal growth, possibly by altering the integrity of the cell membrane, as indicated by scanning electron microscopy and SYTOX Green staining assays. Additionally, LsGRP1(C) induced programmed cell death-like phenomenon in the tested fungal species as indicated by 2',7'-dichlorodihydrofluorescein diacetate and 4',6'-diamidino-2-phenylindole assays. Further immunofluorescence staining showed that LsGRP1(C) was located at the fungal cell surface. According to these observations, we concluded that LsGRP1(C) originated from the plant defense-related protein LsGRP1 would play a role as an antimicrobial peptide and have a potential for practical use.

  4. Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover

    PubMed Central

    Richardson, Gavin D.

    2016-01-01

    Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover occur during normal ageing, quantification of these events remains challenging. This is in part due to the rarity of the process and the fact that multiple cellular sources contribute to myocardial maintenance. Furthermore, DNA duplication within cardiomyocytes often leads to a polyploid cardiomyocyte and only rarely leads to new cardiomyocytes by cellular division. In order to accurately quantify cardiomyocyte turnover discrimination between these processes is essential. The protocol described here employs long term nucleoside labeling in order to label all nuclei which have arisen as a result of DNA replication and cardiomyocyte nuclei identified by utilizing nuclei isolation and subsequent PCM1 immunolabeling. Together this allows the accurate and sensitive identification of the nucleoside labeling of the cardiomyocyte nuclei population. Furthermore, 4′,6-diamidino-2-phenylindole labeling and analysis of nuclei ploidy, enables the discrimination of neo-cardiomyocyte nuclei from nuclei which have incorporated nucleoside during polyploidization. Although this method cannot control for cardiomyocyte binucleation, it allows a rapid and robust quantification of neo-cardiomyocyte nuclei while accounting for polyploidization. This method has a number of downstream applications including assessing the potential therapeutics to enhance cardiomyocyte regeneration or investigating the effects of cardiac disease on cardiomyocyte turnover and ploidy. This technique is also compatible with additional downstream immunohistological techniques, allowing quantification of nucleoside incorporation in all cardiac cell types. PMID:27285379

  5. Combination of cold atmospheric plasma and iron nanoparticles in breast cancer: gene expression and apoptosis study

    PubMed Central

    Jalili, Azam; Irani, Shiva; Mirfakhraie, Reza

    2016-01-01

    Background Current cancer treatments have unexpected side effects of which the death of normal cells is one. In some cancers, iron nanoparticles (NPs) can be subjected to diagnosis and passive targeting treatment. Cold atmospheric plasma (CAP) has a proven induction of selective cell death ability. In this study, we have attempted to analyze the synergy between CAP and iron NPs in human breast adenocarcinoma cells (MCF-7). Materials and methods In vitro cytotoxicity of CAP treatment and NPs in cells measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cell death was shown by 4′,6-diamidino-2-phenylindole and annexin V staining. Fluctuations in BAX and BCL-2 gene expression were investigated by means of real-time polymerase chain reaction. Results MTT assay results showed that combination of plasma and iron NPs decreased the viability of cancer cells significantly (P<0.05). Real-time analysis showed that the combination therapy induced shifting the BAX/BCL-2 ratio in favor of apoptosis. Conclusion Our data indicate that synergy between CAP and iron NPs can be applied in breast cancer treatment selectively. PMID:27729800

  6. Bone-marrow mesenchymal stem cell transplantation to treat diabetic nephropathy in tree shrews.

    PubMed

    Pan, Xing-Hua; Yang, Xiao-Yan; Yao, Xiang; Sun, Xiao-Mei; Zhu, Lu; Wang, Jin-Xiang; Pang, Rong-Qing; Cai, Xue-Min; Dai, Jie-Jie; Ruan, Guang-Ping

    2014-07-01

    Diabetic nephropathy (DN) is a common microvascular complication of diabetes. We used a new DN model in tree shrews to validate the use of bone-marrow mesenchymal stem cell (BM-MSC) transplantation to treat DN. The DN tree shrew model was established by a high-sugar and high-fat diet and four injections of streptozotocin. 4',6-Diamidino-2-phenylindole labelled BM-MSCs were injected into tree shrews. The DN tree shrew model was successfully established. Blood glucose was significantly increased ( p < 0.01) during the entire experiment. DN tree shrews showed dyslipidemia, insulin resistance and increased 24-h proteinuria. At 21 days after BM-MSC transplantation, glucose and levels of triglycerides, total cholesterol and 24-h urine volume were lower than in tree shrews with DN alone ( p < 0.01) but were still higher than control values ( p < 0.01). Levels of creatinine and urea nitrogen as well as 24-h proteinuria were lower for DN tree shrews with BM-MSCs transplantation than DN alone ( p < 0.05). High-sugar and high-fat diet combined with STZ injection can induce a tree shrew model of DN. BM-MSCs injection can home to damaged kidneys and pancreas, for reduced 24-h proteinuria and improved insulin resistance.

