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

  1. 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

  2. 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

  3. Competitive inhibition of Lens culinaris L. copper amine oxidase by amiloride, p-aminobenzamidine, clonidine, 4',6-diamidino-2-phenylindole and gabexate mesylate: a comparative study.

    PubMed

    Ercolini, L; Angelini, R; Federico, R; Rea, G; Venturini, G; Ascenzi, P

    1998-09-01

    The competitive inhibition of Lens culinaris L. copper amine oxidase by amiloride (Ki = 4.1 x 10(-4) M), p-aminobenzamidine (Ki = 6.0 x 10(-4) M), clonidine (Ki = 5.0 x 10(-4) M), 4',6-diamidino-2-phenylindole (DAPI; Ki = 1.9 x 10(-5) M) and gabexate mesylate (Ki = 2.5 x 10(-4) M) has been investigated, at pH 7.0 and 25 degrees C. The affinity of p-aminobenzamidine, clonidine and DAPI for plant and mammalian copper amine oxidase is closely similar. However, values of Ki for amiloride and gabexate mesylate binding to swine kidney copper amine oxidase are lower than those observed for inhibitor binding to Lens culinaris L. cooper amine oxidase. Thus, amiloride and gabexate mesylate may represent useful model compounds for the development of selective inhibitors of mammalian copper amine oxidase, which may be important in view of the potential use of plant copper amine oxidase as drugs. PMID:9825309

  4. An on-line high-performance liquid chromatography-diode-array detector-multi-stage mass spectrometry-deoxyribonucleic acid-4',6-diamidino-2-phenylindole-fluorescence detector system for screening the DNA-binding active compounds in Fufang Banbianlian Injection.

    PubMed

    Li, Sensen; Jiang, Haixiu; Lin, Zongtao; Deng, Shanshan; Guan, Yanqing; Wang, Hong; Chen, Shizhong

    2015-12-11

    Fufang Banbianlian Injection (FBI), a well-known traditional Chinese medicine formula, has been recently approved and extensively used as a newly anti-inflammatory and anti-tumor drug. This prescription comprises an equal ratio of three traditional Chinese herbs, Lobelia chinensis Lour, Scutellaria barbata D. Don and Hedyotis diffusa Willd. The relationships between its chemical compositions and activities have not been understood well yet. To investigate the ingredients and their DNA-binding activities in FBI, an on-line high-performance liquid chromatography-diode-array detector-multi-stage mass spectrometry-deoxyribonucleic acid-4',6-diamidino-2-phenylindole-fluorescence detector (HPLC-DAD-MS(n)-DNA-DAPI-FLD) system was developed using a combination of chromatographic, mass spectrometric and fluorescent detection techniques. 4',6-Diamidino-2-phenylindole (DAPI) specifically binds to three ATT base pairs on the DNA minor groove, and thus can be used as a fluorescent probe for screening active compounds that compete ATT sequences with DAPI. Using this system, 21 of 58 identified or tentatively characterized compounds in FBI showed DNA-binding activities, with most of the active compounds being flavone glycosides. In addition, the structure-activity relationships of these active compounds suggested that conjugated planar structures are favorable for DNA-binding activities, and adjacent hydroxyl groups in flavonoids can significantly improve their activities. This is, to the best of our knowledge, the first application of DAPI as a fluorescent probe for the screening of DNA-binding active compounds in complex samples. PMID:26592560

  5. 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. PMID:22987410

  6. 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

  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. PMID:27304093

  8. 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

  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. Biosynthesis of gold nanoparticles using Sargassum swartzii and its cytotoxicity effect on HeLa cells

    NASA Astrophysics Data System (ADS)

    Dhas, T. Stalin; Kumar, V. Ganesh; Karthick, V.; Govindaraju, K.; Shankara Narayana, T.

    2014-12-01

    In this investigation, biological synthesis of gold nanoparticles (AuNPs) using Sargassum swartzii and its cytotoxicity against human cervical carcinoma (HeLa) cells is reported. The biological synthesis involved the reduction of chloroauric acid led to the formation of AuNPs within 5 min at 60 °C and the formation of AuNPs was confirmed using UV-vis spectrophotometer. The AuNPs were stable; spherical in shape with well-defined dimensions, and the average size of the particle is 35 nm. A zeta potential value of -27.6 mV revealed synthesized AuNPs were highly stable. The synthesized AuNPs exhibited a dose-dependent cytotoxicity against human cervical carcinoma (HeLa) cells. Furthermore, induction of apoptosis was measured by DAPI (4‧,6-Diamidino-2-phenylindole dihydrochloride) staining.

  11. DUAL STAINING OF NATURAL BACTERIOPLANKTON WITH 4,6-DIAMINDO-2-PHENYLINDOLE AND FLUORESCENT OLIGONUCLEOTIDE PROBES TARGETING KINGDOM-LEVEL 16S RRNA SEQUENCES

    EPA Science Inventory

    A method for quantifying eubacterial cell densities in dilute communities of small bacterioplankton is presented. ells in water samples were stained with 4',6-diamidino-2-phenylindole (DAPI), transferred to gelatin-coated slides, and hybridized with rhodamine-labeled oligonucleot...

  12. CHANGES IN ACCESSIBILITY OF DNA TO VARIOUS FLUOROCHROMES DURING SPERMATOGENESIS

    EPA Science Inventory

    Accessibility of mouse testicular and vas deferens (vas) sperm cell DNA to acridine orange, propidium iodide, ellipticine, Hoechst 33342, mithramycin, chromomycin A3, 4'6-diamidino-2-phenylindole (DAPI), and 7-amino-actinomycin D (7-amino-AMD) was determined by flow cytometry. Pe...

  13. High-Level Resistance of Nosema ceranae, a Parasite of the Honeybee, to Temperature and Desiccation▿

    PubMed Central

    Fenoy, S.; Rueda, C.; Higes, M.; Martín-Hernández, R.; del Aguila, C.

    2009-01-01

    Resistance of Nosema ceranae to different exposure conditions has been evaluated by using Sytox green and DAPI (4′,6-diamidino-2-phenylindole) to test spore viability. High thermotolerance at 60 and 35°C and resistance to desiccation were observed. However, a significant decrease in viability after freezing and a rapid degeneration of spores maintained at 4°C were also detected. PMID:19734329

  14. Multiwall carbon nanotubes/polycaprolactone composites for bone tissue engineering application.

    PubMed

    Pan, Lanlan; Pei, Xibo; He, Rui; Wan, Qianbing; Wang, Jian

    2012-05-01

    In this study, the multiwall carbon nanotubes (MWNTs)/polycaprolactone composite scaffolds were fabricated by the solution evaporation technique. The morphology, phase composition and the mechanical properties of the composite scaffolds were characterized and the cellular bioactivity of the scaffolds was assessed by using rat bone-marrow-derived stroma cells (BMSCs). The attachment, proliferation and differentiation of the BMSCs on the composite scaffolds were analyzed by scanning electron microscopy (SEM), 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) nuclear staining and fluorescein diacetate (FDA) and propidium iodide (PI) live/dead staining, methylthiazol tetrazolium (MTT) assay and alkaline phosphatase (ALP) activity assay, respectively. Results showed that mechanical properties of the composite scaffolds were improved with the addition of MWNTs (0.25-2 wt%). BMSCs on the composite scaffolds differentiated down the osteogenic lineage and expressed high levels of bone marker ALP. The scaffolds with low concentration (0.5 wt%) of MWNTs can enhance the proliferation and differentiation of the BMSCs more than that with higher concentration of MWNTs. It is concluded that MWNTs/PCL composite scaffolds have the potential for bone tissue engineering and the relatively low concentration of MWNTs (0.5 wt%) is preferred. PMID:22305638

  15. Benzidine dihydrochloride: risk assessment.

    PubMed

    Littlefield, N A; Nelson, C J; Gaylor, D W

    1984-02-01

    Benzidine, recognized as a bladder carcinogen in man and as a liver carcinogen in experimental animals, is the chemical basis of as many as 200 commercial dyes. Physiological processes can metabolize these dyes to release benzidine, thereby creating a potential exposure hazard. To assess this hazard, both sexes of F1 hybrid (genetically homogeneous) and monohybrid (genetically heterogeneous) mice from a BALB/c male and C57BL/6 female cross were exposed for their respective lifespans to benzidine dihydrochloride in their drinking water at concentrations of 0, 30, 40, 60, 80, 120, and 160 ppm for males, and 0, 20, 30, 40, 60, 80, and 120 ppm for females. Animals were removed from the study when they were dead or moribund. This study was terminated after 33 months of exposure. Using the endpoint of hepatocellular adenomas and carcinomas, the Armitage Doll multistage model was used to describe the tumor rates in the experimental dose range and to obtain the upper confidence level on tumor rates. Linear interpolation was used between zero dose and the upper confidence level of the lowest experimental dosage for predicting potential low dose tumor rates. Dose-response effects on body weight, survival, and liver neoplasms were noted in both stocks. For each of the endpoints, the females were more susceptible than males and the F1 (homogeneous) stock was more susceptible than the monohybrid cross (heterogeneous). The calculated virtually "safe" dose predicted to produce less than one per million F1 female mice with a liver tumor is 0.045 ppb. One part per billion of benzidine dihydrochloride in the drinking water of these mice is estimated to produce liver tumors in less than 2.23 mice per 100,000 population. PMID:6363187

  16. Staining Fission Yeast Filamentous Actin with Fluorescent Phalloidin Conjugates.

    PubMed

    Hagan, Iain M

    2016-01-01

    The Schizosaccharomyces pombe filamentous (F)-actin cytoskeleton drives cell growth, morphogenesis, endocytosis, and cytokinesis. The protocol described here reveals the distribution of F-actin in fixed cells through the use of fluorescently conjugated phalloidin. Simultaneous staining of cell wall landmarks (with calcofluor) and chromatin (with 4',6-diamidino-2-phenylindole, or DAPI) makes this rapid staining procedure highly effective for staging cell cycle progression, monitoring morphogenetic abnormalities, and assessing the impact of environmental and genetic changes on the integrity of the F-actin cytoskeleton. PMID:27250943

  17. 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

  18. 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

  19. 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.

  20. Biogenic terbium oxide nanoparticles as the vanguard against osteosarcoma.

    PubMed

    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 0.102μg/mL) and remained non-toxic up to a concentration of 0.373μg/mL toward primary osteoblasts. Cell viability decreased in a concentration-dependent manner upon exposure to 10nm Tb2O3 NPs in the concentration range 0.023-0.373μg/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. PMID:27288964

  1. 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

  2. Analyzing Cell Death by Nuclear Staining with Hoechst 33342.

    PubMed

    Crowley, Lisa C; Marfell, Brooke J; Waterhouse, Nigel J

    2016-01-01

    The nuclei of healthy cells are generally spherical, and the DNA is evenly distributed. During apoptosis the DNA becomes condensed, but this process does not occur during necrosis. Nuclear condensation can therefore be used to distinguish apoptotic cells from healthy cells or necrotic cells. Dyes that bind to DNA, such as Hoechst 33342 or 4',6-diamidino-2-phenylindole (DAPI), can be used to observe nuclear condensation. These dyes fluoresce at 461 nm when excited by ultraviolet light and can therefore be visualized using conventional fluorescent microscopes equipped with light sources that emit light at ∼350 nm and filter sets that permit the transmission of light at ∼460 nm. This protocol describes staining and visualization of cells stained with Hoechst 33342, but it can be adapted for staining with DAPI or other dyes. PMID:27587774

  3. Effects of rutaecarpine on hydrogen peroxide-induced apoptosis in murine hepa-1c1c7 cells.

    PubMed

    Lee, Sung-Jin; Ahn, Hyunjin; Nam, Kung-Woo; Kim, Kyeong Ho; Mar, Woongchon

    2012-09-01

    The aim of this study was to investigate the inhibitory effects of rutaecarpine on DNA strand breaks and apoptosis induced by hydrogen peroxide (H2O2) in murine Hepa-1c1c7 cells. Oxidative DNA damage was estimated by nuclear condensation assessment, fluorescence-activated cell sorting analysis, and Comet assay. Rutaecarpine inhibited cell death induced by 500 μM H2O2, as assessed by 4',6-diamidino-2-phenylindole (DAPI) staining. Treatment with rutaecarpine reduced the number of DNA strand breaks induced by H2O2, as assessed by DAPI staining and Comet assay, and increased quinone reductase, phosphatidylinositol 3-kinase, and pAkt protein levels, as assessed by western blotting. PMID:24009839

  4. Effects of Rutaecarpine on Hydrogen Peroxide-Induced Apoptosis in Murine Hepa-1c1c7 Cells

    PubMed Central

    Lee, Sung-Jin; Ahn, Hyunjin; Nam, Kung-Woo; Kim, Kyeong Ho; Mar, Woongchon

    2012-01-01

    The aim of this study was to investigate the inhibitory effects of rutaecarpine on DNA strand breaks and apoptosis induced by hydrogen peroxide (H2O2) in murine Hepa-1c1c7 cells. Oxidative DNA damage was estimated by nuclear condensation assessment, fluorescence-activated cell sorting analysis, and Comet assay. Rutaecarpine inhibited cell death induced by 500 μM H2O2, as assessed by 4',6-diamidino-2-phenylindole (DAPI) staining. Treatment with rutaecarpine reduced the number of DNA strand breaks induced by H2O2, as assessed by DAPI staining and Comet assay, and increased quinone reductase, phosphatidylinositol 3-kinase, and pAkt protein levels, as assessed by western blotting. PMID:24009839

  5. Caspofungin Kills Candida albicans by Causing both Cellular Apoptosis and Necrosis

    PubMed Central

    Hao, Binghua; Cheng, Shaoji; 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

  6. 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

  7. 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. PMID:26852140

  8. 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.

  9. 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

  10. 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. PMID:27612768

  11. [Occurrence of Cryptosporidium spp. infection in antillean manatee (Trichechus manatus)].

    PubMed

    Borges, João Carlos Gomes; Alves, Leucio Câmara; Vergara-Parente, Jociery Einhardt; Faustino, Maria Aparecida da Glória; Machado, Erilane de Castro Lima

    2009-01-01

    Cryptosporidiosis is a zoonosis which can affect man and a wide range of domestic and wild animals, mainly immunodeficient individuals. The objective of this paper was reported the occurrence of a Cryptosporidium infection in Antillean manatee. After an unusual behavior of an Antillean manatee kept in captivity at the Centro Mamíferos Aquáticos, ICMBio--FMA, clinical examination and posterior fecal sampling was performed. Fecal samples were examined by the Kinyoun technique, Direct Immunofluorescence Test and also examined by 4',6'-Diamidino-2-Phenylindole (DAPI) staining. At the clinical examination, the animal showed signs of abdominal pain. The results obtained by light and fluorescence microscopy analysis showed the presence of Cryptosporidium spp. oocyst in feces of this manatee. PMID:19500463

  12. Octenidine dihydrochloride: chemical characteristics and antimicrobial properties.

    PubMed

    Assadian, Ojan

    2016-03-01

    The empiric use of antibiotics is being restricted due to the spread of antimicrobial resistance. However, topical antiseptics are less likely to induce resistance, owing to their unspecific mode of action and the high concentrations in which they can be used. One such antiseptic, octenidine dihydrochloride (OCT), can be used either prophylactically or therapeutically on the skin, mucosa and wounds. Evidence to support its use comes from in-vitro, animal and clinical studies on its safety, tolerability and efficacy. This article summarises the physical, chemical and antimicrobial properties of OCT in the context of wound care. PMID:26949863

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

    PubMed

    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 gamma max of ambroxol hydrochloride and levocetirizine dihydrochloride, respectively. Beer's law was obeyed in the concentration range 10-50 mug/ml and 8-24 mug/ml for ambroxol hydrochloride and levocetirizine dihydrochloride respectively. Results of the method were validated statistically and by recovery studies. PMID:20046721

  14. 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

  15. 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....

  16. 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. PMID:25515235

  17. 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. PMID:25300590

  18. 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

  19. Novel energy relay dyes for high efficiency dye-sensitized solar cells

    NASA Astrophysics Data System (ADS)

    Rahman, Md. Mahbubur; Ko, Min Jae; Lee, Jae-Joon

    2015-02-01

    4',6-Diamidino-2-phenylindole (DAPI) and Hoechst 33342 (H33342) were used as novel energy relay dyes (ERDs) for an efficient energy transfer to the N719 dye in I-/I3- based liquid-junction dye-sensitized solar cells (DSSCs). The introduction of the ERDs, either as an additive in the electrolyte or as a co-adsorbent, greatly enhanced the power conversion efficiencies (PCEs), mainly because of an increase in short-circuit current density (Jsc). This was attributed to the effects of non-radiative Förster-type excitation energy transfer as well as the radiative (emission)-type fluorescent energy transfer to the sensitizers. The net PCEs for the N719-sensitized DSSCs with DAPI and H33342 were 10.65% and 10.57%, and showed an improvement of 12.2% and 11.4% over control devices, respectively.4',6-Diamidino-2-phenylindole (DAPI) and Hoechst 33342 (H33342) were used as novel energy relay dyes (ERDs) for an efficient energy transfer to the N719 dye in I-/I3- based liquid-junction dye-sensitized solar cells (DSSCs). The introduction of the ERDs, either as an additive in the electrolyte or as a co-adsorbent, greatly enhanced the power conversion efficiencies (PCEs), mainly because of an increase in short-circuit current density (Jsc). This was attributed to the effects of non-radiative Förster-type excitation energy transfer as well as the radiative (emission)-type fluorescent energy transfer to the sensitizers. The net PCEs for the N719-sensitized DSSCs with DAPI and H33342 were 10.65% and 10.57%, and showed an improvement of 12.2% and 11.4% over control devices, respectively. Electronic supplementary information (ESI) available: Details of the materials and instrumentation, device fabrication, measurement and calculations of the quantum yield (Qd), calculations of the Förster radius (R0), optimization of the ERDs mixed with electrolyte according to Type-A strategy; normalized absorption profiles of the N3, Ru505, and Z907 dyes and the emission profiles of DAPI and H33342

  20. 21 CFR 522.1362 - Melarsomine dihydrochloride for injection.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Melarsomine dihydrochloride for injection. 522.1362 Section 522.1362 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... kilograms (22 pounds). Use alternate sides with each administration. The drug is contraindicated in...

  1. 21 CFR 522.2120 - Spectinomycin dihydrochloride injection.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... dihydrochloride pentahydrate as follows: (1) Subcutaneously in the treatment of 1-to-3-day-old turkey poults at... Arizona group infection. (2) Subcutaneously in the treatment of 1-to-3-day old: (i) Turkey poults at the... 0.1 milliliter (10 milligrams) subcutaneously in nape of neck of 1- to 3-day-old turkey poults as...

  2. 21 CFR 522.2120 - Spectinomycin dihydrochloride injection.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... dihydrochloride pentahydrate as follows: (1) Subcutaneously in the treatment of 1-to-3-day-old turkey poults at... Arizona group infection. (2) Subcutaneously in the treatment of 1-to-3-day old: (i) Turkey poults at the... 0.1 milliliter (10 milligrams) subcutaneously in nape of neck of 1- to 3-day-old turkey poults as...

  3. 21 CFR 522.2120 - Spectinomycin dihydrochloride injection.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... dihydrochloride pentahydrate as follows: (1) Subcutaneously in the treatment of 1-to-3-day-old turkey poults at... Arizona group infection. (2) Subcutaneously in the treatment of 1-to-3-day old: (i) Turkey poults at the... 0.1 milliliter (10 milligrams) subcutaneously in nape of neck of 1- to 3-day-old turkey poults as...

  4. 21 CFR 522.2120 - Spectinomycin dihydrochloride injection.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... dihydrochloride pentahydrate as follows: (1) Subcutaneously in the treatment of 1-to-3-day-old turkey poults at... Arizona group infection. (2) Subcutaneously in the treatment of 1-to-3-day old: (i) Turkey poults at the... 0.1 milliliter (10 milligrams) subcutaneously in nape of neck of 1- to 3-day-old turkey poults as...

  5. 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. PMID:24901627

  6. 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

  7. 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

    2016-01-01

    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

  8. Bioavailability of sulphate and dihydrochloride salts of quinine.

    PubMed

    Sowunmi, A; Salako, L A; Ogunbona, F A

    1994-09-01

    A comparative bioavailability of three formulations of quinine was performed in 6 healthy male adult Africans after intravenous infusion of 600mg quinine hydrochloride in 0.9% saline over 4 hours and after single oral doses of 600mg quinine sulphate capsule, 600mg quinine dihydrochloride plain tablet and 600mg quinine sulphate sugar coated tablet. The drugs were given according to a randomised cross-over design. The quinine sulphate coated tablet was found to contain no quinine. There was no statistical significant difference (P > 0.05) in the plasma Cmax, tmax, AUC and Ka values between the quinine sulphate capsule and quinine dihydrochloride plain tablet, although a considerable degree of inter- and intra-individual variability in the pharmacokinetic parameters was observed. The absolute bioavailability was 64.5 and 64.3% for the quinine sulphate capsule and the quinine dihydrochloride plain tablet respectively. The non-detection of quinine in the sugar coated tablet (obtained from Nigeria) confirms the presence of fake circulating antimalarial drugs in the country. PMID:7604754

  9. Chromatin and Cell Wall Staining of Schizosaccharomyces pombe.

    PubMed

    Hagan, Iain M

    2016-01-01

    Fission yeasts grow by tip extension, maintaining a constant width until they reach a critical size threshold and divide. Division by medial fission-which gives these yeast their name-generates a new end that arises from the site of cytokinesis. The old end, which was produced during the previous cell cycle, initiates progression of the new cell cycle, and in G2, the new end is activated in a process termed new-end takeoff (NETO). In this protocol, the fluorescent stains calcofluor and 4',6-diamidino-2-phenylindole (DAPI) are used to give a rapid and informative assessment of morphogenesis and cell-cycle progression in the fission yeast Schizosaccharomyces pombe Calcofluor reveals the timing of NETO because it stains the birth scars that are generated at new ends by cytokinesis less efficiently than the rest of the cell wall. Intense calcofluor staining of the septum and measurement of cell length are also widely used to identify dividing cells and to gauge the timing of mitotic commitment. Staining nuclei with DAPI identifies mono- and binucleated cells and complements the calcofluor staining procedure to evaluate the stages of the cell cycle and identify mitotic errors. Equally simple DAPI staining procedures reveal chromatin structure in higher resolution, facilitating more accurate staging of mitotic progression and characterization of mitotic errors. PMID:27250942

  10. Effect of fluorochromes on bacterial surface properties and interaction with granular media.

    PubMed Central

    Chen, J; Koopman, B

    1997-01-01

    Simple, efficient, and safe tagging methods are desired in short-term microbial transport studies such as in the study of filtration systems for water and wastewater treatment. Suitability of selected fluorochromes as bacterial tagging agents in transport studies was evaluated on the basis of stability of stained cells and the effect of staining on bacterial surface characteristics and interaction with granular media. Surface properties were characterized by zeta potential and microbial adhesion to hydrocarbons. The effect of staining on interactions between bacteria and porous media was evaluated in terms of removal of bacteria in batch adsorption tests using sand coated with aluminum hydroxide to enhance adsorption. The DNA-specific fluorochrome 4',6-diamidino-2-phenylindole (DAPI) had generally negligible effects on bacterial surface properties and interaction with sand, as indicated in batch adsorption tests using pure cultures (Escherichia coli or Acinetobacter sp.) and wastewater bacteria. Cells stained with DAPI were stable for 48 h at 4 or 20 degrees C. Other nucleic acid fluorochromes tested had different but significant effects on bacterial cells and produced less stable fluorescence. Since transport through porous media is modulated by surface properties, it may be concluded based on these results that the choice of fluorochromes is critical in microbial transport studies. DAPI appeared to be a promising tagging agent. Time dependence of fluorescence of stained cells may limit the use of fluorochrome-tagged cells in long-term transport studies. PMID:9327559

  11. Identification of the High-affinity Substrate-binding Site of the Multidrug and Toxic Compound Extrusion (MATE) Family Transporter from Pseudomonas stutzeri.

    PubMed

    Nie, Laiyin; Grell, Ernst; Malviya, Viveka Nand; Xie, Hao; Wang, Jingkang; Michel, Hartmut

    2016-07-22

    Multidrug and toxic compound extrusion (MATE) transporters exist in all three domains of life. They confer multidrug resistance by utilizing H(+) or Na(+) electrochemical gradients to extrude various drugs across the cell membranes. The substrate binding and the transport mechanism of MATE transporters is a fundamental process but so far not fully understood. Here we report a detailed substrate binding study of NorM_PS, a representative MATE transporter from Pseudomonas stutzeri Our results indicate that NorM_PS is a proton-dependent multidrug efflux transporter. Detailed binding studies between NorM_PS and 4',6-diamidino-2-phenylindole (DAPI) were performed by isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and spectrofluorometry. Two exothermic binding events were observed from ITC data, and the high-affinity event was directly correlated with the extrusion of DAPI. The affinities are about 1 μm and 0.1 mm for the high and low affinity binding, respectively. Based on our homology model of NorM_PS, variants with mutations of amino acids that are potentially involved in substrate binding, were constructed. By carrying out the functional characterization of these variants, the critical amino acid residues (Glu-257 and Asp-373) for high-affinity DAPI binding were determined. Taken together, our results suggest a new substrate-binding site for MATE transporters. PMID:27235402

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

    PubMed Central

    Barhoumi, Aoune; Halas, Naomi J.

    2013-01-01

    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. PMID:20857946

  13. 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

  14. 21 CFR 520.2520g - Trichlorfon, phenothiazine, and piperazine dihydrochloride powder.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... dihydrochloride powder. 520.2520g Section 520.2520g Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... ANIMAL DRUGS § 520.2520g Trichlorfon, phenothiazine, and piperazine dihydrochloride powder. (a) Specifications. Each 54.10 grams (1.91 ounces) of water dispersible powder contains 9.10 grams of trichlorfon,...

  15. 21 CFR 520.2520g - Trichlorfon, phenothiazine, and piperazine dihydrochloride powder.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... dihydrochloride powder. 520.2520g Section 520.2520g Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... ANIMAL DRUGS § 520.2520g Trichlorfon, phenothiazine, and piperazine dihydrochloride powder. (a) Specifications. Each 54.10 grams (1.91 ounces) of water dispersible powder contains 9.10 grams of trichlorfon,...

  16. Seasonal dominance of CL500-11 bacterioplankton (phylum Chloroflexi) in the oxygenated hypolimnion of Lake Biwa, Japan.

    PubMed

    Okazaki, Yusuke; Hodoki, Yoshikuni; Nakano, Shin-ichi

    2013-01-01

    Uncultured bacteria affiliated with the CL500-11 cluster (phylum Chloroflexi) were first reported from the oxygenated hypolimnion of Crater Lake (USA) as a predominant bacterioplankton, although this dominance has not been reported in other environments. In this study, we showed that CL500-11 is also dominant in the oxygenated hypolimnion of Lake Biwa (Japan) and followed its spatiotemporal succession using fluorescent in situ hybridization. CL500-11 cells were almost absent [< 1% of 4',6-diamidino-2-phenylindole (DAPI)-stained cells] at the beginning of the stratification period, dominated (> 10% of DAPI-stained cells; maximum = 16.5%) in the hypolimnion during the stratification period, and decreased to below the detection limit with the collapse of the thermocline. This pattern was observed over two annual cycles. A longitudinal assessment also showed that CL500-11 was the dominant bacterium in the hypolimnion over the whole lake, but was generally undetectable in the stratified epilimnion. These data suggest that CL500-11 is acclimated to the oxygenated hypolimnion and is a potentially important component of the pelagic biogeochemical cycling of the lake. A comparative analysis of 16S rRNA gene sequences revealed that almost all CL500-11 sequences previously deposited in the database were detected from hypolimnion or holomictic water in deep oxic freshwater lakes, suggesting that the bacteria may form one of the common lineages residing in an aerobic hypolimnetic niche. PMID:22809435

  17. Quantification of total bacteria, enterobacteria and lactobacilli populations in pig digesta by real-time PCR.

    PubMed

    Castillo, Marisol; Martín-Orúe, Susana M; Manzanilla, Edgar G; Badiola, Ignacio; Martín, Marga; Gasa, Josep

    2006-04-16

    Jejunum digesta samples were taken from weaning pigs in order to evaluate real-time PCR (qPCR) as a method for quantifying pig gut bacteria. Total bacteria, lactobacilli and enterobacteria were quantified by qPCR and the results were compared with those obtained with traditional methods: 4',6-diamidino-2-phenylindole (DAPI staining) for total bacteria, selective culture for lactobacilli and enterobacteria. Real-time PCR showed higher values in terms of 16S rRNA gene copies than DAPI counts or CFU. Despite the differences, the lactobacilli:enterobacteria ratio was similar between methods (2.5 +/- 0.58 for qPCR and 3.1 +/- 0.71 for selective culture, P = 0.39). Possible reasons for the higher PCR counts are discussed considering both an overestimation with PCR by quantification of dead bacteria or free DNA and also an underestimation with conventional methods. Inherent differences in the pre-treatment of the samples could partially explain the discrepancies observed. Regardless of the numerical differences between methods, values obtained by qPCR and traditional methods showed a significant correlation for lactobacilli and total bacteria. In the light of these results, real-time PCR seems a valid method to quantify microbial shifts in the gastrointestinal tract. PMID:16384658

  18. 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. PMID:23762159

  19. 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

    2016-07-01

    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. PMID:27265326

  20. Fluorometric quantification of polyphosphate in environmental plankton samples: extraction protocols, matrix effects, and nucleic acid interference.

    PubMed

    Martin, Patrick; Van Mooy, Benjamin A S

    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

  1. Bioactive actions of pomegranate fruit extracts on leukemia cell lines in vitro hold promise for new therapeutic agents for leukemia.

    PubMed

    Dahlawi, Haytham; Jordan-Mahy, Nicola; Clench, Malcolm R; Le Maitre, Christine L

    2012-01-01

    Studies suggest that pomegranates contain bioactive chemicals with potential for treatment and prevention of cancer. Pomegranate juice extracts (PJE) have been shown to inhibit cellular proliferation and tumor growth and induce cell death via apoptosis in a number of cancer cell lines. However, to date, few studies have investigated the potential of PJE in the treatment of leukemia. We investigated the potential effect of PJE on induction of apoptosis and inhibition of cellular proliferation in 8 leukemia cell lines (4 lymphoid and 4 myeloid) and nontumor hematopoietic stem cells (control cells). Apoptosis was assessed by 2 methods: Annexin V-FITC/propidium iodide staining with flow cytometric analysis and 4'-6-diamidino-2-phenylindole (DAPI) morphological assessment. Cell cycle stage was investigated using propidum iodide staining of DNA content and flow cytometric analysis. Live cell counts were also performed using a trypan exclusion assay. PJE significantly induced apoptosis in all cell lines, including nontumor control cells, although lymphoid cells and 2 of the myeloid cell lines were more sensitive. Furthermore, PJE induced cell cycle arrest. These results were confirmed by DAPI analysis and viable cell counts using trypan blue exclusion assay. Our results provide evidence that PJE contain bioactive compounds that could be used in the treatment of leukemia. PMID:22098126

  2. Co-release of acetylcholine and gamma-aminobutyric acid by a retinal neuron

    SciTech Connect

    O'Malley, D.M.; Masland, R.H.

    1989-05-01

    Rabbit retinas were vitally stained with 4',6-diamidino-2-phenylindole (DAPI), a fluorescent compound that selectively accumulates within the cholinergic amacrine cells. The retinas were then incubated in vitro in the presence of radioactive gamma-aminobutyric acid (GABA) and autoradiographed. The cells that accumulated DAPI were found to accumulate GABA, confirming immunohistochemical evidence that the cholinergic amacrine cells contain GABA. Incubation of retinas in the presence of elevated concentrations of K+ caused them to release acetylcholine and GABA, and autoradiography showed depletion of radioactive GABA from the cholinergic amacrine cells. This indicates that the cholinergic amacrine cells can secrete acetylcholine and GABA. Retinas were double-labeled with (14C)GABA and (3H)acetylcholine, allowing simultaneous measurement of their release. The release of (14C)GABA was found to be independent of extracellular Ca2+. Radioactive GABA synthesized endogenously from (14C)glutamate behaved the same way as radioactive GABA accumulated from the medium. In the same experiments the simultaneously measured release of (3H)acetylcholine was strongly Ca2+-dependent, indicating that the releases of acetylcholine and GABA are controlled by different mechanisms. Synaptic vesicles immunologically isolated from double-labeled retinas contained much (3H)acetylcholine and little or no (14C)GABA. These results suggest that the cholinergic amacrine cells release acetylcholine primarily by vesicle exocytosis and release GABA primarily by means of a carrier.

  3. Nonperturbative Imaging of Nucleoid Morphology in Live Bacterial Cells during an Antimicrobial Peptide Attack

    PubMed Central

    Bakshi, Somenath; Choi, Heejun; Rangarajan, Nambirajan; Barns, Kenneth J.; Bratton, Benjamin P.

    2014-01-01

    Studies of time-dependent drug and environmental effects on single, live bacterial cells would benefit significantly from a permeable, nonperturbative, long-lived fluorescent stain specific to the nucleoids (chromosomal DNA). The ideal stain would not affect cell growth rate or nucleoid morphology and dynamics, even during laser illumination for hundreds of camera frames. In this study, time-dependent, single-cell fluorescence imaging with laser excitation and a sensitive electron-multiplying charge-coupled-device (EMCCD) camera critically tested the utility of “dead-cell stains” (SYTOX orange and SYTOX green) and “live-cell stains” (DRAQ5 and SYTO 61) and also 4′,6-diamidino-2-phenylindole (DAPI). Surprisingly, the dead-cell stains were nearly ideal for imaging live Escherichia coli, while the live-cell stains and DAPI caused nucleoid expansion and, in some cases, cell permeabilization and the halting of growth. SYTOX orange performed well for both the Gram-negative E. coli and the Gram-positive Bacillus subtilis. In an initial application, we used two-color fluorescence imaging to show that the antimicrobial peptide cecropin A destroyed nucleoid-ribosome segregation over 20 min after permeabilization of the E. coli cytoplasmic membrane, reminiscent of the long-term effects of the drug rifampin. In contrast, the human cathelicidin LL-37, while similar to cecropin A in structure, length, charge, and the ability to permeabilize bacterial membranes, had no observable effect on nucleoid-ribosome segregation. Possible underlying causes are suggested. PMID:24907320

  4. A high-throughput method for detection of DNA in chloroplasts using flow cytometry

    PubMed Central

    Rowan, Beth A; Oldenburg, Delene J; Bendich, Arnold J

    2007-01-01

    Background The amount of DNA in the chloroplasts of some plant species has been shown recently to decline dramatically during leaf development. A high-throughput method of DNA detection in chloroplasts is now needed in order to facilitate the further investigation of this process using large numbers of tissue samples. Results The DNA-binding fluorophores 4',6-diamidino-2-phenylindole (DAPI), SYBR Green I (SG), SYTO 42, and SYTO 45 were assessed for their utility in flow cytometric analysis of DNA in Arabidopsis chloroplasts. Fluorescence microscopy and real-time quantitative PCR (qPCR) were used to validate flow cytometry data. We found neither DAPI nor SYTO 45 suitable for flow cytometric analysis of chloroplast DNA (cpDNA) content, but did find changes in cpDNA content during development by flow cytometry using SG and SYTO 42. The latter dye provided more sensitive detection, and the results were similar to those from the fluorescence microscopic analysis. Differences in SYTO 42 fluorescence were found to correlate with differences in cpDNA content as determined by qPCR using three primer sets widely spaced across the chloroplast genome, suggesting that the whole genome undergoes copy number reduction during development, rather than selective reduction/degradation of subgenomic regions. Conclusion Flow cytometric analysis of chloroplasts stained with SYTO 42 is a high-throughput method suitable for determining changes in cpDNA content during development and for sorting chloroplasts on the basis of DNA content. PMID:17381841