  7. Novel anatomic structures in the brain and spinal cord of rabbit that may belong to the Bonghan system of potential acupuncture meridians.

    PubMed

    Lee, Byung-Cheon; Kim, Sungkwang; Soh, Kwang-Sup

    2008-09-01

    Novel threadlike structures of 20 to 40 microm in diameter were observed running afloat in the cerebrospinal fluid of the brain ventricles and the spinal central canal of a rabbit. We developed an effective in situ staining technique using hematoxylin to visualize the threadlike structure. The presence of the rod-shaped nuclei in the threadlike structure was confirmed by various nucleus specific staining dyes such as 4',6'-diamidino-2-phenylindole, propidium iodide and yoyo-1. The threadlike structure was surrounded by a cellular membrane, whose presence was visualized by using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate staining. The location, diameter, optical transparency and the presence of rod-shaped nuclei in and the surrounding membranes of the threadlike structure were consistent with a nerve Bonghan duct. The Bonghan duct was claimed to be the extension of the physical substance of acupuncture meridians and to be a distinct type of circulatory system present in mammals. Although Bonghan theory has not been reproduced for a long time, recently, some portions of the Bonghan duct network were confirmed in various organs in mammals including blood vessels, lymphatic vessels and enteric organs. The novel threadlike structure in the central nervous system, more specifically in brain ventricles, is one in a series of findings in an attempt to rediscover the Bonghan duct network.

  8. Meniscus reconstruction through coculturing meniscus cells with synovium-derived stem cells on small intestine submucosa--a pilot study to engineer meniscus tissue constructs.

    PubMed

    Tan, Yunbing; Zhang, Yuanyuan; Pei, Ming

    2010-01-01

    The purpose of this study was to investigate the feasibility of coculturing meniscus cells (MCs) and synovium-derived stem cells (SDSCs) on small intestine submucosa (SIS) to establish an innovative method to engineer in vitro meniscus constructs. About 0.9 million cells (MCs, prelabeled SDSCs [with Vybrant DiI], and a coculture of MCs and prelabeled SDSCs [50:50]) were mixed with fibrin gel and seeded onto freeze-dried SIS discs (5 mm diameter x 1-2 mm thickness) individually. The tissue constructs were incubated in a serum-free defined medium supplemented with 10 ng/mL transforming growth factor beta-1 and 500 ng/mL insulin-like growth factor I for 1 month. One day after cell seeding, samples for scanning electron microscopy were prepared to evaluate cell attachment on the SIS surface. During incubation, fluorescent microscopy was used to trace cell migration (with 4',6-diamidino-2-phenylindole as a counterstain) on SIS scaffold. The tissue constructs were assessed using histology, immunostaining, biochemical analysis, real-time polymerase chain reaction, and compressive modulus. All three groups of cells attached well on SIS. The coculture with SDSCs yielded MC-based tissue constructs with greater cell survival and differentiation into chondrogenic phenotypes, which exhibited higher glycosaminoglycan, collagen II, and Sox 9 but relatively low collagen I, resulting in the concomitant increase in equilibrium modulus. This pilot study demonstrates the advantages of coculturing MCs with SDSCs on SIS for meniscus tissue engineering and regeneration.

  9. Isolation and evaluation of biological efficacy of quercetol in human hepatic carcinoma cells

    PubMed Central

    Ali, Huma; Dixit, Savita; Ali, Daoud; Alkahtane, Abdullah A; Alarifi, Saud; Ali, Bahy A; Alkahtani, Saad

    2016-01-01

    Quercetol is a polyphenolic molecule present in vegetables and fruits, and is beneficial to human and animal health. The current work aimed to test cytotoxic and apoptotic effects of quercetol on HepG2 cells. Quercetol was isolated from Ocimum sanctum and characterized by gas chromatography–tandom mass spectrometry (GC-MS/MS), nuclear magnetic resonance spectroscopy, and Fourier transform infrared spectroscopy. Quercetol (50–600 μg/mL) was examined for cytotoxic activity by tetrazolium salt and neutral red uptake tests and comet assay for genotoxicity, using HepG2 cells, over 24 hours. Data from 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide and neutral red uptake tests demonstrated quercetol-induced cytotoxicity in HepG2 cells in a concentration-dependent manner. With 4′,6-diamidino-2-phenylindole staining, a significant induction of chromosomal condensation was observed at 300 μg/mL of quercetol. DNA fragmentation analysis showed that quercetol produced cell death in HepG2 cells in a concentration-dependent manner. Thus, our study suggests that an environmentally relevant concentration of quercetol, which was a chemically standardized extract from O. sanctum, induced cell death and DNA damage in HepG2 cells. PMID:26792982

  10. Toward a Cytological Characterization of the Rice Genome

    PubMed Central

    Cheng, Zhukuan; Buell, C. Robin; Wing, Rod A.; Gu, Minghong; Jiang, Jiming

    2001-01-01

    Rice (Oryza sativa L.) will be the first major crop, as well as the first monocot plant species, to be completely sequenced. Integration of DNA sequence-based maps with cytological maps will be essential to fully characterize the rice genome. We have isolated a set of 24 chromosomal arm-specific bacterial artificial chromosomes to facilitate rice chromosome identification. A standardized rice karyotype was constructed using meiotic pachytene chromosomes of O. sativa spp. japonica rice var. Nipponbare. This karyotype is anchored by centromere-specific and chromosomal arm-specific cytological landmarks and is fully integrated with the most saturated rice genetic linkage maps in which Nipponbare was used as one of the mapping parents. An ideogram depicting the distribution of heterochromatin in the rice genome was developed based on the patterns of 4',6-diamidino-2-phenylindole staining of the Nipponbare pachytene chromosomes. The majority of the heterochromatin is distributed in the pericentric regions with some rice chromosomes containing a significantly higher proportion of heterochromatin than other chromosomes. We showed that pachytene chromosome-based fluorescence in situ hybridization analysis is the most effective approach to integrate DNA sequences with euchromatic and heterochromatic features. PMID:11731505

  11. Single Walled Carbon Nanotubes Exhibit Dual-Phase Regulation to Exposed Arabidopsis Mesophyll Cells

    PubMed Central

    2011-01-01

    Herein we are the first to report that single-walled carbon nanotubes (SWCNTs) exhibit dual-phase regulation to Arabidopsis mesophyll cells exposed to different concentration of SWCNTs. The mesophyll protoplasts were prepared by enzyme digestion, and incubated with 15, 25, 50, 100 μg/ml SWCNTs for 48 h, and then were observed by optical microscopy and transmission electron microscopy, the reactive oxygen species (ROS) generation was measured. Partial protoplasts were stained with propidium iodide and 4'-6- diamidino-2-phenylindole, partial protoplasts were incubated with fluorescein isothiocyanate-labeled SWCNTs, and observed by fluorescence microscopy. Results showed that SWCNTs could traverse both the plant cell wall and cell membrane, with less than or equal to 50 μg/ml in the culture medium, SWCNTs stimulated plant cells to grow out trichome clusters on their surface, with more than 50 μg/ml SWCNTs in the culture medium, SWCNTs exhibited obvious toxic effects to the protoplasts such as increasing generation of ROS, inducing changes of protoplast morphology, changing green leaves into yellow, and inducing protoplast cells' necrosis and apoptosis. In conclusion, single walled carbon nanotubes can get through Arabidopsis mesophyll cell wall and membrane, and exhibit dose-dependent dual-phase regulation to Arabidopsis mesophyll protoplasts such as low dose stimulating cell growth, and high dose inducing cells' ROS generation, necrosis or apoptosis. PMID:27502666

  12. Development and Optimization of a Novel 384-Well Anti-Malarial Imaging Assay Validated for High-Throughput Screening

    PubMed Central

    Duffy, Sandra; Avery, Vicky M.

    2012-01-01

    With the increasing occurrence of drug resistance in the malaria parasite, Plasmodium falciparum, there is a great need for new and novel anti-malarial drugs. We have developed a 384-well, high-throughput imaging assay for the detection of new anti-malarial compounds, which was initially validated by screening a marine natural product library, and subsequently used to screen more than 3 million data points from a variety of compound sources. Founded on another fluorescence-based P. falciparum growth inhibition assay, the DNA-intercalating dye 4′,6-diamidino-2-phenylindole, was used to monitor changes in parasite number. Fluorescent images were acquired on the PerkinElmer Opera High Throughput confocal imaging system and analyzed with a spot detection algorithm using the Acapella data processing software. Further optimization of this assay sought to increase throughput, assay stability, and compatibility with our high-throughput screening equipment platforms. The assay typically yielded Z'-factor values of 0.5–0.6, with signal-to-noise ratios of 12. PMID:22232455

  13. Flow sorting of the Y sex chromosome in the dioecious plant Melandrium album

    SciTech Connect

    Veuskens, J.; Jacobs, M.; Negrutiu, I.