  5. Application of flow cytometry and fluorescent in situ hybridization for assessment of exposures to airborne bacteria.

    PubMed Central

    Lange, J L; Thorne, P S; Lynch, N

    1997-01-01

    Current limitations in the methodology for enumeration and identification of airborne bacteria compromise the precision and accuracy of bioaerosol exposure assessment. In this study, flow cytometry and fluorescent in situ hybridization (FISH) were evaluated for the assessment of exposures to airborne bacteria. Laboratory-generated two-component bioaerosols in exposures chambers and complex native bioaerosols in swine barns were sampled with two types of liquid impingers (all-glass impinger-30 and May 3-stage impinger). Aliquots of collection media were processed and enumerated by a standard culture technique, microscopy, or flow cytometry after nucleic acid staining with 4',6-diamidino-2-phenylindole (DAPI) and identified taxonomically by FISH. DAPI-labeled impinger samples yielded comparable estimates of bioaerosol concentrations when enumerated by microscopy or flow cytometry. The standard culture method underestimated bioaerosol concentrations by 2 orders of magnitude when compared to microscopy or flow cytometry. In the FISH method, aliquots of collection media were incubated with a probe universally complementary to eubacteria, a probe specific for several Pseudomonas species, and a probe complementary to eubacteria for detection of nonspecific binding. With these probes, FISH allowed quantitative identification of Pseudomonas aeruginosa and Escherichia coli bioaerosols in the exposure chamber without measurable nonspecific binding. Impinger samples from the swine barn demonstrated the efficacy of the FISH method for the identification of eubacteria in a complex organic dust. This work demonstrates the potential of emerging molecular techniques to complement traditional methods of bioaerosol exposure assessment. PMID:9097451

  6. Bioimpedance modeling to monitor astrocytic response to chronically implanted electrodes.

    PubMed

    McConnell, G C; Butera, R J; Bellamkonda, R V

    2009-10-01

    The widespread adoption of neural prosthetic devices is currently hindered by our inability to reliably record neural signals from chronically implanted electrodes. The extent to which the local tissue response to implanted electrodes influences recording failure is not well understood. To investigate this phenomenon, impedance spectroscopy has shown promise for use as a non-invasive tool to estimate the local tissue response to microelectrodes. Here, we model impedance spectra from chronically implanted rats using the well-established Cole model, and perform a correlation analysis of modeled parameters with histological markers of astroglial scar, including glial fibrillary acid protein (GFAP) and 4',6-diamidino-2- phenylindole (DAPI). Correlations between modeled parameters and GFAP were significant for three parameters studied: Py value, R(o) and |Z|(1 kHz), and in all cases were confined to the first 100 microm from the interface. Py value was the only parameter also correlated with DAPI in the first 100 microm. Our experimental results, along with computer simulations, suggest that astrocytes are a predominant cellular player affecting electrical impedance spectra. The results also suggest that the largest contribution from reactive astrocytes on impedance spectra occurs in the first 100 microm from the interface, where electrodes are most likely to record electrical signals. These results form the basis for future approaches where impedance spectroscopy can be used to evaluate neural implants, evaluate strategies to minimize scar and potentially develop closed-loop prosthetic devices. PMID:19721187

  7. In situ detection of bacteria in continuous-flow cultures of seawater sediment suspensions with fluorescently labelled rRNA-directed oligonucleotide probes.

    PubMed

    Bruns, A; Berthe-Corti, L

    1998-10-01

    rRNA-targeted and fluorescently labelled oligonucleotide probes were used to study the composition of natural bacterial populations in continuous-flow cultures of seawater sediment suspensions. The cultures were run as enrichment cultures with increasing dilution rates, and hexadecane as the sole carbon source. Total cell numbers were analysed by counting DAPI (4',6-diamidino-2-phenylindole)-stained cells. To differentiate the population composition, oligonucleotide probes for eubacteria, for Cytophaga/Flavobacteria, and for four subclasses of the Proteobacteria (alpha, beta, gamma and delta) were used. About 40-80% of the DAPI-stained cells could be detected with the EUB338 probe. Moreover, it was possible to detect a shift in the composition of the natural bacterial population with increasing dilution rate of the continuous culture, from large amounts of Cytophaga/Flavobacteria to large numbers of members of the gamma-Proteobacteria. The cell recovery rate for bacteria labelled with specific oligonucleotide probes was analysed with defined cell numbers of Rhodospirillum rubrum, Comamonas testosteroni and Desulfovibrio vulgaris subsp. vulgaris introduced into the seawater sediment suspension, and was determined to be 13.9-33.5%. The standard deviation determined for this method applied to sediment suspensions was +/- 8.3%. The results suggest that the application of the in situ hybridization technique allows a good insight into the structure of populations growing in sediment suspensions. PMID:9802019

  8. Bioimpedance modeling to monitor astrocytic response to chronically implanted electrodes

    NASA Astrophysics Data System (ADS)

    McConnell, G. C.; Butera, R. J.; Bellamkonda, R. V.

    2009-10-01

    The widespread adoption of neural prosthetic devices is currently hindered by our inability to reliably record neural signals from chronically implanted electrodes. The extent to which the local tissue response to implanted electrodes influences recording failure is not well understood. To investigate this phenomenon, impedance spectroscopy has shown promise for use as a non-invasive tool to estimate the local tissue response to microelectrodes. Here, we model impedance spectra from chronically implanted rats using the well-established Cole model, and perform a correlation analysis of modeled parameters with histological markers of astroglial scar, including glial fibrillary acid protein (GFAP) and 4',6-diamidino-2- phenylindole (DAPI). Correlations between modeled parameters and GFAP were significant for three parameters studied: Py value, Ro and |Z|1 kHz, and in all cases were confined to the first 100 µm from the interface. Py value was the only parameter also correlated with DAPI in the first 100 µm. Our experimental results, along with computer simulations, suggest that astrocytes are a predominant cellular player affecting electrical impedance spectra. The results also suggest that the largest contribution from reactive astrocytes on impedance spectra occurs in the first 100 µm from the interface, where electrodes are most likely to record electrical signals. These results form the basis for future approaches where impedance spectroscopy can be used to evaluate neural implants, evaluate strategies to minimize scar and potentially develop closed-loop prosthetic devices.

  9. 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. PMID:27150102

  10. Flow Cytometric Analysis of Marine Bacteria with Hoechst 33342 †

    PubMed Central

    Monger, Bruce C.; Landry, Michael R.

    1993-01-01

    We investigated the accuracy and precision of flow cytometric (FCM) estimates of bacterial abundances using 4′, 6-diamidino-2-phenylindole (DAPI) and Hoechst 33342 (HO342, a bisbenzamide derivative) on paraformaldehyde-fixed seawater samples collected from two stations near Oahu, Hawaii. The accuracy of FCM estimates was assessed against direct counts by using epifluorescence microscopy. DAPI and HO342 differ in two aspects of their chemistry that make HO342 better suited for staining marine heterotrophic bacteria for FCM analysis. These differences are most important in studies of open-ocean ecosystems that require dual-beam FCM analysis to clearly separate heterotrophic bacterial populations from populations of photosynthetic Prochlorococcus spp. Bacterial populations were easier to distinguish from background fluorescence when stained with HO342 than when stained with DAPI, because HO342 has a higher relative fluorescence quantum yield. A substantially higher coefficient of variation of blue fluorescence, which was probably due to fluorescent complexes formed by DAPI with double-stranded RNA, was observed for DAPI-stained populations. FCM estimates averaged 2.0 and 12% higher than corresponding epifluorescence microscopy direct counts for HO342 and DAPI-stained samples, respectively. A paired-sample t test between FCM estimates and direct counts found no significant difference for HO342-stained samples but a significant difference for DAPI-stained samples. Coefficients of variation of replicate FCM abundance estimates ranged from 0.63 to 2.9% (average, 1.5%) for natural bacterial concentrations of 6 × 105 to 15 × 105 cells ml-1. PMID:16348898

  11. 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.

  12. 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

  13. 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. PMID:24861204

  14. Study on performance of magnetic fluorescent nanoparticles as gene carrier and location in pig kidney cells

    NASA Astrophysics Data System (ADS)

    Wang, Yan; Cui, Haixin; Sun, Changjiao; Du, Wei; Cui, Jinhui; Zhao, Xiang

    2013-03-01

    We evaluated the performance of green fluorescent magnetic Fe3O4 nanoparticles (NPs) as gene carrier and location in pig kidney cells. When the mass ratio of NPs to green fluorescent protein plasmid DNA reached 1:16 or above, DNA molecules can be combined completely with NPs, which indicates that the NPs have good ability to bind negative DNA. Atomic force microscopy (AFM) experiments were carried out to investigate the binding mechanism between NPs and DNA. AFM images show that individual DNA strands come off of larger pieces of netlike agglomerations and several spherical nanoparticles are attached to each individual DNA strand and interact with each other. The pig kidney cells were labelled with membrane-specific red fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate and nucleus-specific blue fluorescent dye 4',6-diamidino-2-phenylindole dihydrochloride. We found that green fluorescent nanoparticles can past the cell membrane and spread throughout the interior of the cell. The NPs seem to locate more frequently in the cytoplasm than in the nucleus.

  15. Flow-cytometry techniques in radiation biology

    SciTech Connect

    McCarthy, K.F.; Hale, M.L.

    1988-01-01

    Considerable evidence exists that all blood cells are derived from HSC. These cells are of interest to radiobiologists because they are highly sensitive to low doses of ionizing radiation. Hematopoietic stem cells (HSC) are present in the marrow at a concentration of approximately 2-3 HSC per 1000 nucleated marrow cells. In the past, only clonogenic assays requiring 8-13 days and ten irradiated recipient rodents were available for assaying HSC. Because of the importance of HSC in the post-irradiation syndrome, the authors developed a new rapid method based on flow cytometry not only to assay but also to purify and characterize HSC. This new method makes extensive use of non-clonal antibodies conjugated to fluorescent phycobiliproteins through the sulfhydryls of the hinge region of the IgG molecule. An optical bench arrangement with a dye laser and an argon laser was used for dual excitation of the phycobiliprotein-monoclonal antibody conjugates and various cellular and DNA probes. Using 4', 6-diamidino 2-phenylindole dihydrochloride (DAP) exclusion to identify viable cells, it was possible to follow regeneration of post-irradiated rat marrow HSC.

  16. Automated image analysis and in situ hybridization as tools to study bacterial populations in food resources, gut and cast of Lumbricus terrestris L.

    PubMed

    Schönholzer, Frank; Hahn, Dittmar; Zarda, Boris; Zeyer, Josef

    2002-01-01

    An image analysis procedure was developed for bacterial cells after staining with the DNA-intercalating dye 4'-6-diamidino-2-phenylindole (DAPI), and after in situ hybridization with Cy3-labeled, rRNA-targeted oligonucleotide probes. DAPI- and Cy3-images were captured separately from the same scenery with a cooled digital video camera with three CCD chips for the basic colors red (R), green (G) and blue (B). Using the appropriate filter sets, images of DAPI-stained cells were captured with the red channel shut down, while Cy3-stained cells were captured with the green and blue channels shut down. DAPI images and Cy3 images were subsequently merged to produce virtual color (RGB)-images. Processing of all color channels allowed to specifically enumerate DAPI-stained and hybridized bacteria, to measure their cell sizes, to subsequently calculate their biovolumes and to estimate their biomass. Using this procedure, significant differences were detected in bacterial populations in food resources, digestive tract and cast of the earthworm L. terrestris L. In leaves, bacteria were on average ten times more abundant and two times larger than in soil. In the digestive tract of L. terrestris, however, numbers and volumes of bacteria were comparable to those in soil indicating the disruption of cells originating from leaves before arriving in the foregut. Passage through the digestive tract of L. terrestris significantly reduced bacterial populations belonging to the alpha-, beta- and gamma-subdivisions of Proteobacteria. While these populations did not recover during incubation of cast, populations of the delta-subdivision of Proteobacteria and the Cytophaga-Flavobacterium cluster of the CFB phylum increased in cast. These results suggest a large impact of passage through the digestive tract of L. terrestris on bacterial community structure and demonstrate the usefulness of our image analysis procedure for the determination of cell sizes and biovolumes and thus biomass of

  17. 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. PMID:22706058

  18. 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. PMID:25209888

  19. 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. PMID:22941421

  20. 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

  1. Modulation of Cyclins, p53 and Mitogen-Activated Protein Kinases Signaling in Breast Cancer Cell Lines by 4-(3,4,5-Trimethoxyphenoxy)benzoic Acid

    PubMed Central

    Lee, Kuan-Han; Ho, Wen-Yueh; Wu, Shu-Jing; Omar, Hany A.; Huang, Po-Jui; Wang, Clay C. C.; Hung, Jui-Hsiang

    2014-01-01

    Despite the advances in cancer therapy and early detection, breast cancer remains a leading cause of cancer-related deaths among females worldwide. The aim of the current study was to investigate the antitumor activity of a novel compound, 4-(3,4,5-trimethoxyphenoxy)benzoic acid (TMPBA) and its mechanism of action, in breast cancer. Results indicated the relatively high sensitivity of human breast cancer cell-7 and MDA-468 cells towards TMPBA with IC50 values of 5.9 and 7.9 μM, respectively compared to hepatocarcinoma cell line Huh-7, hepatocarcinoma cell line HepG2, and cervical cancer cell line Hela cells. Mechanistically, TMPBA induced apoptotic cell death in MCF-7 cells as indicated by 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining, cell cycle analysis and the activation of caspase-3. Western blot analysis revealed the ability of TMPBA to target pathways mediated by mitogen-activated protein (MAP) kinases, 5′ adenosine monophosphate-activated protein kinase (AMPK), and p53, of which the concerted action underlined its antitumor efficacy. In addition, TMPBA induced alteration of cyclin proteins’ expression and consequently modulated the cell cycle. Taken together, the current study underscores evidence that TMPBA induces apoptosis in breast cancer cells via the modulation of cyclins and p53 expression as well as the modulation of AMPK and mitogen-activated protein kinases (MAPK) signaling. These findings support TMPBA’s clinical promise as a potential candidate for breast cancer therapy. PMID:24406729

  2. Anacardic acid induces mitochondrial-mediated apoptosis in the A549 human lung adenocarcinoma cells.

    PubMed

    Seong, Yeong-Ae; Shin, Pyung-Gyun; Kim, Gun-Do

    2013-03-01

    Anacardic acid (AA) is a constituent of the cashew nut shell and is known as an inhibitor of nuclear factor-κB (NF-κB). We investigated the cytotoxicity of AA on cancer cells and more experiments to reveal the cell death mechanism focused on A549 lung adenocarcinoma cells for our interest in lung cancer. To examine the molecular mechanism of cell death in AA treated A549 cells, we performed experiments such as transmission electron microscopy (TEM), western blot analysis, fluorescence-activated cell sorting (FACS), genomic DNA extraction and staining with 4',6-diamidino-2-phenylindole (DAPI). For the first time we revealed that AA induces caspase-independent apoptosis with no inhibition of cytotoxicity by pan-caspase inhibitor, Z-VAD-fmk, in A549 cells. Our results showed the possibility of mitochondrial-mediated apoptosis through the activation of apoptosis-inducing factor (AIF) and an intrinsic pathway executioner such as cytochrome c. This study will be helpful in revealing the cell death mechanisms and in developing potential drugs for lung cancer using AA. PMID:23314312

  3. Endosymbiotic Bacteria in the Parasitic Ciliate Ichthyophthirius multifiliis▿

    PubMed Central

    Sun, H. Y.; Noe, J.; Barber, J.; Coyne, R. S.; Cassidy-Hanley, D.; Clark, T. G.; Findly, R. C.; Dickerson, H. W.

    2009-01-01

    Endosymbiotic bacteria were identified in the parasitic ciliate Ichthyophthirius multifiliis, a common pathogen of freshwater fish. PCR amplification of DNA prepared from two isolates of I. multifiliis, using primers that bind conserved sequences in bacterial 16S rRNA genes, generated an ∼1,460-bp DNA product, which was cloned and sequenced. Sequence analysis demonstrated that 16S rRNA gene sequences from three classes of bacteria were present in the PCR product. These included Alphaproteobacteria (Rickettsiales), Sphingobacteria, and Flavobacterium columnare. DAPI (4′,6-diamidino-2-phenylindole) staining showed endosymbionts dispersed throughout the cytoplasm of trophonts and, in most, but not all theronts. Endosymbionts were observed by transmission electron microscopy in the cytoplasm, surrounded by a prominent, electron-translucent halo characteristic of Rickettsia. Fluorescence in situ hybridization demonstrated that bacteria from the Rickettsiales and Sphingobacteriales classes are endosymbionts of I. multifiliis, found in the cytoplasm, but not in the macronucleus or micronucleus. In contrast, F. columnare was not detected by fluorescence in situ hybridization. It likely adheres to I. multifiliis through association with cilia. The role that endosymbiotic bacteria play in the life history of I. multifiliis is not known. PMID:19820157

  4. Morphological structure of propagules and electrophoretic karyotype analysis of false smut Villosiclava virens in rice.

    PubMed

    Fu, Rongtao; Ding, Lei; Zhu, Jun; Li, Ping; Zheng, Ai-Ping

    2012-04-01

    The target pathogen Villosiclava virens (teleomorph: claviceps oryzae-sativae) was isolated from the infected rice, where it caused false smut. In our study, the forming processes of the chlamydospores, chlamydospore balls, conidiospores, and secondary conidiospores during the asexual reproduction were observed more precisely and in greater detail than previous descriptions. The microstructure of the infected rice kernel showed that the outer dense chlamydospores piled around the false smut balls grown on XBZ medium; moreover the sclerotia consisting of dense mycelium were found. The different morphology was observed across the different growing conditions. In addition, we observed the nuclear numbers of both the conidiospores and hyphae using 4',6-diamidino-2-phenylindole (DAPI) staining. Because the fungus has small chromosomes and the numbers were not previously known, we analyzed the electrophoretic karyotype using a pulsed field gel electrophoresis (PFGE) technique. The results showed that V. virens has at least 10 chromosomes ranging in size from 0.6 kb to 6 Mb. The V. virens genome size is estimated to be 23 Mb. Here, we report the morphological characteristics of the fungus and the process of asexual spores forming asexual propagules, along with the first analyze the molecular karyotype of V. virens. These results supply a foundation for further study of the pathogenicity and biology of this devastating pathogen. PMID:22538655

  5. 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

  6. A Molecular Portrait of Arabidopsis Meiosis

    PubMed Central

    Ma, Hong

    2006-01-01

    Meiosis is essential for eukaryotic sexual reproduction and important for genetic diversity among individuals. Efforts during the last decade in Arabidopsis have greatly expanded our understanding of the molecular basis of plant meiosis, which has traditionally provided much information about the cytological description of meiosis. Through both forward genetic analysis of mutants with reduced fertility and reverse genetic studies of homologs of known meiotic genes, we now have a basic knowledge about genes important for meiotic recombination and its relationship to pairing and synapsis, critical processes that ensure proper homolog segregation. In addition, several genes affecting meiotic progression, spindle assembly, chromosome separation, and meiotic cytokinesis have also been uncovered and characterized. It is worth noting that Arabidopsis molecular genetic studies are also revealing secrets of meiosis that have not yet been recognized elsewhere among eukaryotes, including gene functions that might be unique to plants and those that are potentially shared with animals and fungi. As we enter the post-genomics era of plant biology, there is no doubt that the next ten years will see an even greater number of discoveries in this important area of plant development and cell biology. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; DSB, double strand break; DSBR, double strand break repair; SC, synaptonemal complex; TEM, transmission electron microscopy PMID:22303228

  7. 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. PMID:23896652

  8. Nuclear DNA Content Variation in Life History Phases of the Bonnemasoniaceae (Rhodophyta)

    PubMed Central

    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. PMID:24465835

  9. Human pathogenic Cryptosporidium species bioanalytical detection method with single oocyst detection capability.

    PubMed

    Connelly, John T; Nugen, Sam R; Borejsza-Wysocki, Wlodek; Durst, Richard A; Montagna, Richard A; Baeumner, Antje J

    2008-05-01

    A bioanalytical detection method for specific detection of viable human pathogenic Cryptosporidium species, C. parvum, C. hominis, and C. meleagridis is described. Oocysts were isolated from water samples via immunomagnetic separation, and mRNA was extracted with oligo-dT magnetic beads, amplified using nucleic acid sequence-based amplification (NASBA), and then detected in a nucleic acid hybridization lateral flow assay. The amplified target sequence employed was hsp70 mRNA, production of which is stimulated via a brief heat shock. The described method was capable of detecting one oocyst in 10 μL using flow-cytometer-counted samples. Only viable oocysts were detected, as confirmed using 4',6-diamidino-2-phenylindole and propidium iodide (DAPI/PI) staining. The detection system was challenged by detecting oocysts in the presence of large numbers of common waterborne microorganisms and packed pellet material filtered from environmental water samples. When the method was compared with EPA Method 1622 for C. parvum detection, highly comparable results were obtained. Since the described detection system yields unambiguous results within 4.5 h, it is an ideal method for monitoring the safety of drinking water. PMID:18311563

  10. 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.

  11. Chicken chromobox proteins: cDNA cloning of CHCB1, -2, -3 and their relation to W-heterochromatin.

    PubMed

    Yamaguchi, K; Hidema, S; Mizuno, S

    1998-07-10

    Three clones for chicken chromobox proteins were obtained from liver and ovary cDNA libraries. pCHCB1 and pCHCB2 encode polypeptides showing 96 and 95% identity with mouse M31 and M32, respectively, which are homologues of Drosophila heterochromatin protein 1 (HP1), and pCHCB3 encodes a polypeptide whose sequences of chromobox and C-terminal region show high-level similarities with those of mouse M33, Drosophila polycomb (Pc) protein, and Xenopus Pc homologue. When these cDNAs were expressed in female chicken embryonic fibroblasts (CEFs) as GFP-fused or HA-tagged proteins, all three proteins were found to be localized in nuclei. Among them, CHCB1 associates with brightly stained spots with 4', 6-diamidino-2-phenylindole (DAPI), suggesting its accumulation on heterochromatins. One of those spots was identified as W-heterochromatin. When CHCB1 lacking the N-terminal basic/acidic region or a part of the chromobox region was overexpressed in CEFs, W-heterochromatin became partially or extensively decondensed in the majority of nuclei. Overexpression of CHCB3 lacking a part of the chromobox did not cause decondensation of W-heterochromatin. Specific antisera raised against a part of CHCB1 or CHCB2, produced in Escherichia coli, detected protein species having apparent molecular masses of 25 kDa or 22 plus 23 kDa, respectively, in the subnuclear fraction containing the majority of chromatin from female chicken MSB-1 cells. PMID:9665828

  12. Environmental inactivation of Cryptosporidium parvum oocysts in waste stabilization ponds.

    PubMed

    Reinoso, Roberto; Bécares, Eloy

    2008-11-01

    The survival of Cryptosporidium parvum oocysts in a waste stabilization pond system in northwestern Spain and the effects of sunlight and the depth and type of pond on oocyst viability were 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). All tested factors had significant effects (P < 0.01) over time on C. parvum oocyst viability. Sunlight exposure was the most influential factor for oocyst inactivation. A 40% reduction was observed after 4 days exposure to sunlight conditions compared with dark conditions. The type of pond also caused a significant reduction in C. parvum oocyst viability (P < 0.01). Inactivation rates reflected that the facultative pond was the most aggressive environment for oocysts placed both at the surface (presence of sunlight) and at the bottom (absence of sunlight) of the pond, followed by the maturation pond and the anaerobic pond. The mean inactivation rates of oocysts in the ponds ranged from 0.0159 to 0.3025 day(-1). PMID:18345476

  13. The different fate of satellite cells on conductive composite electrospun nanofibers with graphene and graphene oxide nanosheets.

    PubMed

    Mahmoudifard, Matin; Soleimani, Masoud; Hatamie, Shadie; Zamanlui, Soheila; Ranjbarvan, Parviz; Vossoughi, Manouchehr; Hosseinzadeh, Simzar

    2016-04-01

    Electrospinning of composite polymer solutions provides fantastic potential to prepare novel nanofibers for use in a variety of applications. The addition of graphene (G) and graphene oxide (GO) nanosheets to bioactive polymers was found to enhance their conductivity and biocompatibility. Composite conductive nanofibers of polyaniline (PANI) and polyacrylonitrile (PAN) with G and GO nanosheets were prepared by an electrospinning process. The fabricated membranes were investigated by physical and chemical examinations including scanning electron microscopy (SEM), Raman spectroscopy, x-ray diffraction (XRD) and tensile assay. The muscle satellite cells enriched by a pre-plating technique were cultured in the following and their proliferation and differentiation behavior studied by MTT, Real-Time PCR assays and 4', 6-diamidino-2-phenylindole (DAPI) staining. The cultured cells on composite nanofibrous PAN/PANI-CSA/G confirmed a higher proliferation and differentiation value compared to other groups including PAN/PANI-CSA/GO and PAN/PANI-CSA scaffolds. Furthermore, the higher stiffness of the former scaffold showed a lower cell spreading as a function of stem cell activation into more proliferative cells. It is supposed that the enhanced conductivity value in addition to relative higher stiffness of the PAN/PANI-CSA/G composite nanofibers plays a favorable role for proliferation and differentiation of satellite cells. PMID:26962722

  14. In-vitro cytotoxicity assessment of carbon-nanodot-conjugated Fe-aminoclay (CD-FeAC) and its bio-imaging applications.

    PubMed

    Kang, Kyoung Suk; Lee, Hyun Uk; Kim, Moon Il; Park, So Young; Chang, Sung-Jin; Park, Ji-Ho; Huh, Yun Suk; Lee, Jouhahn; Yang, Mino; Lee, Young-Chul; Park, Hyun Gyu

    2015-01-01

    We have investigated the cytotoxic assay of Fe-aminoclay (FeAC) nanoparticles (NPs) and simultaneous imaging in HeLa cells by photoluminescent carbon nanodots (CD) conjugation. Non-cytotoxic, photostable, and CD NPs are conjugated with cationic FeAC NPs where CD NPs play a role in bio-imaging and FeAC NPs act as a substrate for CD conjugation and help to uptake of NPs into cancer cells due to positively charged surface of FeAC NPs in physiological media. As increase of CD-FeAC NPs loading in HeLa cell in vitro, it showed slight cytotoxicity at 1000 μg/mL but no cytotoxicity for normal cells up to concentration of 1000 μg/mL confirmed by two 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and neutral red (NR) assays, with further observations by 4',6-diamidino-2-phenylindole (DAPI) stained confocal microscopy images, possessing that CD-FeAC NPs can be used as potential drug delivery platforms in cancer cells with simultaneous imaging. Graphical abstract CD conjugation with organo-building blocks of delaminated FeAC NPs. PMID:26612177

  15. 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

  16. 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. PMID:24718345

  17. 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.

  18. 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.

  19. [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. PMID:27371841

  20. The ancestral carnivore karyotype (2n = 38) lives today in ringtails.

    PubMed

    Nash, William G; Menninger, Joan C; Padilla-Nash, Hesed M; Stone, Gary; Perelman, Polina L; O'Brien, Stephen J

    2008-01-01

    Chromosome painting was used to investigate the conservation of high-resolution longitudinal 4',6-diamidino-2-phenylindole (DAPI)/G bands in Carnivore chromosomes. Cat (Felis catus) and raccoon dog (Nyctereutes procyonoides) painting probes were hybridized to the ringtail (Bassaricus astutus), dwarf mongoose (Helogale parvula), and Malagasy civet (Fossa fossa) to identify homologous chromosome elements. The patterns of chromosome segment homology among Carnivore species allowed us to reconstruct and propose the disposition of a high-resolution banded ancestral carnivore karyotype (ACK). Three bi-armed chromosomes consistently found among Caniformia species are represented as 6 homologous acrocentric chromosomes among Feliformia species of Carnivora. However, reexamination of the most basal of Feliformia species, the African palm civet Nandinia, revealed the presence of the 3 heretofore Caniformia bi-armed chromosomes. Because these 3 bi-armed chromosomes are found in both Caniformia and Feliformia lineages, they are presumed ancestral for all Carnivora, suggesting that the ACK chromosome number would be 38, rather than the previously supposed 42. Banded chromosomes of the ACK are used to evaluate the consistency between recently determined molecular phylogenetic relationships and postulated cytogenetic dynamics in the same Carnivore species. PMID:18339652

  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. PMID:26473703

  2. 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.

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

    USGS Publications Warehouse

    Simmons, O. D., III; Sobsey, M.D.; Schaefer, F. W., III; 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.

  4. Candidacidal mechanism of the arenicin-3-derived peptide NZ17074 from Arenicola marina.

    PubMed

    Wang, Xiaojie; Wang, Xiumin; Teng, Da; Zhang, Yong; Mao, Ruoyu; Xi, Di; Wang, Jianhua

    2014-09-01

    The candidacidal mechanisms of NZ17074, which is a variant of arenicin-3 from Arenicola marina, against human pathogenic fungus Candida albicans are reported in this work. The minimum inhibitory concentration (MIC) of NZ17074 toward C. albicans was 4 μg/ml, and this peptide exerted marked candidacidal activity in an energy-dependent and salt-sensitive manner. The flow cytometric analysis using propidium iodide (PI) showed that the plasma membrane of cells treated with NZ17074 was perturbed and that the cells were arrested in the G2/M phase. The dihydrorhodamine-123 (DHR-123) staining showed that the reactive oxygen species (ROS) production of C. albicans increased after exposure to NZ17074. Typical cellular disruption events, such as mitochondrial degradation, nuclear fragmentation, nuclear membrane disruption, and chromatin condensation, were further revealed through rhodamine 123 (RH123) staining, 4',6-diamidino-2-phenylindole (DAPI) staining, and transmission electron microscopy. In addition, the intracellular localization of this peptide was concentration dependent: it was located in the membrane at low concentrations (4 to 8 μg/ml) and penetrated into the cytoplasm at high concentrations (16 to 32 μg/ml). Our results suggested that NZ17074 exerts its candidacidal effects by disrupting the cell membrane, inducing apoptosis, and interrupting the cell cycle. These findings showed the potential of NZ17074 as a new candidacidal peptide, in addition to its antibacterial activities. PMID:24816779

  5. Novel tissue-engineered vascular patches based on decellularized canine aortas and their recellularization in vitro

    NASA Astrophysics Data System (ADS)

    Tu, Qiufen; Zhang, Yi; Ge, Dongxia; Wu, Jiang; Chen, Huaiqing

    2008-11-01

    Decellularized allo- or xenogeneic vascular grafts have been found to give more promising results than some biodegradable synthetic polymers. However, owing to absence of well-organized cells, especially confluent endothelial cells, their long-term patency is limited. Seeding vessel-originated cells on these grafts may overcome the deficiency. In this study, canine aortas were decellularized and cross-linked. 4',6-Diamidino-2-phenylindole (DAPI) and Masson' trichrome staining showed complete removal of cell debris, while structure integrity of extracellular matrix (ECM) was remained. Human umbilical vein endothelial cells (HUVECs) and human umbilical artery smooth muscle cells (HUASMCs) were seeded on these decellularized aorta patches in three manners, ECs alone (EC/O), SMCs alone (SMC/O) and ECs on SMCs layer (EC/SMC). In EC/O and SMC/O, scanning electron microscopy (SEM) examination indicated both cells could form confluent layers on the decellularized patches when seeded at high density, but their morphology and alignment changed with seeding density. In EC/SMC, ECs could grow well on SMCs layer, but their morphology, alignment, and confluence degree were deeply influenced by the density of SMCs beneath.

  6. (-)-Nortrachelogenin from Partrinia scabiosaefolia elicits an apoptotic response in Candida albicans.

    PubMed

    Lee, Heejeong; Woo, Eun-Rhan; Lee, Dong Gun

    2016-05-01

    This study analyzes the antifungal properties of (-)-nortrachelogenin and elucidates its mode of action against pathogenic fungi. We performed susceptibility tests against several pathogenic fungi and verified the absence of hemolysis against human erythrocytes. Its antifungal activity increased reactive oxygen species (ROS) in response to intracellular stress and increased concentrations of both intracellular and extracellular trehalose without causing hemolysis. In addition, a cell wall regeneration study indicated its action on the cytoplasmic membrane. A cell surface study using 3,3(')-dipropylthiacarbocyanine iodide [DiSC3(5)] and 1,6-diphenyl-1,3,5-hexatriene (DPH) demonstrated dissipation of the cytoplasmic membrane at high concentrations. Our study revealed a disturbance in the membrane at higher concentrations and externalization of phosphatidylserine in a dose-dependent manner, affecting other intracellular responses. Furthermore, we investigated the late stage of apoptosis using TUNEL and 4('),6-diamidino-2-phenylindole (DAPI) assays. (-)-Nortrachelogenin-treated cells underwent apoptosis which was triggered by mitochondrial dysfunction via depolarization of the mitochondrial membrane, release of cytochrome c and calcium ion signaling, resulting in the activation of metacaspases. Different concentrations of (-)-nortrachelogenin induced membrane disruption and caspase-dependent apoptosis. PMID:26880798

  7. 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.

  8. Hydrothermal synthesis of highly luminescent blue-emitting ZnSe(S) quantum dots exhibiting low toxicity.

    PubMed

    Mirnajafizadeh, Fatemeh; Ramsey, Deborah; McAlpine, Shelli; Wang, Fan; Reece, Peter; Stride, John Arron

    2016-07-01

    Highly luminescent quantum dots (QDs) that emit in the visible spectrum are of interest to a number of imaging technologies, not least that of biological samples. One issue that hinders the application of luminescent markers in biology is the potential toxicity of the fluorophore. Here we show that hydrothermally synthesized ZnSe(S) QDs have low cytotoxicity to both human colorectal carcinoma cells (HCT-116) and human skin fibroblast cells (WS1). The QDs exhibited a high degree of crystallinity, with a strong blue photoluminescence at up to 29% quantum yield relative to 4',6-diamidino-2-phenylindole (DAPI) without post-synthetic UV-irradiation. Confocal microscopy images obtained of HCT-116 cells after incubation with the QDs highlighted the stability of the particles in cell media. Cytotoxicity studies showed that both HCT-116 and WS1 cells retain 100% viability after treatment with the QDs at concentrations up to 0.5g/L, which makes them of potential use in biological imaging applications. PMID:27127041

  9. Curcumin Triggers DNA Damage and Inhibits Expression of DNA Repair Proteins in Human Lung Cancer Cells.

    PubMed

    Ting, Chien-Yi; Wang, Hsin-Ell; Yu, Chien-Chih; Liu, Hsin-Chung; Liu, Yu-Chang; Chiang, I-Tsang

    2015-07-01

    The study goal was to evaluate the effects of curcumin on DNA damage and expression of DNA-repair proteins in human lung cancer. Thus, NCI-H460 cells were used to study the effects of curcumin on DNA damage and repair in vitro. We investigated curcumin induces DNA damage by comet the assay and 4',6-diamidino-2-phenylindole (DAPI) staining. The DNA damage/repair-related protein levels were examined and monitored by western blotting and confocal microscopy. Curcumin significantly increased the length of comet tails and DNA condensation in NCI-H460 cells. Curcumin reduced expression of DNA-repair proteins such as 14-3-3 protein sigma (14-3-3σ), O6-methylguanine-DNA methyltransferase (MGMT), breast cancer susceptibility gene 1 (BRCA1), and mediator of DNA damage checkpoint 1 (MDC1). Curcumin also increased phosphorylation of p53 and Histone H2A.X (S140) in the nuclei of NCI-H460 cells. Taken together, our findings indicated that curcumin triggered DNA damage and inhibited expression of DNA-repair-associated proteins in NCI-H460 cells. PMID:26124332

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

    PubMed

    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

  11. 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.