    1995-12-01

    The preparation of stable chromosome suspensions and flow cytometric sorting of both the Y sex chromosome of the white campion, Melandrium album, and the deleted Y chromosome of an asexual mutant, 5K63, is described. The principle has been to maintain transformed roots in vitro, synchronize and block mitosis, reduce cells to protoplasts, and lyse these to release chromosomes. Such in vitro material, unlike many cell suspensions, showed a stable karyotype. Factors critical to producing high-quality chromosome suspensions from protoplasts include osmolality of isolation solutions and choice of spindle toxin and of lysis buffer. Agrobacterium rhizogenes transformed young growing root cultures were synchronized at G1/S with 50 {mu}M aphidicolin for 24 h and released to a mitotic block with 30 {mu}M oryzalin for 11 h. Protoplast preparations from such tissue routinely had metaphase indices reaching 15%. Suspensions of intact metaphase chromosomes, with few chromatids, were obtained by lysing swollen mitotic protoplasts in a citric acid/disodium phosphate buffer. Except for the presence of clumps of autosomal chromosomes near the X and Y chromosome zones, monoparametric histograms of fluorescence intensities of suspensions stained with 4{prime},6-diamidino-2-phenylindole showed profiles similar to theoretical flow karyotypes. Two types of Y chromosomes, one full-length and one partially deleted (from the asexual mutant), could be sorted at 90% purity (21-fold enrichment of Y). These results are discussed in the context of sex determination and differentiation in higher plants. 45 refs., 6 figs., 2 tabs.

  14. Variations in cell morphology in the canine cruciate ligament complex.

    PubMed

    Smith, K D; Vaughan-Thomas, A; Spiller, D G; Clegg, P D; Innes, J F; Comerford, E J

    2012-08-01

    Cell morphology may reflect the mechanical environment of tissues and influence tissue physiology and response to injury. Normal cruciate ligaments (CLs) from disease-free stifle joints were harvested from dog breeds with a high (Labrador retriever) and low (Greyhound) risk of cranial cruciate ligament (CCL) rupture. Antibodies against the cytoskeletal components vimentin and alpha tubulin were used to analyse cell morphology; nuclei were stained with 4',6-diamidino-2-phenylindole, and images were collected using conventional and confocal microscopy. Both cranial and caudal CLs contained cells of heterogenous morphologies. Cells were arranged between collagen bundles and frequently had cytoplasmic processes. Some of these processes were long (type A cells), others were shorter, thicker and more branched (type B cells), and some had no processes (type C cells). Processes were frequently shown to contact other cells, extending longitudinally and transversely through the CLs. Cells with longer processes had fusiform nuclei, and those with no processes had rounded nuclei and were more frequent in the mid-substance of both CLs. Cells with long processes were more commonly noted in the CLs of the Greyhound. As contact between cells may facilitate direct communication, variances in cell morphology between breeds at a differing risk of CCL rupture may reflect differences in CL physiology.

  15. Pulsed Electromagnetic Field Stimulation Promotes Anti-cell Proliferative Activity in Doxorubicin-treated Mouse Osteosarcoma Cells

    PubMed Central

    MURAMATSU, YOSHITAKA; MATSUI, TAKUYA; DEIE, MASATAKA; SATO, KEIJI

    2017-01-01

    Aim: We aimed to investigate the synergistic effects of pulsed electromagnetic field (PEMF) and doxorubicin therapy in a mouse osteosarcoma cell line (LM8 cells) in vitro. Materials and Methods: The effects of PEMF (5 mT, 200 Hz) of different durations and doxorubicin on the proliferative activity of LM8 cells were measured by the MTT assay. Apoptotic-related factors such as cell-cycle phase, mitochondrial membrane potential, and caspase 3/7 activity were investigated using 4’,6-diamidino-2-phenylindole staining and apoptosis kits. Identification of intracellular signaling molecules induced by the combination was comprehensively explored using a stress and apoptosis-related protein array kit. Results: PEMF enhanced the inhibition of cell proliferation mediated by doxorubicin but did not affect the cell cycle, mitochondrial membrane potential, or doxorubicin-induced G2/M arrest. The combination of PEMF and doxorubicin altered a few signaling molecules. PEMF tended to reduce the doxorubicin-induced decrease of phosphorylated BAD, while reducing the increased expression of total IĸB and phosphorylated-CHK1 induced by doxorubicin. Conclusion: Our results indicate that combination of PEMF and doxorubicin could be a novel chemotherapeutic strategy. PMID:28064222

  16. Isolation and evaluation of biological efficacy of quercetol in human hepatic carcinoma cells.

    PubMed

    Ali, Huma; Dixit, Savita; Ali, Daoud; Alkahtane, Abdullah A; Alarifi, Saud; Ali, Bahy A; Alkahtani, Saad

    2016-01-01

    Quercetol is a polyphenolic molecule present in vegetables and fruits, and is beneficial to human and animal health. The current work aimed to test cytotoxic and apoptotic effects of quercetol on HepG2 cells. Quercetol was isolated from Ocimum sanctum and characterized by gas chromatography-tandom mass spectrometry (GC-MS/MS), nuclear magnetic resonance spectroscopy, and Fourier transform infrared spectroscopy. Quercetol (50-600 μg/mL) was examined for cytotoxic activity by tetrazolium salt and neutral red uptake tests and comet assay for genotoxicity, using HepG2 cells, over 24 hours. Data from 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide and neutral red uptake tests demonstrated quercetol-induced cytotoxicity in HepG2 cells in a concentration-dependent manner. With 4',6-diamidino-2-phenylindole staining, a significant induction of chromosomal condensation was observed at 300 μg/mL of quercetol. DNA fragmentation analysis showed that quercetol produced cell death in HepG2 cells in a concentration-dependent manner. Thus, our study suggests that an environmentally relevant concentration of quercetol, which was a chemically standardized extract from O. sanctum, induced cell death and DNA damage in HepG2 cells.