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

    PubMed

    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

  13. Biodegradation potentiality of psychrophilic bacterial strain Oleispira antarctica RB-8(T).

    PubMed

    Gentile, G; Bonsignore, M; Santisi, S; Catalfamo, M; Giuliano, L; Genovese, L; Yakimov, M M; Denaro, R; Genovese, M; Cappello, S

    2016-04-15

    The present study is focused on assessing the growth and hydrocarbon-degrading capability of the psychrophilic strain Oleispira antarctica RB-8(T). This study considered six hydrocarbon mixtures that were tested for 22days at two different cultivation temperatures (4 and 15°C). During the incubation period, six sub-aliquots of each culture at different times were processed for total bacterial abundance and GC-FID (gas chromatography-flame ionization detection) hydrocarbon analysis. Results from DNA extraction and DAPI (4',6-diamidino-2-phenylindole) staining showed a linear increase during the first 18days of the experiment in almost all the substrates used; both techniques showed a good match, but the difference in values obtained was approximately one order of magnitude. GC-FID results revealed a substantial hydrocarbon degradation rate in almost all hydrocarbon sources and in particular at 15°C rather than 4°C (for commercial oil engine, oily waste, fuel jet, and crude oil). A more efficient degradation was observed in cultures grown with diesel and bilge water at 4°C. PMID:26912198

  14. 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

  15. 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

  16. Cytolethal Distending Toxin Damages the Oral Epithelium of Gingival Explants

    PubMed Central

    Damek-Poprawa, M.; Haris, M.; Volgina, A.; Korostoff, J.; DiRienzo, J.M.

    2011-01-01

    The cytolethal distending toxin (Cdt), expressed by the periodontal pathogen Aggregatibacter actinomycetemcomitans, inhibits the proliferation of cultured epithelial cells by arresting the cell cycle. The gingival epithelium is an early line of defense against microbial assault. When damaged, bacteria collectively gain entry into underlying connective tissue where microbial products can affect infiltrating inflammatory cells, leading to the destruction of the attachment apparatus. Histological evaluation of rat and healthy human gingival tissue exposed ex vivo to the Cdt for 36 and 18 hours, respectively, revealed extensive detachment of the keratinized outer layer and distention of spinous and basal cells in the oral epithelium. Treated human tissue also exhibited disruption of rete pegs and dissolution of cell junctions. Cells in the connective tissue appeared unaffected. Primary gingival epithelial cells, but not gingival fibroblasts, isolated from the same healthy human tissue were cell-cycle-arrested when treated with the toxin. These findings provide new evidence that the Cdt severely damages the oral epithelium, ex vivo, by specifically targeting epithelial cells, in situ. The Cdt shows preferential targeting of the epithelium as opposed to connective tissue in animal and human gingival explant models. Abbreviations: cytolethal distending toxin (Cdt), connective tissue (CT), 4′,6-diamidino-2-phenylindole (DAPI), human gingival epithelial cells (HGEC), human gingival explants (HGX), human gingival fibroblasts (HGF), junctional epithelium (JE), oral epithelium (OE), rete pegs (RP), sulcular epithelium (SE) PMID:21471326

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

    PubMed Central

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

    2016-01-01

    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. PMID:26839163

  18. Polyphosphate Is a Novel Pro-inflammatory Regulator of Mast Cells and Is Located in Acidocalcisomes*

    PubMed Central

    Moreno-Sanchez, David; Hernandez-Ruiz, Laura; Ruiz, Felix A.; Docampo, Roberto

    2012-01-01

    Polyphosphate (polyP) is a pro-inflammatory agent and a potent modulator of the human blood-clotting system. The presence of polyP of 60 phosphate units was identified in rat basophilic leukemia (RBL-2H3) mast cells using specific enzymatic assays, urea-polyacrylamide gel electrophoresis of cell extracts, and staining of cells with 4,6-diamidino-2-phenylindole (DAPI), and the polyP-binding domain of Escherichia coli exopolyphosphatase. PolyP co-localizes with serotonin- but not with histamine-containing granules. PolyP levels greatly decreased in mast cells stimulated to degranulate by IgE. Mast cell granules were isolated and found to be acidic and decrease their polyP content upon alkalinization. In agreement with these results, when RBL-2H3 mast cells were loaded with the fluorescent calcium indicator fura-2 acetoxymethyl ester to measure their intracellular Ca2+ concentration ([Ca2+]i), they were shown to possess a significant amount of Ca2+ stored in an acidic compartment different from lysosomes. PolyP derived from RBL-2H3 mast cells stimulated bradykinin formation, and it was also detected in human basophils. All of these characteristics of mast cell granules, together with their known elemental composition, and high density, are similar to those of acidocalcisomes. The results suggest that mast cells polyP could be an important mediator of their pro-inflammatory and pro-coagulant activities. PMID:22761438

  19. Phloroglucinol induces apoptosis through the regulation of insulin-like growth factor 1 receptor signaling pathways in human colon cancer HT-29 cells

    PubMed Central

    KANG, MI-HYE; KIM, IN-HYE; NAM, TAEK-JEONG

    2014-01-01

    Phloroglucinol is a polyphenol compound with free radical scavenging, anti-inflammatory and antitumor activity. In this study, we investigated the anticancer effects of phloroglucinol on insulin-like growth factor-1 receptor (IGF-1R) signaling in HT-29 human colon cancer cells. Apoptosis was evaluated using 4′,6-diamidino-2-phenylindole (DAPI) staining, which clearly demonstrated cell shrinkage and condensed nuclei. Treatment with a pan-caspase inhibitor reduced the expression of phosphatidylinositol-3-kinase (PI3K)/Akt, which could induce apoptosis through IGF-1R signaling pathways. Treatment with phloroglucinol significantly inhibited the expression of Ras, Raf, mitogen-activated protein kinase (MEK), extracellular-signal regulated kinase (ERK) phosphorylation, PI3K and Akt. Phloroglucinol also decreased mammalian target of rapamycin (mTOR) and expression of its downstream effectors p70S6 kinase and translation initiation factors elF4B and RPS6. These results demonstrate that IGF-1R activates PI3K/Akt/mTOR and Ras/ERK-MAPK downstream signaling pathways, which has important implications for understanding the roles of cell growth pathways in colon cancer cell tumorigenesis. PMID:24970012

  20. β-lapachone suppresses the proliferation of human malignant melanoma cells by targeting specificity protein 1.

    PubMed

    Bang, Woong; Jeon, Young-Joo; Cho, Jin Hyoung; Lee, Ra Ham; Park, Seon-Min; Shin, Jae-Cheon; Choi, Nag-Jin; Choi, Yung Hyun; Cho, Jung-Jae; Seo, Jae-Min; Lee, Seung-Yeop; Shim, Jung-Hyun; Chae, Jung-Il

    2016-02-01

    β-lapachone (β-lap), a novel natural quinone derived from the bark of the Pink trumpet tree (Tabebuia avellanedae) has been demonstrated to have anticancer activity. In this study, we investigated whether β-lap exhibits anti-proliferative effects on two human malignant melanoma (HMM) cell lines, G361 and SK-MEL-28. The effects of β-lap on the HMM cell lines were investigated using 3-(4,5-dimethylthiazol-2-yl)‑5-(3-carboxymethoxyphenyl)‑2-(4-sulfophenyl-2H-tetrazolium (MTS) assay, 4',6-diamidino-2-phenylindole (DAPI) staining, Annexin V and Dead cell assay, mitochondrial membrane potential (MMP) assay and western blot analysis. We demonstrated that β-lap significantly induced apoptosis and suppressed cell viability in the HMM cells. Intriguingly, the transcription factor specificity protein 1 (Sp1) was significantly downregulated by β-lap in a dose- and time-dependent manner. Furthermore, β-lap modulated the protein expression level of the Sp1 regulatory genes including cell cycle regulatory proteins and apoptosis-associated proteins. Taken together, our findings indicated that β-lap modulates Sp1 transactivation and induces apoptotic cell death through the regulation of cell cycle- and apoptosis-associated proteins. Thus, β-lap may be used as a promising anticancer drug for cancer prevention and may improve the clinical outcome of patients with cancer. PMID:26718788

  1. Benzidine dihydrochloride: toxicological assessment in mice during chronic exposures.

    PubMed

    Littlefield, N A; Nelson, C J; Frith, C H

    1983-01-01

    Although benzidine is recognized as a bladder carcinogen in humans and a liver carcinogen in laboratory animals, its toxicological effects appear to be extended to several other endpoints. This economically important chemical is the base for over 200 dyes and is used extensively in manufacturing. In a chronic lifespan study lasting 33 months, both sexes of F1 hybrid (genetically homogeneous) and monohybrid cross (genetically heterogeneous) mice from BALB/c male and C57BL/6 female crosses were exposed to benzidine dihydrochloride in their drinking water at concentrations of 0, 20, 30, 40, 60, 80, and 120 ppm for the females, and 0, 30, 40, 60, 80, 120, and 160 ppm for males. Animals were removed from the study when they were dead or moribund. In addition to hepatocellular carcinomas, there were several other toxicological end-points identified that appeared to be related to the administration of benzidine. Dose-response trends were noted for pigmentation of the spleen, hepatic cytological alterations, hyperplasia of the bile ducts, megakaryocytosis of the bone marrow, vacuolization of the brain, adenoma of the Harderian gland, atrophy of the ovaries, and angioma of the uterus. Also, dose-related effects were noted with respect to time to lung tumor and time to mortality due to reticulum-cell sarcomas. PMID:6366243

  2. 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. PMID:27377012

  3. 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

  4. Automatic stage identification of Drosophila egg chamber based on DAPI images.

    PubMed

    Jia, Dongyu; Xu, Qiuping; Xie, Qian; Mio, Washington; Deng, Wu-Min

    2016-01-01

    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

  5. 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. PMID:25176372

  6. 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. PMID:19863484

  7. 40 CFR 721.9750 - 2-Chloro-4,6-bis(substituted)-1,3,5-triazine, dihydrochloride.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...-triazine, dihydrochloride. 721.9750 Section 721.9750 Protection of Environment ENVIRONMENTAL PROTECTION... New Uses for Specific Chemical Substances § 721.9750 2-Chloro-4,6-bis(substituted)-1,3,5-triazine... identified generically as 2-chloro-4,6-bis(substituted)-1,3,5-triazine, dihydrochloride (PMN P-91-659)...

  8. 21 CFR 520.1242c - Levamisole hydrochloride and piperazine dihydrochloride.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... an aqueous solution which contains in each fluid ounce 0.36 gram of levamisole hydrochloride and piperazine dihydrochloride equivalent to 3.98 grams of piperazine base. (2) The drug is a soluble powder which when reconstituted with water contains in each fluid ounce 0.45 gram of levamisole...

  9. 21 CFR 520.1242c - Levamisole hydrochloride and piperazine dihydrochloride.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... an aqueous solution which contains in each fluid ounce 0.36 gram of levamisole hydrochloride and piperazine dihydrochloride equivalent to 3.98 grams of piperazine base. (2) The drug is a soluble powder which when reconstituted with water contains in each fluid ounce 0.45 gram of levamisole...

  10. 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

  11. 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

    2016-09-01

    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. PMID:27522151

  12. 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

  13. Structural optimization of pyridine-type DAPY derivatives to exploit the tolerant regions of the NNRTI binding pocket.

    PubMed

    Chen, Wenmin; Zhan, Peng; Daelemans, Dirk; Yang, Jiapei; Huang, Boshi; De Clercq, Erik; Pannecouque, Christophe; Liu, Xinyong

    2016-10-01

    Based on the crystallographic studies of diarylpyrimidines (DAPYs), we embarked on incorporating the hydrophilic piperidyl or morpholinyl group into the known DAPY derivatives bearing the pyridine moiety as a core structure, with the double aim to exploit additional interactions with the HIV-1 NNRTI binding pocket (NNIBP), as well as to improve the compound solubility. The antiviral evaluation result show that the most potent compounds I-8b2, I-8b3, I-8b4 and I-8c3 exhibited anti-HIV-1 (IIIB) strain activity ranging from 7.4 nM to 9.4 nM (SI = 168-1283), superior to FDA-approved drugs of nevirapine (NVP), lamivudine (3TC) and delavirdine (DLV), and comparable to etravirine (ETV), zidovudine (AZT) and efavirenz (EFV). Additionally, compounds I-8c2 and I-8c3 showed moderate activity against NNRTI resistant strains baring mutations K103N and Y181C with EC50 values of 6.2 μM and 6.8 μM, respectively. Preliminary structure-activity relationships (SARs), reverse transcriptase inhibition efficacy and molecular modeling of selected compounds are also presented. These outcomes support our design hypothesis and demonstrate that the piperidyl group modified pyridine-typed DAPY derivatives are highly potent NNRTIs with improved water solubility. PMID:27267005

  14. 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. PMID:22206469

  15. Cytotoxic and apoptotic activities of Amorphophallus campanulatus tuber extracts against human hepatoma cell line

    PubMed Central

    Ansil, P.N.; Wills, P.J.; Varun, R.; Latha, M.S.

    2014-01-01

    Amorphophallus campanulatus (Roxb.) Blume belonging to the family of Araceae, is a perennial herb commonly known as elephant foot yam. Its tuber has been traditionally used for the treatment of liver diseases, abdominal tumors, piles. The aim of the present study was to evaluate the dose-dependent cytotoxic and apoptosis inducing effects of the sub fractions of Amorphophallus campanulatus tuber methanolic extract (ACME) namely petroleum ether fraction (PEF), chloroform fraction (CHF), ethyl acetate fraction (EAF) and methanolic fraction (MeF) on human liver cancer cell line, PLC/PRF/5. Antiproliferative effects of the sub fractions of ACME were studied by MTT assay. Apoptotic activity was assessed by 4′,6-diamidino-2-phenylindole (DAPI), annexin V- fluorescein isothiocyanate (FITC) and 5,5’,6,6’ tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) fluorescent staining. The chemotherapeutic drug, 5-flurouracil (5-FU) was used as positive drug control. The sub fractions of ACME were found to produce considerable cytotoxicity in human liver cancer cell line, PLC/PRF/5. In addition, the extracts were found to induce apoptosis and were substantiated by DAPI, annexin V-FITC and JC-1 fluorescent staining. A pronounced results of cytotoxic and apoptotic activities were observed in the cells treated with 5-FU and CHF, whereas, EAF and MeF treated cells exhibited a moderate result and the least effect were observed in PEF treated cells. Furthermore, these findings confirm that the sub fractions of ACME dose-dependently suppress the proliferation of PLC/PRF/5 cells by inducing apoptosis. PMID:25657798

  16. Cytotoxic and apoptotic activities of Amorphophallus campanulatus tuber extracts against human hepatoma cell line.

    PubMed

    Ansil, P N; Wills, P J; Varun, R; Latha, M S

    2014-01-01

    Amorphophallus campanulatus (Roxb.) Blume belonging to the family of Araceae, is a perennial herb commonly known as elephant foot yam. Its tuber has been traditionally used for the treatment of liver diseases, abdominal tumors, piles. The aim of the present study was to evaluate the dose-dependent cytotoxic and apoptosis inducing effects of the sub fractions of Amorphophallus campanulatus tuber methanolic extract (ACME) namely petroleum ether fraction (PEF), chloroform fraction (CHF), ethyl acetate fraction (EAF) and methanolic fraction (MeF) on human liver cancer cell line, PLC/PRF/5. Antiproliferative effects of the sub fractions of ACME were studied by MTT assay. Apoptotic activity was assessed by 4',6-diamidino-2-phenylindole (DAPI), annexin V- fluorescein isothiocyanate (FITC) and 5,5',6,6' tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) fluorescent staining. The chemotherapeutic drug, 5-flurouracil (5-FU) was used as positive drug control. The sub fractions of ACME were found to produce considerable cytotoxicity in human liver cancer cell line, PLC/PRF/5. In addition, the extracts were found to induce apoptosis and were substantiated by DAPI, annexin V-FITC and JC-1 fluorescent staining. A pronounced results of cytotoxic and apoptotic activities were observed in the cells treated with 5-FU and CHF, whereas, EAF and MeF treated cells exhibited a moderate result and the least effect were observed in PEF treated cells. Furthermore, these findings confirm that the sub fractions of ACME dose-dependently suppress the proliferation of PLC/PRF/5 cells by inducing apoptosis. PMID:25657798

  17. 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. PMID:24817642

  18. Acellular porcine corneal matrix as a carrier scaffold for cultivating human corneal epithelial cells and fibroblasts in vitro

    PubMed Central

    Zhang, Ju; Zhang, Can-Wei; Du, Li-Qun; Wu, Xin-Yi

    2016-01-01

    AIM To investigate the feasibility of corneal anterior lamellar reconstruction with human corneal epithelial cells and fibroblasts, and an acellular porcine cornea matrix (APCM) in vitro. METHODS The scaffold was prepared from fresh porcine corneas which were treated with 0.5% sodium dodecyl sulfate (SDS) solution and the complete removal of corneal cells was confirmed by hematoxylin-eosin (HE) staining and 4′, 6-diamidino-2-phenylindole (DAPI) staining. Human corneal fibroblasts and epithelial cells were cultured with leaching liquid extracted from APCM, and then cell proliferative ability was evaluated by MTT assay. To construct a human corneal anterior lamellar replacement, corneal fibroblasts were injected into the APCM and cultured for 3d, followed by culturing corneal epithelial cells on the stroma construction surface for another 10d. The corneal replacement was analyzed by HE staining, and immunofluorescence staining. RESULTS Histological examination indicated that there were no cells in the APCM by HE staining, and DAPI staining did not detect any residual DNA. The leaching liquid from APCM had little influence on the proliferation ability of human corneal fibroblasts and epithelial cells. At 10d, a continuous 3 to 5 layers of human corneal epithelial cells covering the surface of the APCM was observed, and the injected corneal fibroblasts distributed within the scaffold. The phenotype of the construction was similar to normal human corneas, with high expression of cytokeratin 12 in the epithelial cell layer and high expression of vimentin in the stroma. CONCLUSION Corneal anterior lamellar replacement can be reconstructed in vitro by cultivating human corneal epithelial cells and fibroblasts with an acellular porcine cornea matrix. This laid the foundation for the further transplantation in vivo. PMID:26949602

  19. 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. PMID:11685534

  20. Cytogenetic characterization of species of the family Heptapteridae (Teleostei: Siluriformes) from the Cuiabá and Ivaí River Basins, Brazil.