  17. Visualization of the network of primo vessels and primo nodes above the pia mater of the brain and spine of rats by using Alcian blue.

    PubMed

    Lee, Ho-Sung; Lee, Byung-Cheon

    2012-10-01

    By spraying and injecting Alcian blue into the lateral ventricle, we were able to visualize the network of the nerve primo vascular system above the pia mater of the brain and spine of rats. Staining these novel structures above the pia mater with 4',6-diamidino-2-phenylindole demonstrated that they coexisted in cellular and extracellular DNA forms. The cellular primo node consisted of many cells surrounded by rod-shaped nuclei while the extracellular primo node had a different morphology from that of a general cell in terms of DNA signals, showing granular DNA in a threadlike network of extracellular DNA. Also, differently from F-actin in general cells, the F-actin in the primo vessel was short and rod-shaped. Light and transmission electron microscopic images of the PN showed that the nerve primo vascular system above the pia mater of the brain and spine was a novel dynamic network, suggesting the coexistence of DNA and extracellular DNA. Based on these data, we suggest that a novel dynamic system with a certain function exists above the pia mater of the central nerve system. We also discuss the potential of this novel network system in the brain and spine as related to acupuncture meridians and neural regeneration.

  18. Gibberellin Produced in the Cotyledon Is Required for Cell Division during Tissue Reunion in the Cortex of Cut Cucumber and Tomato Hypocotyls1

    PubMed Central

    Asahina, Masashi; Iwai, Hiroaki; Kikuchi, Akira; Yamaguchi, Shinjiro; Kamiya, Yuji; Kamada, Hiroshi; Satoh, Shinobu

    2002-01-01

    Cucumber (Cucumis sativus) hypocotyls were cut to one-half of their diameter transversely, and morphological and histochemical analyses of the process of tissue reunion in the cortex were performed. Cell division in the cortex commenced 3 d after cutting, and the cortex was nearly fully united within 7 d. 4′,6-Diamidino-2-phenylindole staining and 5-bromo-2′-deoxyuridine labeling experiments indicate that nDNA synthesis occurred during this process. In addition, specific accumulation of pectic substances was observed in the cell wall of attached cells in the reunion region of the cortex. Cell division during tissue reunion was strongly inhibited when the cotyledon was removed. This inhibition was reversed by applying gibberellin (GA, 10−4 m GA3) to the apical tip of the cotyledon-less plant. Supporting this observation, cell division in the cortex was inhibited by treatment of the cotyledon with 10−4 m uniconazole-P (an inhibitor of GA biosynthesis), and this inhibition was also reversed by simultaneous application of GA. In contrast to the essential role of cotyledon, normal tissue reunion in cut hypocotyls was still observed when the shoot apex was removed. The requirement of GA for tissue reunion in cut hypocotyls was also evident in the GA-deficient gib-1 mutant of tomato (Lycopersicon esculentum). Our results suggest that GA, possibly produced in cotyledons, is essential for cell division in reuniting cortex of cut hypocotyls. PMID:12011351

  19. Esculetin, a Coumarin Derivative, Exhibits Anti-proliferative and Pro-apoptotic Activity in G361 Human Malignant Melanoma

    PubMed Central

    Jeon, Young-Joo; Jang, Jeong-Yun; Shim, Jung-Hyun; Myung, Pyung Keun; Chae, Jung-Il

    2015-01-01

    Background: Although esculetin, a coumarin compound, is known to induce apoptosis in human cancer cells, the effects and molecular mechanisms on the apoptosis in human malignant melanoma (HMM) cells are not well understood yet. In this study, we investigated the anti-proliferative effects of esculetin on the G361 HMM cells. Methods: We analyzed the anti-proliferative effects and molecular mechanisms of esculetin on G361 cells by a 3-(4,5-dimethylthiazol- 2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, 4′,6-diamidino-2-phenylindole staining and Western blotting. Results: Esculetin exhibited significant anti-proliferative effects on the HMM cells in a dose-dependent manner. Interestingly, we found that esculetin induced nuclear shrinkage and fragmentation, typical apoptosis markers, by suppression of Sp1 transcription factor (Sp1). Notably, esculetin modulated Sp1 downstream target genes including p27, p21 and cyclin D1, resulted in activation of apoptosis signaling molecules such as caspase-3 and PARP in G361 HMM cells. Conclusions: Our results clearly demonstrated that esculetin induced apoptosis in the HMM cells by downregulating Sp1 protein levels. Thus, we suggest that esculetin may be a potential anti-proliferative agent that induces apoptotic cell death in G361 HMM cells. PMID:26151043

  20. Formation of volutin granules in Corynebacterium glutamicum.

    PubMed

    Pallerla, Srinivas Reddy; Knebel, Sandra; Polen, Tino; Klauth, Peter; Hollender, Juliane; Wendisch, Volker F; Schoberth, Siegfried M

    2005-02-01

    Volutin granules are intracellular storages of complexed inorganic polyphosphate (poly P). Histochemical staining procedures differentiate between pathogenic corynebacteria such as Corynebacterum diphtheriae (containing volutin) and non-pathogenic species, such as C. glutamicum. Here we report that strains ATCC13032 and MH20-22B of the non-pathogenic C. glutamicum also formed subcellular entities (18-37% of the total cell volume) that had the typical characteristics of volutin granules: (i) volutin staining, (ii) green UV fluorescence when stained with 4',6-diamidino-2-phenylindole, (iii) electron-dense and rich in phosphorus when determined with transmission electron microscopy and X-ray microanalysis, and (iv) 31P NMR poly P resonances of isolated granules dissolved in EDTA. MgCl2 addition to the growth medium stimulated granule formation but did not effect expression of genes involved in poly P metabolism. Granular volutin fractions from lysed cells contained polyphosphate glucokinase as detected by SDS-PAGE/MALDI-TOF, indicating that this poly P metabolizing enzyme is present also in intact poly P granules. The results suggest that formation of volutin is a more widespread phenomenon than generally accepted.

  1. Vacuolar localization of phosphorus in hyphae of Phialocephala fortinii, a dark septate fungal root endophyte.

    PubMed

    Saito, Katsuharu; Kuga-Uetake, Yukari; Saito, Masanori; Peterson, R Larry

    2006-07-01

    Phialocephala fortinii is a dark septate fungal endophyte that colonizes roots of many host species. Its effect on plant growth varies from being pathogenic to beneficial. The basic biology of this species has received little research, and thus the main objectives of this study were to determine cytological features of hyphae, including the nature of the vacuolar system, and whether polyphosphate was present in vacuoles. Both living hyphae and hyphae that had been rapidly frozen and freeze substituted before embedding were studied. A complex system of vacuoles, including a motile tubular vacuolar system, elongated vacuoles, and spherical vacuoles, was demonstrated in living hyphae by the fluorescent probe Oregon Green 488 carboxylic acid diacetate, using laser scanning confocal microscopy. The motile tubular vacuolar system was more prevalent at the hyphal tip than in more distal regions, whereas elongated vacuoles and spherical vacuoles were more abundant distal to the tip. All vacuoles contained polyphosphate as shown by labelling embedded samples with recombinant polyphosphate binding domain of Escherichia coli exopolyphosphatase, containing Xpress tag at the N-terminal end, followed by anti-Xpress antibody and a secondary antibody conjugated either to a fluorescent probe for laser scanning confocal microscopy or colloidal gold for transmission electron microscopy. The polyphosphate was dispersed in vacuoles. This was confirmed by staining embedded samples with 4',6-diamidino-2-phenylindole and viewing with UV light using epifluorescence microscopy. These cytological methods showed that the tubular vacuolar system had lower concentrations of polyphosphate than the spherical vacuoles. Lipid bodies were present around vacuoles.

  2. Dihydropyrimidine based hydrazine dihydrochloride derivatives as potent urease inhibitors.

    PubMed

    Khan, Ajmal; Hashim, Jamshed; Arshad, Nuzhat; Khan, Ijaz; Siddiqui, Naureen; Wadood, Abdul; Ali, Muzaffar; Arshad, Fiza; Khan, Khalid Mohammed; Choudhary, M Iqbal

    2016-02-01

    Four series of heterocyclic compounds 4-dihydropyrimidine-2-thiones 7-12 (series A), N,S-dimethyl-dihydropyrimidines 13-18 (series B), hydrazine derivatives of dihydropyrimidine 19-24 (series C), and tetrazolo dihydropyrimidine derivatives 25-30 (series D), were synthesized and evaluated for in vitro urease inhibitory activity. The series B-D were first time examined for urease inhibition. Series A and C were found to be significantly active with IC50 values between 34.7-42.9 and 15.0-26.0 μM, respectively. The structure-activity relationship showed that the free S atom and hydrazine moiety are the key pharmacophores against urease enzyme. The kinetic studies of the active series A (7-12) and C (19-24) were carried out to determine their modes of inhibition and dissociation constants Ki. Compounds of series A (7-12) and series C (19-24) showed a mixed-type of inhibition with Ki values ranging between 15.76-25.66 and 14.63-29.42 μM, respectively. The molecular docking results showed that all the active compounds of both series have significant binding interactions with the active sites specially Ni-ion of the urease enzyme. Cytotoxicity of all series A-D was also evaluated against mammalian mouse fibroblast 3T3 cell lines, and no toxicity was observed in cellular model.