    PubMed

    Barbosa, L M; Ferreira, G E B; Prizon, A C; Portela-Castro, A L B; Martins-Santos, I C

    2015-01-01

    We cytogenetically characterized three species of Heptapteridae (Pimelodella sp, Pimelodella taenioptera, and Imparfinis schubarti) by investigating the distribution of constitutive heterochromatin and nucleolar organizer regions by silver nitrate impregnation (Ag-NOR) and fluorescence in situ hybridization. Pimelodella sp showed had a diploid number (2n) = 46 chromosomes, 26m + 10sm + 10st, and FN = 92; P. taenioptera, 2n = 52 chromosomes, 26m + 22sm + 4st, and FN = 104; and I. schubarti, 2n = 58 chromosomes, 28m + 28sm + 2st, and FN = 116. The two Pimelodella species had Ag-NORs sites on the submetacentric pair 14, located on the short arm in terminal position. In I. schubarti, the Ag-NORs sites were in an interstitial position on the long arm of the metacentric pair 1. C-banding revealed that Pimelodella sp contained a small amount of constitutive heterochroma-tin, whereas P. taenioptera contained a higher number of heterochromatic regions, in the pericentromeric, interstitial, and telomeric positions. I. schubarti had markers in centromeric and telomeric regions of a few chromosomes, and a large pericentromeric block on pair 1. Fluorochrome chromomycin A3 (CMA3) staining revealed positive signals on pair 14 in both Pimelodella species. Treatment with 4ꞌ,6-diamidino- 2-phenylindole (DAPI) revealed no markings in P. taenioptera, but an interstitial marking on the long arm of pair 14 in Pimelodella sp. In I. schubarti, positive signals of CMA3 were detected in the first pair, but negative signals were detected for DAPI staining. These results con-tribute to the karyotypic description of the less-studied species in the Brazilian Midwest. PMID:26345795

  1. Intravenous injection of mesenchymal stem cells is effective in treating liver fibrosis

    PubMed Central

    Zhao, Wei; Li, Jun-Jie; Cao, Da-Yong; Li, Xiao; Zhang, Lin-Ying; He, Yong; Yue, Shu-Qiang; Wang, De-Sheng; Dou, Ke-Feng

    2012-01-01

    AIM: To compare the influence of different transplant sites in bone marrow mesenchymal stem cell (MSC)-based therapy for liver fibrosis. METHODS: MSCs isolated from Sprague Dawley (SD) rats were induced into hepatocyte-like cells. Liver fibrosis in SD rats was induced with carbon tetrachloride. Following hepatocyte induction in vitro, 4’,6-diamidino-2-phenylindole (DAPI)-labeled MSCs were transplanted by intravenous, intrahepatic, and intraperitoneal injection. Histopathological staining, immunohistochemistry, and biochemical analysis were used to compare the morphological and functional liver regeneration among different MSC injection modalities. The expression differences of interleukins, growth factor, extracellular matrix, matrix metalloproteinases, and tissue inhibitor of metalloproteinase were examined by real-time reverse transcription-polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA). RESULTS: Four days after exposure to hepatocyte differentiation medium, MSCs that did not express hepatocyte markers could express α-fetoprotein, albumin, and cytokeratin 18. The results of histopathological staining, immunohistochemistry, and biochemical analysis indicated that intravenous injection is more effective at rescuing liver failure than other injection modalities. DAPI-labeled cells were found around liver lobules in all three injection site groups, but the intravenous group had the highest number of cells. PCR and ELISA analysis indicated that interleukin-10 (IL-10) was highest in the intravenous group, whereas il1β, il6, tnfα and tgfβ, which can be regulated by IL10 and are promoters of liver fibrosis, were significantly lower than in the other groups. CONCLUSION: MSC administration is able to protect against liver fibrosis. Intravenous injection is the most favorable treatment modality through promotion of IL10 expression. PMID:22416179

  2. 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. PMID:26781342

  3. Microbiology and Geochemistry of Acidic Cave Biofilms in the Frasassi Caves, Italy

    NASA Astrophysics Data System (ADS)

    Meyer, K. M.; Cleaveland, L. C.; Galdenzi, S.; Macalady, J. L.

    2003-12-01

    Acidic (pH 2-4) and extremely acidic (pH 0-1) biofilms in sulfidic regions of the Frasassi cave system are relatively simple, chemoautotrophic microbial communities. As such, they serve as model systems to test relationships between microbial diversity and physical and geochemical factors. Both biofilm types are isolated from surface sources of C and N and are ultimately powered by oxidation of H2S present in the cave atmosphere. pH 2-4 biofilms consist of cells in close association with sub-um to sub-mm mineral grains (primarily CaSO4) coating cave walls. Direct counts of cells stained with 4',6-diamidino-2-phenylindole, hydrochloride (DAPI) yield a biomass estimate of 7.5 x 106 to 1.3 x 107 cells per cm3. The great majority of these cells are either dormant (contain few ribosomes) or cells which do not hybridize with either bacteria- or archaea-specific oligonucleotide probes. Sparse clusters of short rod and coccus-shaped cells hybridized with a bacteria-specific Fluorescent In Situ Hybridization (FISH) probe. Polymerase Chain Reaction (PCR) amplification of 16S rDNA was successful with bacteria-specific primers as well as with several sets of archaeal-specific primers, suggesting that some of the "dormant" cells are archaea. Extremely acidic biofilms (snottites) drip from macroscopic (1-2 cm length), reddish CaSO4 crystals on the cave walls. DAPI-staining and FISH revealed abundant bacterial rods, bacterial filaments, and fungi in the snottites. Future work will characterize the acidic cave wall biofilms, as well as neutral-pH cave stream biofilms, using 16S rDNA clone libraries in order to determine whether pH is an important factor controlling microbial diversity.

  4. Functionally Cloned pdrM from Streptococcus pneumoniae Encodes a Na+ Coupled Multidrug Efflux Pump

    PubMed Central

    Hashimoto, Kohei; Ogawa, Wakano; Nishioka, Toshihiro; Tsuchiya, Tomofusa; Kuroda, Teruo

    2013-01-01

    Multidrug efflux pumps play an important role as a self-defense system in bacteria. Bacterial multidrug efflux pumps are classified into five families based on structure and coupling energy: resistance−nodulation−cell division (RND), small multidrug resistance (SMR), major facilitator (MF), ATP binding cassette (ABC), and multidrug and toxic compounds extrusion (MATE). We cloned a gene encoding a MATE-type multidrug efflux pump from Streptococcus pneumoniae R6, and designated it pdrM. PdrM showed sequence similarity with NorM from Vibrio parahaemolyticus, YdhE from Escherichia coli, and other bacterial MATE-type multidrug efflux pumps. Heterologous expression of PdrM let to elevated resistance to several antibacterial agents, norfloxacin, acriflavine, and 4′,6-diamidino-2-phenylindole (DAPI) in E. coli KAM32 cells. PdrM effluxes acriflavine and DAPI in a Na+- or Li+-dependent manner. Moreover, Na+ efflux via PdrM was observed when acriflavine was added to Na+-loaded cells expressing pdrM. Therefore, we conclude that PdrM is a Na+/drug antiporter in S. pneumoniae. In addition to pdrM, we found another two genes, spr1756 and spr1877,that met the criteria of MATE-type by searching the S. pneumoniae genome database. However, cloned spr1756 and spr1877 did not elevate the MIC of any of the investigated drugs. mRNA expression of spr1756, spr1877, and pdrM was detected in S. pneumoniae R6 under laboratory growth conditions. Therefore, spr1756 and spr1877 are supposed to play physiological roles in this growth condition, but they may be unrelated to drug resistance. PMID:23555691

  5. Identification in situ and dynamics of bacteria on limnetic organic aggregates (lake snow).

    PubMed Central

    Weiss, P; Schweitzer, B; Amann, R; Simon, M

    1996-01-01

    Microbial assemblages on large organic aggregates (lake snow) of Lake Constance, Germany, were analyzed with rRNA-directed fluorescent oligonucleotide probes specific for the domain Bacteria and the alpha-, beta-, and gamma-subclasses of the class Proteobacteria. Lake snow aggregates were either collected in situ by SCUBA diving or in a sediment trap at 50 m or formed of natural lake water incubated in rolling cylinders under simulated in situ conditions. For the latter aggregates, the time course of the microbial colonization was also examined. The natural aggregates and those made in rolling cylinders were composed of the particulate organic material present in the lake and thus reflected the composition of the ambient plankton community. All types of lake snow aggregates examined were heavily colonized by microbial cells and harbored between 0.5 x 10(6) and > 2 x 10(6) cells aggregate -1. Between 55 and 100% of the microbial cells stained with 4', 6-diamidino-2-phenylindole (DAPI) could be visualized with the domain Bacteria-specific probe. In most samples, beta-subclass proteobacteria dominated the microbial community, constituting 27 to 42% of total cells as counted by DAPI staining, irrespective of the composition of the aggregates. During the time course experiments with the laboratory-made aggregates, the fraction of beta-subclass proteobacteria usually increased over time. Except for a few samples, alpha- and gamma-subclass proteobacteria were far less abundant than beta-subclass proteobacteria, constituting 11 to 25 and 9 to 33% of total cells, respectively. Therefore, we assume that a specific aggregate-adapted microbial community was established on the aggregates. Because the compositions of the microbial assemblages on natural and laboratory-made aggregates were similar, we conclude that aggregates made in rolling cylinders are good model system with which to examine the formation and microbial colonization of macroscopic organic aggregates. PMID

  6. Visualization and enumeration of marine planktonic archaea and bacteria by using polyribonucleotide probes and fluorescent in situ hybridization.

    PubMed

    DeLong, E F; Taylor, L T; Marsh, T L; Preston, C M

    1999-12-01

    Fluorescent in situ hybridization (FISH) using rRNA-specific oligonucleotide probes has emerged as a popular technique for identifying individual microbial cells. In natural samples, however, the signal derived from fluor-labeled oligonucleotide probes often is undetectable above background fluorescence in many cells. To circumvent this difficulty, we applied fluorochrome-labeled polyribonucleotide probes to identify and enumerate marine planktonic archaea and bacteria. The approach greatly enhanced the sensitivity and applicability of FISH with seawater samples, allowing confident identification and enumeration of planktonic cells to ocean depths of 3,400 m. Quantitative whole-cell hybridization experiments using these probes accounted for 90 to 100% of the total 4',6-diamidino-2-phenylindole (DAPI)-stained cells in most samples. As predicted in a previous study (R. Massana, A. E. Murray, C. M. Preston, and E. F. DeLong, Appl. Environ. Microbiol. 63:50-56, 1997), group I and II marine archaea predominate in different zones in the water column, with maximal cell densities of 10(5)/ml. The high cell densities of archaea, extending from surface waters to abyssal depths, suggest that they represent a large and significant fraction of the total picoplankton biomass in coastal ocean waters. The data also show that the vast majority of planktonic prokaryotes contain significant numbers of ribosomes, rendering them easily detectable with polyribonucleotide probes. These results imply that the majority of planktonic cells visualized by DAPI do not represent lysed cells or "ghosts," as was suggested in a previous report. PMID:10584017

  7. 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

  8. Cyto/Genotoxic Effects of Pistacia atlantica Resin, a Traditional Gum.

    PubMed

    Rahbar Saadat, Yalda; Barzegari, Abolfazl; Zununi Vahed, Sepideh; Saeedi, Nazli; Eskandani, Morteza; Omidi, Yadollah; Barar, Jaleh

    2016-06-01

    In recent years, many researchers have focused on native plants to search for a new source of natural components with medical approach, especially by means of anticancer potential. One of these natural components is Saqez, the resin of Pistacia atlantica sub-kurdica with the local name of Baneh. It has been reported as an anticancer and apoptosis inducer component; therefore, in this research, we aimed to evaluate the solvated resin's possible cyto/genotoxic effects. The cell viability was assessed using MTT assay. Flow cytometry analysis was performed to distinguish the role of apoptosis and necrosis in cell toxicity, which was further confirmed by Comet and DNA ladder assay, and 4,6-diamidino2-phenylindole (DAPI) staining. Pistacia atlantica's resin decreased the growth of the treated cells in a dose- and time-dependent manner, and single-strand DNA breaks have been observed through comet assay. Moreover, morphological changes of DAPI-stained cells showed fragmentation in the nucleus of resin-treated cells. In addition, early and late apoptosis in the treated cells was determined by flow cytometry analysis, also DNA ladder assay showed fragmentation in DNA of the treated cells. This study has revealed that the resin has significant cyto/genotoxic effects on cancerous and noncancerous cell lines. Our results show that apoptosis and necrosis are the dominant mechanisms by which the resin affects cell lines. Although the resin of P. atlantica is the main source of mastic gum and has been used for a long time as a natural remedy for different diseases, it is necessary to perform thorough analysis due to its cyto/genotoxicity in vivo. PMID:27196631

  9. 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.

  10. Identification of Putative Fallopian Tube Stem Cells

    PubMed Central

    Snegovskikh, Victoria; Mutlu, Levent; Massasa, Effi

    2014-01-01

    Stem cells are used to repair and regenerate multiple tissues in the adult. We have previously shown that stem cells play a significant role in mediating endometrial repair and tissue regeneration. We hypothesized that the oviduct may possess a similar population of stem cells that contribute to the maintenance of this tissue. Here we identify label-retaining cells (LRCs) in the murine oviduct which indicate the presence of a stem/progenitor cell population in this tissue as well. Two-day-old CD-1 mice were injected intraperitoneally with 5-bromo-2-deoxyuridine (BrdU) or vehicle control. Female animals (n = 36 for each group) were killed at 6 weeks post injection. Reproductive tracts were removed, specimens were embedded in paraffin, and 5-µ sections were prepared. Oviduct was identified by hematoxylin and eosin staining and morphology. Immunofluorescence studies were performed on serial sections tissues (n = 12 per animal) using antibodies against BrdU. Confocal microscopy was used to identify 4′,6-diamidino-2-phenylindole (DAPI)- and BrdU-stained nuclei. In the group of mice exposed to BrdU, we identified a population of LRCs in all specimens and not in controls. The putative stem cells are located at the base of each villi, suggesting the location of the stem cell niche. The number of DAPI-stained nuclei divided by the number of LRCs; LRCs constituted 0.5% of all nucleated cells. The oviduct contains a population of progenitor cells, likely used in the repair and regeneration of fallopian tube. Defective or insufficient stem cell reserve may underlie common tubal diseases, including hydrosalpinx and ectopic pregnancy. PMID:25305130

  11. 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. PMID:22252896

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

    PubMed Central

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

    2012-01-01

    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 as small molecule capable of improving 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 known nucleic acid binders and then using virtual screening to optimize them for bioactivity, may be generally applicable for designing small molecules that target other RNAs in human genomic sequence. PMID:22252896

  13. 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

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

    PubMed

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

    2013-05-01

    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 cm(2)). The solvent system was ethyl acetate:methanol:glacial acetic acid in the proportion of 8:1:1, (v/v/v). R f 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

  15. 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

  16. Application of anodized titanium for enhanced recruitment of endothelial progenitor cells

    PubMed Central

    2012-01-01

    Objectives To study the efficacy of an effective anodized titanium surface with enhanced attachment of endothelial progenitor cell (EPC). Background In-stent restenosis is a major obstacle for vascular patency after catheter-based intravascular interventions. Recently, stents that capture EPCs have been paid attention in order to make a functional endothelialized layer at the site of stent-induced endothelial denudation. Anodized titanium has been shown to enhance stem cell attachment. Anodization is a quick and inexpensive method, which can provide suitable stent surface. Methods Surface topography was examined by high-resolution scanning electron microscopy (SEM). Substrates were co-cultured with EPCs at second passage in 24-well culture plates. Evaluation of cell growth, proliferation, viability, surface cytotoxicity and cell adhesion was performed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and 4,6-diamidino-2-phenylindole dihydrochloride staining. For platelet attachment, platelets added to substrates were evaluated under SEM. Results The average MTT values for tissue culture polystyrene plate, unanodized and anodized titanium with nanostructure were equal to 0.49, 0.16 and 0.72, respectively (P < 0.05). The surface had no cytotoxic effects on cells. The average cell attachment results showed that 9,955 ± 461.18, 3,300 ± 197.98 and 11,359 ± 458.10 EPCs were attached per well of tissue culture polystyrene plate, unanodized and anodized titanium surfaces, respectively (P < 0.05). Conclusions Anodized titanium surfaces can be potentially applied for devices that need enhanced recruitment of EPCs. This unique property makes these anodized surfaces good and cheap candidates for designing cardiovascular medical devices as endovascular stents. PMID:22676440

  17. Corosolic acid analogue, a natural triterpenoid saponin, induces apoptosis on human hepatocarcinoma cells through mitochondrial pathway in vitro.

    PubMed

    Qu, Liping; Zhang, Huiqing; Yang, Yanlong; Yang, Geliang; Xin, Hailiang; Ling, Changquan

    2016-08-01

    Context 2a,-3a,-24-Trihydroxyurs-12-en-28-oic acid (TEO, a corosolic acid analogue) is a triterpenoid saponin isolated from Actinidia valvata Dunn (Actinidiaceae), a well-known traditional Chinese medicine. Objective This study investigated the anti-proliferation and inducing apoptosis effects of TEO in three human hepatocellular carcinoma (HCC) cell lines. Materials and methods Cytotoxic activity of TEO was determined by the MTT assay at various concentrations from 2.5 to 40 μg/mL in BEL-7402, BEL-7404 and SMMC-7721 cell lines. Cell morphology was assessed by acridine orange/ethidium bromide and 4'-6-diamidino-2-phenylindole dihydrochloride staining and fluorescence microscopy. Cell-cycle distribution and DNA damage were determined by flow cytometry and comet assay. Mitochondrial dysfunction was assessed by JC-1 staining and transmission electron microscopy. Apoptosis changes were explored by Western blot, TNF-α and caspase-3, -8, -9 assays. Results TEO exhibited inhibition effects on BEL-7402, BEL-7404 and SMMC-7721 cells treated for 24 h, the IC50 values were 34.6, 30.8 and 30.5 μg/mL, respectively. TEO (40 μg/mL)-treated three cell lines increased by more than 21% in the G1 phase and presented the morphological change and DNA damage. TEO also declined the mitochondrial membrane potential and altered mitochondrial ultra-structure. Furthermore, caspase-3, caspase-8, caspase-9 and TNF-α were also activated. Mechanism investigation showed that TEO could decrease anti-apoptotic Bcl-2 protein expression, increase proapoptotic Bax and Bid proteins expressions and increase Bax/Bcl-2 ratio. Conclusion Our results demonstrate for the first time that TEO inhibited growth of HCC cell lines and induced G1 phase arrest. Moreover, proapoptotic effects of TEO were mediated through the activation of TNF-α, caspases and mitochondrial pathway. PMID:26810384

  18. 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.

  19. Cardiotoxicity of emetine dihydrochloride by calcium channel blockade in isolated preparations and ventricular myocytes of guinea-pig hearts.

    PubMed Central

    Lemmens-Gruber, R.; Karkhaneh, A.; Studenik, C.; Heistracher, P.

    1996-01-01

    1. The cardiotoxic effects of emetine dihydrochloride on mechanical and electrical activity were studied in isolated preparations (papillary muscles, sinoatrial and atrioventricular nodes, ventricular myocytes) of the guinea-pig heart. 2. Force of contraction was measured isometrically, action potentials and maximum rate of rise of the action potential were recorded by means of the intracellular microelectrode technique. Single channel L-type calcium current (Ba2+ ions as charge carrier) was studied with the patch-clamp technique in the cell-attached mode. 3. Emetine dihydrochloride (8-256 microM) reduced force of contraction in papillary muscles and spontaneous activity of sinoatrial and atrioventricular nodes concentration-dependently; the negative inotropic effect was abolished when the extracellular Ca2+ concentration was increased. 4. Maximum diastolic potential, action potential amplitude, maximum rate of rise of the action potential and the slope of the slow diastolic depolarization were decreased by emetine in sinoatrial as well as atrioventricular noes, while action potential duration was prolonged in both preparations (1-64 microM). 5. The amplitude of the L-type calcium single channel current was not altered by emetine dihydrochloride, while average open state probability was decreased concentration-dependently (10, 30 and 60 microM). 6. The most prominent effect of emetine dihydrochloride on single channel current was an increase of sweeps without activity. 7. At 60 microM, emetine dihydrochloride caused a decrease of the mean open time an increase of the mean closed time. The number of openings per record and number of bursts per record were reduced. 8. It is concluded that emetine dihydrochloride produces an L-type calcium channel block which might contribute to its cardiac side effects. PMID:8789394

  20. 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

  1. 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. PMID:25142820

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

    PubMed

    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

  3. Solid-State Characterization and Interconversion of Recrystallized Amodiaquine Dihydrochloride in Aliphatic Monohydric Alcohols.

    PubMed

    Sirikun, Wiriyaporn; Chatchawalsaisin, Jittima; Sutanthavibul, Narueporn

    2016-04-01

    Amodiaquine dihydrochloride monohydrate (AQ-DM) was obtained by recrystallizing amodiaquine dihydrochloride dihydrate (AQ-DD) in methanol, ethanol, and n-propanol. Solid-state characterization of AQ-DD and AQ-DM was performed using X-ray powder diffractometry, Fourier transform infrared spectroscopy, thermogravimetry, and differential scanning calorimetry. All recrystallized samples were identified as AQ-DM. Crystal habits of AQ-DD and AQ-DM were shown to be needle-like and rhombohedral crystals, respectively. When AQ-DD and AQ-DM were exposed to various relative humidity in dynamic vapor sorption apparatus, no solid-state interconversion was observed. However, AQ-DM showed higher solubility than AQ-DD when exposed to bulk water during solubility study, while excess AQ-DM was directly transformed back to a more stable AQ-DD structure. Heating AQ-DM sample to temperatures ≥190°C induced initial change to metastable amorphous form (AQ-DA) which was rapidly recrystallized to AQ-DD upon ≥80%RH moisture exposure. AQ-DD was able to be recrystallized in alcohols (C1-C3) as AQ-DM solid-state structure. In summary, AQ-DM was shown to have different solubility, moisture and temperature stability, and interconversion pathways when compared to AQ-DD. Thus, when AQ-DM was selected for any pharmaceutical applications, these critical transformation and property differences should be observed and closely monitored. PMID:26206402

  4. Anti-cancer Activity of Osmanthus matsumuranus Extract by Inducing G2/M Arrest and Apoptosis in Human Hepatocellular Carcinoma Hep G2 Cells

    PubMed Central

    Jin, Soojung; Park, Hyun-Jin; Oh, You Na; Kwon, Hyun Ju; Kim, Jeong-Hwan; Choi, Yung Hyun; Kim, Byung Woo

    2015-01-01

    Background: Osmanthus matsumuranus, a species of Oleaceae, is found in East Asia and Southeast Asia. The bioactivities of O. matsumuranus have not yet been fully understood. Here, we studied on the molecular mechanisms underlying anti-cancer effect of ethanol extract of O. matsumuranus (EEOM). Methods: Inhibitory effect of EEOM on cell growth and proliferation was determined by WST assay in various cancer cells. To investigate the mechanisms of EEOM-mediated cytotoxicity, HepG2 cells were treated with various concentration of EEOM and analyzed the cell cycle arrest and apoptosis induction by flow cytometry, Western blot analysis, 4,6-diamidino-2-phenylindole (DAPI) staining and DNA fragmentation. Results: EEOM showed the cytotoxic activities in a dose-dependent manner in various cancer cell lines but not in normal cells, and HepG2 cells were most susceptible to EEOM-induced cytotoxicity. EEOM induced G2/M arrest in HepG2 cells associated with decreased expression of cyclin-dependent kinase 1 (CDK1), cyclin A and cylcin B, and increased expression of phospho-checkpoint kinase 2, p53 and CDK inhibitor p21. Immunofluorescence staining showed that EEOM-treated HepG2 increased doublet nuclei and condensed actin, resulting in cell rounding. Furthermore, EEOM-mediated apoptosis was determined by Annexin V staining, chromatin condensation and DNA fragmentation. EEOM caused upregulation of FAS and Bax, activation of caspase-3, -8, -9, and fragmentation of poly ADP ribose polymerase. Conclusions: These results suggest that EEOM efficiently inhibits proliferation of HepG2 cells by inducing both G2/M arrest and apoptosis via intrinsic and extrinsic pathways, and EEOM may be used as a cancer chemopreventive agent in the food or nutraceutical industry. PMID:26734586

  5. 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

  6. 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

  7. 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. PMID:25545914

  8. Neutrophil extracellular traps formation by bacteria causing endometritis in the mare.

    PubMed

    Rebordão, M R; Carneiro, C; Alexandre-Pires, G; Brito, P; Pereira, C; Nunes, T; Galvão, A; Leitão, A; Vilela, C; Ferreira-Dias, G

    2014-12-01

    Besides the classical functions, neutrophils (PMNs) are able to release DNA in response to infectious stimuli, forming neutrophil extracellular traps (NETs) and killing pathogens. The pathogenesis of endometritis in the mare is not completely understood. The aim was to evaluate the in vitro capacity of equine PMNs to secrete NETs by chemical activation, or stimulated with Streptococcus equi subspecies zooepidemicus (Szoo), Escherichia coli (Ecoli) or Staphylococcus capitis (Scap) strains obtained from mares with endometritis. Ex vivo endometrial mucus from mares with bacterial endometritis were evaluated for the presence of NETs. Equine blood PMNs were used either without or with stimulation by phorbol-myristate-acetate (PMA), a strong inducer of NETs, for 1-3h. To evaluate PMN ability to produce NETs when phagocytosis was impaired, the phagocytosis inhibitor cytochalasin (Cyt) was added after PMA. After the addition of bacteria, a subsequent 1-h incubation was carried out in seven groups. NETs were visualized by 4',6-diamidino-2-phenylindole (DAPI) and anti-histone. Ex vivo samples were immunostained for myeloperoxidase and neutrophil elastase. A 3-h incubation period of PMN + PMA increased NETs (p < 0.05). Bacteria + 25 nM PMA and bacteria + PMA + Cyt increased NETs (p<0.05). Szoo induced fewer NETs than Ecoli or Scap (p < 0.05). Ex vivo NETs were present in mares with endometritis. Scanning electron microscopy showed the spread of NETs formed by smooth fibers and globules that can be aggregated in thick bundles. Formation of NETs and the subsequent entanglement of bacteria suggest that equine NETs might be a complementary mechanism in fighting some of the bacteria causing endometritis in the mare. PMID:25218891

  9. 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.

  10. 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

  11. 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

  12. 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. PMID:23735683

  13. 13-acetoxysarcocrassolide induces apoptosis on human gastric carcinoma cells through mitochondria-related apoptotic pathways: p38/JNK activation and PI3K/AKT suppression.

    PubMed

    Su, Ching-Chyuan; Chen, Jeff Yi-Fu; Din, Zhong-Hao; Su, Jui-Hsin; Yang, Zih-Yan; Chen, Yi-Jen; Wang, Robert Y L; Wu, Yu-Jen

    2014-10-01

    13-acetoxysarcocrassolide (13-AC), an active compound isolated from cultured Formosa soft coral Sarcophyton crassocaule, was found to possess anti-proliferative and apoptosis-inducing activities against AGS (human gastric adenocarcinoma cells) gastric carcinoma cells. The anti-tumor effects of 13-AC were determined by MTT assay, colony formation assessment, cell wound-healing assay, TUNEL/4,6-Diamidino-2-phenylindole (DAPI) staining, Annexin V-fluorescein isothiocyanate/propidium iodide (PI) staining and flow cytometry. 13-AC inhibited the growth and migration of gastric carcinoma cells in a dose-dependent manner and induced both early and late apoptosis as assessed by flow cytometer analysis. 13-AC-induced apoptosis was confirmed through observation of a change in ΔΨm, up-regulated expression levels of Bax and Bad proteins, down-regulated expression levels of Bcl-2, Bcl-xl and Mcl-1 proteins, and the activation of caspase-3, caspase-9, p38 and JNK. Furthermore, inhibition of p38 and JNK activity by pretreatment with SB03580 (a p38-specific inhibitor) and SP600125 (a JNK-specific inhibitor) led to rescue of the cell cytotoxicity of 13-AC-treated AGS cells, indicating that the p38 and the JNK pathways are also involved in the 13-AC-induced cell apoptosis. Together, these results suggest that 13-AC induces cell apoptosis against gastric cancer cells through triggering of the mitochondrial-dependent apoptotic pathway as well as activation of the p38 and JNK pathways. PMID:25342459

  14. Bladder cancer cells re-educate TAMs through lactate shuttling in the microfluidic cancer microenvironment

    PubMed Central

    Liu, Pengfei; Cao, Yanwei; Wang, Yonghua; Yang, Xuecheng; Xu, Xiaodong; Wang, Xinsheng; Niu, Haitao

    2015-01-01

    Background In the present study, we aimed to investigate the influence of lactate shuttling on the functional polarization and spatial distribution of transitional cell carcinoma of the bladder (TCCB) cells and macrophages. Methods We designed a microfluidic coculture chip for real-time integrative assays. The effect of lactate shuttling on the re-education of macrophages by TCCB cells was explored by measuring the levels of NO using a total NO assay kit and by evaluating the protein expression of iNOS, p-NFkB-p65, Arg-1 and HIF-1α via cell immunofluorescence and western blotting. Additionally, we examined TCCB cell viability using acridine orange/ethidium bromide (AO/EB) and MitoTracker staining. Moreover, the concentration distributions of lactate and large signaling proteins in the culture chambers were measured using 4′,6-diamidino-2-phenylindole (DAPI) and fluorescein isothiocyanate-dextran (FITC-dextran). Furthermore, the recruitment of macrophages and the influence of macrophages on BC metastasis were observed via light microscopy. Results We confirmed that TCCB cells reprogrammed macrophages into an M2 phenotype. Moreover, lactate inhibited M1 polarization and induced M2 polarization of macrophages, but blockade of cancer cell-macrophage lactate flux significantly inhibited the re-education of macrophages by TCCB cells. In addition, lactate diffused faster and deeper than large signaling proteins in the microfluidic tumor microenvironment. Furthermore, lactate alone induced the migration of macrophages, and M1, but not M2, macrophages reduced the motility of TCCB cells. Conclusions TCCB cells reprogrammed macrophages into an M2 phenotype in a manner that depended on cancer cell-TAM lactate flux. Furthermore, the lactate shuttle may be a determinant of the density of TAMs in tumor tissue. PMID:26474279

  15. 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

  16. SP600125 enhances the anti-apoptotic capacity and migration of bone marrow mesenchymal stem cells treated with tumor necrosis factor-α.

    PubMed

    Wei, Bo; Bai, Xizhuang; Chen, Kang; Zhang, Xiaonan

    2016-07-01

    Osteoarthritis (OA) and rheumatoid arthritis (RA) are chronic disorders associated with inflammation of joints characterized by damage to the underlying cartilage and bone. Bone marrow mesenchymal stem cells (BMSCs) are candidates for regeneration of bone and cartilage, which is inhibited by inflammatory cytokines in OA and RA, in particular tumor necrosis factor-α (TNF-α). This study aimed to investigate if the c-Jun N-terminal kinases (JNK)-specific inhibitor SP600125 could enhance the anti-apoptosis and migration of BMSCs treated with TNF-α. The level of apoptosis was evaluated via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)/4',6-diamidino-2-phenylindole (DAPI) staining, annexin V/propidium iodide (PI) staining and western blotting. Migration of BMSCs was assessed using transwell migration chambers. We showed that the survival capacity and migration of BMSCs was significantly inhibited by TNF-α, which was blocked by pretreatment with SP600125. In the presence of SP600125, expression of cleaved caspase-9/-3 and p53 as well as the ratio of Bax to Bcl-2 was significantly decreased compared to treatment with TNF-α alone. Our results therefore indicate that SP600125 improves the migration capacity of TNF-α-treated BMSCs and exerts a significant effect on the viability of TNF-α-treated BMSCs through reducing the up-regulation of p53, caspase-9/-3 and the Bcl-2 family induced by TNF-α. These findings suggest that SP600125 is of potential use in promoting the regeneration of bone and cartilage in OA and RA. PMID:27233606

  17. Effect of alpha-ketoglutarate on growth and metabolism of cells cultured on three-dimensional cryogel matrix.

    PubMed

    Singh, Deepti; Vishnoi, Tanushree; Kumar, Ashok

    2013-01-01

    Alpha- ketoglutarate (α-KG) is a well-known intermediate of Kreb's cycle and thus one of the important candidates in the role of cellular metabolism. The aim was to investigate the effect of α-KG on the growth and proliferation of different cells (fibroblasts and chondrocytes). Further change in the consumption of glucose and release of ammonia in the media, upon the addition of α-KG was also examined. NIH3T3 and chondrocytes were seeded on two different macroporous cryogel matrices synthesized by poly (hydroxyethylmethaacrylate)-gelatin (HG) and alginate-gelatin (AG). Higher proliferation was observed for both the cell lines in 2-D as well as 3-D where α-KG was added to the media compared to the controls. It was evident that the chondrocytes were metabolically more active in the case of α-KG containing samples as early extracellular matrix (ECM) accumulation was observed. In comparison to the 6-8 weeks duration required by the chondrocytes for ECM accumulation in normal in-vitro culture conditions, α-KG containing samples showed an earlier accumulation within 3 weeks. In order to further validate the results, scanning electron microscopic (SEM) analysis was performed showed high ECM deposition and cells embedded in the matrix. Homogenous distribution of cells on both the synthesized matrix was reported using 4'-6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) staining. The results clearly showed reduction in ammonia concentration in α-KG containing samples thus leading to decreased ammonia toxicity that builds up during long cell culture conditions thereby enhancing the proliferation and metabolic activity. Thus α-KG can be used potentially for long batch-cultures for the production of vaccines or antibody as well as can play an important role as a bioactive molecule for in vitro neo-cartilage generation. PMID:23781146

  18. Estimates of nuclear DNA content in 98 species of brown algae (Phaeophyta)

    PubMed Central

    Phillips, Naomi; Kapraun, Donald F.; Gómez Garreta, Amelia; Ribera Siguan, M. Antonia; Rull, Jorde L.; Salvador Soler, Noemi; Lewis, Raymond; Kawai, Hiroshi

    2011-01-01

    Background and aims Brown algae are critical components of marine ecosystems around the world. However, the genome of only one species of the class has so far been sequenced. This contrasts with numerous sequences available for model organisms such as higher plants, flies or worms. The present communication expands our coverage of DNA content information to 98 species of brown algae with a view to facilitating further genomic investigations of the class. Methodology The DNA-localizing fluorochrome DAPI (4′,6-diamidino-2-phenylindole) and the red blood cell (chicken erythrocyte) standard were used to estimate 2C values by static microspectrophotometry. Principal results 2C DNA contents are reported for 98 species of brown algae, almost doubling the number of estimates available for the class. The present results also expand the reported DNA content range to 0.2–3.6 pg, with several species of Fucales and Laminariales containing apparent polyploid genomes with 2C = 1.8–3.6 pg. Conclusions The data provide DNA content values for 12 of the 19 recognized orders of brown algae spanning the breadth of the class. Despite earlier contentions concerning DNA content and the presence of oogamy, the present results do not support a correlation between phylogenetic placement and genome size. The closest sister groups to the brown algae have genome sizes on the order of 0.3 pg (e.g. Schizocladiophyceae), suggesting that this may be the ancestral genome size. However, DNA content ranges widely across the class. PMID:22476472

  19. 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

  20. Determination of the Biomasses of Small Bacteria at Low Concentrations in a Mixture of Species with Forward Light Scatter Measurements by Flow Cytometry

    PubMed Central

    Robertson, B. R.; Button, D. K.; Koch, A. L.

    1998-01-01

    The forward light scatter intensity of bacteria analyzed by flow cytometry varied with their dry mass, in accordance with theory. A standard curve was formulated with Rayleigh-Gans theory to accommodate cell shape and alignment. It was calibrated with an extinction-culture isolate of the small marine organism Cycloclasticus oligotrophus, for which dry weight was determined by CHN analysis and 14C-acetate incorporation. Increased light scatter intensity due to formaldehyde accumulation in preserved cells was included in the standard curve. When differences in the refractive indices of culture media and interspecies differences in the effects of preservation were taken into account, there was agreement between cell mass obtained by flow cytometry for various bacterial species and cell mass computed from Coulter Counter volume and buoyant density. This agreement validated the standard curve and supported the assumption that cells were aligned in the flow stream. Several subpopulations were resolved in a mixture of three species analyzed according to forward light scatter and DNA-bound DAPI (4′,6-diamidino-2-phenylindole) fluorescence intensity. The total biomass of the mixture was 340 μg/liter. The lowest value for mean dry mass, 0.027 ± 0.008 pg/cell, was for the subpopulation of C. oligotrophus containing cells with a single chromosome. Calculations from measurements of dry mass, Coulter Counter volume, and buoyant density revealed that the dry weight of the isolate was 14 to 18% of its wet weight, compared to 30% for Escherichia coli. The method is suitable for cells with 0.005 to about 1.2 pg of dry weight at concentrations of as low as 103 cells/ml and offers a unique capability for determining biomass distributions in mixed bacterial populations. PMID:9758817

  1. 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. PMID:26091260

  2. 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

  3. Fluorescence In Situ Hybridization Method Using a Peptide Nucleic Acid Probe for Identification of Salmonella spp. in a Broad Spectrum of Samples ▿

    PubMed Central

    Almeida, C.; Azevedo, N. F.; Fernandes, R. M.; Keevil, C. W.; Vieira, M. J.