  3. Formulation and evaluation of Cetirizine dihydrochloride orodispersible tablet.

    PubMed

    Subramanian, S; Sankar, V; Manakadan, Asha Asokan; Ismail, Sareena; Andhuvan, G

    2010-04-01

    Cetirizine orodispersible tablets were prepared to achieve quick onset of action and for maximum bioavailability. Tablets were prepared using cetirizine along with camphor and mannitol in the proportion of 1:1:1, 1:1:3, and 1:1:6. The flow property of granules was found to be good for the formulation CZ2 (1:1:3). The hardness and friability of all the formulations were found to be within the standard limit for orodispersible tablets. Disintegration time was found to be rapid in formulation CZ2 (1:1:3).The in vitro dissolution time was found to be 100% in 11 minutes for the formulation CZ2 (1:1:3).

  4. HPLC-ELSD analysis of spectinomycin dihydrochloride and its impurities.

    PubMed

    Zhou, Junyi; Zhang, Lin; Wang, Yan; Yan, Chao

    2011-08-01

    A simple, rapid and reliable reversed-phase ion-pair chromatography method by HPLC coupled to an evaporative light scattering detector (ELSD) has been developed to simultaneously determine chloride, spectinomycin and its related substances in a sample. The column was a TSKgel ODS-100V. The mobile phase was ACN/aqueous solution of 15 mM ammonium acetate adjusted with TFA to pH 3.0 (2:98 v/v), in an isocratic mode. The drift tube temperature was set at 50°C and the nebulizing gas flow rate of air was 3.5 L/min for ELSD detection. Almost all of the reported degradation compounds of spectinomycin such as actinamine, actinospectinoic acid and biosynthesis intermediates such as dihydrospectinomycin diastereoisomers were baseline separated. MS was utilized for the identification of spectinomycin and its seven related substances. The method for the assay of spectinomycin was successfully validated with respect to accuracy, precision (RSD less than 2%), linearity (throughout the linear range 0.025-3 mg/mL, r=0.9993), sensitivity (LOD: 100 ng on column) and robustness. The experimental results demonstrated that the simultaneous determination of chloride, spectinomycin and related substances is feasible in a single run, which suggests applicability in routine assays.

  5. 21 CFR 522.1362 - Melarsomine dihydrochloride for injection.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... with class 4 (very severe) heartworm disease (Caval Sydrome). Not for use in breeding animals and....1362 Section 522.1362 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS IMPLANTATION OR INJECTABLE DOSAGE FORM...

  6. 21 CFR 522.1362 - Melarsomine dihydrochloride for injection.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... with class 4 (very severe) heartworm disease (Caval Sydrome). Not for use in breeding animals and....1362 Section 522.1362 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS IMPLANTATION OR INJECTABLE DOSAGE FORM...

  7. 21 CFR 522.1362 - Melarsomine dihydrochloride for injection.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... with class 4 (very severe) heartworm disease (Caval Sydrome). Not for use in breeding animals and....1362 Section 522.1362 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS IMPLANTATION OR INJECTABLE DOSAGE FORM...

  8. ENHANCED DAPI STAINING FOR CRYPTOSPORIDIUM IN WATER SAMPLES

    EPA Science Inventory

    The U.S. Environmental Protection Agency's Method 1623 is used to detect and quantify the presence of {ital Cryptosporidium} spp. oocysts in water. The protocol consists of concentrating a sample, staining this concentrate with a fluorescent antibody, and examining the sample mi...

  9. Morphogenesis of human embryonic stem cells into mature neurons under in vitro culture conditions

    PubMed Central

    Shroff, Geeta

    2016-01-01

    AIM To describe the morphogenesis of different neuronal cells from the human embryonic stem cell (hESC) line, SCT-N, under in vitro culture conditions. METHODS The directed neuronal cell line was produced from a single, spare, pre-implantation stage fertilized ovum that was obtained during a natural in vitro fertilization process. The hESCs were cultured and maintained as per our proprietary in-house technology in a Good Manufacturing Practice, Good Laboratory Practice and Good Tissue Practice compliant laboratory. The cell line was derived and incubated in aerobic conditions. The cells were examined daily under a phase contrast microscope for their growth and differentiation. RESULTS Different neural progenitor cells (NPCs) and differentiating neurons were observed under the culture conditions. Multipotent NPCs differentiated into all three types of nervous system cells, i.e., neurons, oligodendrocytes and astrocytes. Small projections resembling neurites or dendrites, and protrusion coming out of the cells, were observed. Differentiating cells were observed at day 18 to 20. The differentiating neurons, neuronal bodies, axons, and neuronal tissue were observed on day 21 and day 30 of the culture. On day 25 and day 30, prominent neurons, axons and neuronal tissue were observed under phase contrast microscopy. 4’, 6-diamidino-2-phenylindole staining also indicated the pattern of differentiating neurons, axonal structure and neuronal tissue. CONCLUSION This study describes the generation of different neuronal cells from an hESC line derived from biopsy of blastomeres at the two-cell cleavage stage from a discarded embryo. PMID:27909687

  10. Measurement of the effects of cadmium stress on protozoan grazing of bacteria (bacterivory) in activated sludge by fluorescence microscopy

    SciTech Connect

    Hoffman, R.L.; Atlas, R.M.

    1987-10-01

    The effect of cadmium stress on protozoan bacterivory in sewage sludge was measured by experimentally exposing sludge communities to 0 to 150 mg of Cd per liter for up to 6 h and then determining the rates of protozoan grazing on bacteria, using a double-staining technique and epifluorescence microscopy. Bacterivory was measured by incubating the sludge with fluorescently labeled bacterium-sized latex beads and directly observing ingestion of the beads and bacterial cells in the sludge by epifluorescence microscopy of preserved samples. Staining with 4',6-diamidino-2-phenylindole and acridine orange permitted the simultaneous determination of protozoan numbers and bacterivory activity as estimated by the number of bacterial cells and bacterium-sized latex beads ingested by the representative ciliate Aspidisca costata. Enumeration with latex beads proved to be an effective way of estimating bacterivory in sludges subjected to heavy-metal stress. This technique should prove useful for determining the effects of other chemical stresses on protozoan numbers and bacterivory in organic-rich environments. Although the number of protozoa declined significantly only after exposure to 100 mg of Cd per liter for 4 h, grazing, as indicated by bead ingestion, was significantly inhibited by Cd concentrations of > 25 mg/liter in < 1 h, and exposure to 100 mg of Cd per liter effectively stopped protozoan grazing within 1 h of exposure. Protozoan ingestion of latex beads and bacteria was inversely correlated to Cd concentration and exposure time. The reduction of protozoan bacterivory by Cd provides a possible explanation for the increase in suspended bacteria in the effluents of metal-stressed treatment facilities.

  11. Green synthesis of silver and gold nanoparticles from Gymnema sylvestre leaf extract: study of antioxidant and anticancer activities

    NASA Astrophysics Data System (ADS)

    Nakkala, Jayachandra Reddy; Mata, Rani; Bhagat, Ekta; Sadras, Sudha Rani

    2015-03-01

    The present study reports the biological synthesis of silver and gold nanoparticles from Gymnema sylvestre leaf extract and their in vitro free radical scavenging efficacy as well as antiproliferative effect in Hep2 cells. The formation of silver (GYAgNPs) and gold nanoparticles (GYAuNPs) was confirmed by UV-visible spectroscopy. The average size of synthesized GYAgNPs and GYAuNPs was found to be 33 and 26 nm, respectively, by DLS particle size analyzer. TEM analysis indicated spherical shape of GYAgNPs and GYAuNPs and in EDX analysis they produced strong signal for silver and gold, respectively. Both GYAgNPs and GYAuNPs exhibited strong in vitro free radical quenching ability and their activity was comparable to that of GYLE. The cytotoxic effect of GYAgNPs and GYAuNPs in Hep2 cells was examined by MTT assay in which GYAgNPs displayed an IC50 value of 121 µg ml-1, while GYAuNPs produced up to 38 % of inhibition at the maximum concentration of 250 µg ml-1 used in this study. Distinct morphological changes were observed in Hep2 cells following treatment with GYAgNPs and GYAuNPs at 24 h, and orange-colored apoptotic bodies were located by acridine orange and ethidium bromide double-staining technique. Also, there was increase in the levels of reactive oxygen species in treated cells as indicated by 2',7'-dichlorofluorescin diacetate staining. Further, nuclear changes like chromatin condensation/fragmentation were also observed by propidium iodide and 4',6-diamidino-2-phenylindole dilactate staining methods. These findings support that the antiproliferative effects of GYAgNPs and GYAuNPs in Hep2 cells are mediated through induction of apoptosis.

  12. Effects of a simulated martian UV flux on the cyanobacterium, Chroococcidiopsis sp. 029.

    PubMed

    Cockell, Charles S; Schuerger, Andrew C; Billi, Daniela; Friedmann, E Imre; Panitz, Corinna

    2005-04-01

    Dried monolayers of Chroococcidiopsis sp. 029, a desiccation-tolerant, endolithic cyanobacterium, were exposed to a simulated martian-surface