    2010-01-01

    A fluorescence in situ hybridization (FISH) method for the rapid detection of Salmonella spp. using a novel peptide nucleic acid (PNA) probe was developed. The probe theoretical specificity and sensitivity were both 100%. The PNA-FISH method was optimized, and laboratory testing on representative strains from the Salmonella genus subspecies and several related bacterial species confirmed the predicted theoretical values of specificity and sensitivity. The PNA-FISH method has been successfully adapted to detect cells in suspension and is hence able to be employed for the detection of this bacterium in blood, feces, water, and powdered infant formula (PIF). The blood and PIF samples were artificially contaminated with decreasing pathogen concentrations. After the use of an enrichment step, the PNA-FISH method was able to detect 1 CFU per 10 ml of blood (5 × 109 ± 5 × 108 CFU/ml after an overnight enrichment step) and also 1 CFU per 10 g of PIF (2 × 107 ± 5 × 106 CFU/ml after an 8-h enrichment step). The feces and water samples were also enriched according to the corresponding International Organization for Standardization methods, and results showed that the PNA-FISH method was able to detect Salmonella immediately after the first enrichment step was conducted. Moreover, the probe was able to discriminate the bacterium in a mixed microbial population in feces and water by counter-staining with 4′,6-diamidino-2-phenylindole (DAPI). This new method is applicable to a broad spectrum of samples and takes less than 20 h to obtain a diagnosis, except for PIF samples, where the analysis takes less than 12 h. This procedure may be used for food processing and municipal water control and also in clinical settings, representing an improved alternative to culture-based techniques and to the existing Salmonella PNA probe, Sal23S10, which presents a lower specificity. PMID:20453122

  4. Reversibly Switching Bilayer Permeability and Release Modules of Photochromic Polymersomes Stabilized by Cooperative Noncovalent Interactions.

    PubMed

    Wang, Xiaorui; Hu, Jinming; Liu, Guhuan; Tian, Jie; Wang, Huijuan; Gong, Ming; Liu, Shiyong

    2015-12-01

    We report on the fabrication of photochromic polymersomes exhibiting photoswitchable and reversible bilayer permeability from newly designed poly(ethylene oxide)-b-PSPA (PEO-b-PSPA) diblock copolymers, where SPA is spiropyran (SP)-based monomer containing a unique carbamate linkage. Upon self-assembling into polymersomes, SP moieties within vesicle bilayers undergo reversible phototriggered isomerization between hydrophobic spiropyran (SP, λ2 > 450 nm irradiation) and zwitterionic merocyanine (MC, λ1 < 420 nm irradiation) states. For both SP and MC polymersomes, their microstructures are stabilized by multiple cooperative noncovalent interactions including hydrophobic, hydrogen bonding, π-π stacking, and paired electrostatic (zwitterionic) interactions, with the latter two types being exclusive for MC polymersomes. Control experiments using analogous block copolymers of hydrophobic SP monomer with a carbonate linkage (SPO) and conventional spiropyran methacrylate monomer (SPMA) containing a single ester functionality were then conducted, revealing that carbamate-incurred hydrogen bonding interactions in PEO-b-PSPA are crucial for polymersome stabilization in the zwitterionic MC state. Moreover, reversible phototriggered SP-to-MC polymersome transition is accompanied by membrane polarity and permeability switching from being nonimpermeable to selectively permeable toward noncharged, charged, and zwitterionic small molecule species below critical molar masses. Intriguingly, UV-actuated MC polymersomes possess two types of release modules: (1) sustained release upon short UV irradiation duration by taking advantage of the unexpectedly slow spontaneous MC-to-SP transition kinetics (t1/2 > 20 h) under dark conditions; (2) on-demand and switchable release under alternated UV-vis light irradiation. We further demonstrate photoswitchable spatiotemporal release of 4',6-diamidino-2-phenylindole (DAPI, cell nuclei-staining dye) within living HeLa cells. PMID:26583385

  5. 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.

  6. 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

  7. 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. PMID:23159396

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

    USGS Publications Warehouse

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

  9. Effect of Diuron on aquatic bacteria in laboratory-scale wastewater treatment ponds with special reference to Aeromonas species studied by colony hybridization.

    PubMed

    Sumpono; Perotti, P; Belan, A; Forestier, C; Lavedrine, B; Bohatier, J

    2003-01-01

    Six laboratory-scale wastewater treatment ponds were filled with sediment and water obtained from a reference pond (a wastewater treatment plant located in a rural environment at Montel-de-Gelat, Puy-de-Dôme, France). They were kept at 20 degrees C, with alternative light and dark periods (12 h-12 h), and fed with raw effluent supplied weekly. Three of them were treated with Diuron (dissolved in DMSO) at a final concentration 10 mg/l, while the other three received only DMSO. Physico-chemical parameters, total bacteria, cultivable bacteria, and Aeromonas spp. were measured periodically until 41 days after the Diuron contamination. Total bacteria were treated with 4,6-diamidino 2-phenylindole (DAPI) and counted by epifluoroscence microscopy. The cultivable bacteria were quantified on plate count agar medium and Aeromonas spp. using colony hybridization. In the contaminated pilots, biochemical oxygen demand (BOD5), chemical oxygen demand (COD), suspended solids (SS), volatile suspended solids (VSS), ammonium, phosphorus, and bacteria increased, but dissolved oxygen decreased. The abundance of total bacteria, cultivable bacteria (multiplied by 30), and Aeromonas spp. increased for two weeks after Diuron introduction, reverting to initial values three weeks later. The percentage of cultivable bacteria relative to total bacteria was 0.2% in controls and 1.2% in treated pilots, while the percentage of Aeromonas spp. relative to cultivable bacteria decreased from 6-10% to 2%. Our results suggest that Diuron, which acts on the photosystem II of phototrophs, supports the development of cultivable bacteria through new carbon sources derived from the decomposition of photosynthetic micro-organisms, but does not specifically support Aeromonas spp. PMID:12656266

  10. 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

  11. DNA characterization and karyotypic evolution in the bee genus Melipona (Hymenoptera, Meliponini).

    PubMed

    Rocha, Marla Piumbini; Pompolo, Silvia Das Graças; Dergam, Jorge Abdala; Fernandes, Anderson; Campos, Lucio Antonio De Oliveira

    2002-01-01

    We analyzed patterns of heterochromatic bands in the Neotropical stingless bee genus Melipona (Hymenoptera, Meliponini). Group I species (Melipona bicolor bicolor, Melipona quadrifasciata, Melipona asilvae, Melipona marginata, Melipona subnitida) were characterized by low heterochromatic content. Group II species (Melipona capixaba, Melipona compressipes, Melipona crinita, Melipona seminigra fuscopilosa e Melipona scutellaris) had high heterochromatic content. All species had 2n = 18 and n = 9. In species of Group I heterochromatin was pericentromeric and located on the short arm of acrocentric chromosomes, while in Group II species heterochromatin was distributed along most of the chromosome length. The most effective sequential staining was quinacrine mustard (QM)/distamycin (DA)/chromomycin A3(CMA3)/4-6-diamidino-2-phenylindole (DAPI). Heterochromatic and euchromatic bands varied extensively within Group I. In Group II species euchromatin was restricted to the chromosome tips and it was uniformly GC+. Patterns of restriction enzymes (EcoRI, DraI, HindIII) showed that heterochromatin was heterogeneous. In all species the first pair of homologues was of unequal size and showed heteromorphism of a GC+ pericentromeric heterochromatin. In M. asilvae (Group I) this pair bore NOR and in M. compressipes (Group II) it hybridized with a rDNA FISH probe. As for Group I species the second pair was AT+ in M. subnitida and neutral for AT and GC in the remaining species of this group. Outgroup comparison indicates that high levels of heterochromatin represent a derived condition within Melipona. The pattern of karyotypic evolution sets Melipona in an isolated position within the Meliponini. PMID:12184485

  12. A NIR-remote controlled upconverting nanoparticle: an improved tool for living cell dye-labeling.

    PubMed

    Zheng, Bin; Gong, Xiaoqun; Wang, Hanjie; Wang, Sheng; Wang, Huiquan; Li, Wei; Tan, Jian; Chang, Jin

    2015-10-23

    In living cells, due to the selective permeability and complicated cellular environment, the uptake efficiency and fluorescence decay of organic dyes during dye-labeling may be influenced, which may eventually result in poor fluorescent imaging. In this work, a protocol of UCNs@mSiO2-(FA and Azo) core-shell nanocarriers was designed and prepared successfully. The core-shell nanocarriers were assembled from two parts, including a mesoporous silica shell surface modified by folate (FA) and azobenzene (Azo), and an upconverting nanocrystal (UCN) core. The mesoporous silica shell is used for loading organic dyes and conjugating folate which helps to enhance the cellular uptake of nanocarriers. The UCN core works as a transducer to convert near infrared (NIR) light to local UV and visible light to activate a back-and-forth wagging motion of azobenzene molecules on the surface, while the azobenzene acts as a molecular impeller for propelling the release of organic dyes. The nanocarriers of loading organic dyes can maintain the stability of the fluorescent imaging effect better than free organic dyes. The experimental results show that with the help of the nanoparticle, cell uptake efficiency of the model dyes of rhodamine and 4', 6-diamidino-2-phenylindole (DAPI) was significantly improved. The release of dyes can only be triggered by NIR light exposure and their quantity is highly dependent on the duration of NIR light exposure, thus realizing NIR-regulated dye release spatiotemporally. Our work may open a novel avenue for precisely controlling UCN-based living cell imaging in biotechnology and diagnostics, as well as studying cell dynamics, cell-cell interactions, and tissue morphogenesis. PMID:26422130

  13. Etomidate induces cytotoxic effects and gene expression in a murine leukemia macrophage cell line (RAW264.7).

    PubMed

    Wu, Rick Sai-Chuen; Wu, King-Chuen; Yang, Jai-Sing; Chiou, Shang-Ming; Yu, Chun-Shu; Chang, Shu-Jen; Chueh, Fu-Shin; Chung, Jing-Gung

    2011-06-01

    Etomidate is an important tool in the arsenal of the emergency physician, and it has been used in a variety of scenarios for both intubation and procedural sedation. In the present study, we investigated the cytotoxicity of etomidate including induction of apoptosis, and levels of protein and gene expressions associated with apoptotic cell death in murine leukemia RAW264.7 cells in vitro. Cytotoxic and apoptotic responses to etomidate of RAW264.7 cells, including cell morphological changes and cell viability were examined and measured by phase-contrast microscopy and flow cytometric assay, respectively. Results indicated that etomidate increased apoptotic cell morphological changes and reduced cell viability in RAW264.7 cells. 4',6-Diamidino-2-phenylindole (DAPI) staining also showed that etomidate induced the formation of apoptotic bodies, a characteristic of apoptosis. Results from Western blotting indicated that etomidate enhanced the levels of cytochrome c, apoptosis-inducing factor (AIF), endonuclease G (Endo G), caspase-9, caspase-3 active form and Bax proteins, but it inhibited the expression of Bcl-xl, leading to apoptosis. DNA microarray assay indicated that etomidate increased the expression of 17 genes (LOC676175; Gm14636; 2810021G02Rik; Iltifb; Olfr1167; Ttc30b; Olfr766; Gas5; Rgs1; LOC280487; V1rd4; Hist1h2bc; V1rj3; Gm10366; Olfr192; Gm10002 and Cspp1) and reduced the expression of 15 genes: (Gm10152; Gm5334; Olfr216; Lcn9; Gm10683; Gm5100; Tdgf1; Cypt2; Gm5595; 1700018F24Rik; Gm10417; Maml2; Olfr591; Trdn and Apol7c). In conclusion, etomidate induced cytotoxic and apoptotic effects the in murine leukemia RAW264.7 cells in vitro. PMID:21737642

  14. Adaptive epithelial cytoplasm segmentation and epithelial unit separation in immunoflurorescent images

    NASA Astrophysics Data System (ADS)

    Ramachandran, Janakiramanan; Scott, Richard; Ajemba, Peter; Karvir, Hrishikesh; Khan, Faisal; Zeineh, Jack; Donovan, Michael; Fernandez, Gerardo

    2012-02-01

    Tissue segmentation is one of the key preliminary steps in the morphometric analysis of tissue architecture. In multi-channel immunoflurorescent biomarker images, the primary segmentation steps consist of segmenting the nuclei (epithelial and stromal) and epithelial cytoplasm from 4',6-diamidino-2-phenylindole (DAPI) and cytokeratin 18 (CK18) biomarker images respectively. The epithelial cytoplasm segmentation can be very challenging due to variability in cytoplasm morphology and image staining. A robust and adaptive segmentation algorithm was developed for the purpose of both delineating the boundaries and separating thin gaps that separate the epithelial unit structures. This paper discusses novel methods that were developed for adaptive segmentation of epithelial cytoplasm and separation of epithelial units. The adaptive segmentation was performed by computing the non-epithelial background texture of every CK18 biomarker image. The epithelial unit separation was performed using two complementary techniques: a marker based, center-initialized watershed transform and a boundary initialized fast marching-watershed segmentation. The adaptive segmentation algorithm was tested on 926 CK18 biomarker biopsy images (326 patients) with limited background noise and 1030 prostatectomy images (374 patients) with noisy to very noisy background. The segmentation performance was measured using two different methods, namely; stability and background textural metrics. It was observed that the database of 1030 noisy prostatectomy images had a lower mean value (using stability and three background texture performance metrics) compared to the biopsy dataset of 926 images that had limited background noise. The average of all four performance metrics yielded 94.32% accuracy for prostatectomy images compared to 99.40% accuracy for biopsy images.

  15. Bacterioplankton community structure in a maritime antarctic oligotrophic lake during a period of holomixis, as determined by denaturing gradient gel electrophoresis (DGGE) and fluorescence in situ hybridization (FISH).

    PubMed

    Pearce, D A

    2003-07-01

    The bacterioplankton community structure in Moss Lake, a maritime Antarctic oligotrophic lake, was determined with vertical depth in the water column, during the ice-free period on Signy Island in the South Orkney Islands. Bacterioplankton community structure was determined using a combination of direct counting of 4',6-diamidino-2-phenylindole (DAPI) stained cells, PCR amplification of 16S rRNA gene fragments, denaturing gradient gel electrophoresis (DGGE) and in situ hybridization with group-specific, fluorescently labeled oligonucleotide probes. Using PCR amplification of 16S rRNA gene fragments and DGGE, the bacterioplankton community composition was shown to be constant with vertical depth in the water column. Specific bacterioplankton species identified through cloning and sequencing the DGGE products obtained were Flavobacterium xinjiangensis (a Flavobacterium), Leptothrix discophora (a beta-Proteobacterium), and a number of uncultured groups: two beta-Proteobacteria, an unclassified Proteobacterium, three sequences from Actinobacteria, and a Cyanobacterium. Fluorescence in situ hybridization (FISH), however, demonstrated that there were minor but significant fluctuations in different groups of bacteria with vertical depth in the water column. It showed that the beta-Proteobacteria accounted for between 26.4 and 71.5%, the alpha-Proteobacteria 2.3-10.6%, the gamma-Proteobacteria 0-29.6%, and the Cytophaga-Flavobacterium group 1.8-23.5% of cells hybridizing to a universal probe. This study reports the first description of the community structure of an oligotrophic Antarctic freshwater lake as determined by PCR-dependent and PCR-independent molecular techniques. It also suggests that the bacterioplankton community of Moss Lake contains classes of bacteria known to be important in freshwater systems elsewhere in the world. PMID:12739078

  16. 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

  17. Insulin-like growth factor II blocks apoptosis of N-myc2-expressing woodchuck liver epithelial cells.

    PubMed Central

    Yang, D; Faris, R; Hixson, D; Affigne, S; Rogler, C E

    1996-01-01

    N-myc2 and insulin-like growth factor II (IGF-II) are coordinately overexpressed in the great majority of altered hepatic foci, which are the earliest precancerous lesions observed in the liver of woodchuck hepatitis virus carrier woodchucks, and these genes continue to be overexpressed in hepatocellular carcinomas (HCCs). We have investigated the function of these genes in woodchuck hepatocarcinogenesis by using a woodchuck liver epithelial cell line (WC-3). WC-3 cells react positively with a monoclonal antibody (12.8.5) against woodchuck oval cells, suggesting a lineage relationship with oval cells. Overexpression of N-myc2 in three WC-3 cell lines caused their morphological transformation and increased their growth rate and saturation density in medium containing 10% serum. Removal of serum from the medium increased cell death of the N-myc2-expressing lines, whereas cell death in control lines was minimal. The death of N-myc2-expressing WC-3 cells was accompanied by nucleosomal fragmentation of cellular DNA, and DAPI (4',6-diamidino-2-phenylindole) staining revealed condensation and fragmentation of the nuclei, suggesting that N-myc2-expressing WC-3 cells undergo apoptosis in the absence of serum. In colony regression assays, conducted in the absence of serum, control colonies were stable, while N-myc2-expressing colonies regressed to various degrees. Addition of recombinant human IGF-II to the serum-free medium blocked both cell death and colony regression in all the N-myc2-expressing lines. Therefore, coordinate overexpression of N-myc2 and IGF-II in woodchuck altered hepatic foci may allow cells which otherwise might die to survive and progress to hepatocellular carcinoma. PMID:8709253

  18. 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. PMID:23817366

  19. 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

  20. A NIR-remote controlled upconverting nanoparticle: an improved tool for living cell dye-labeling

    NASA Astrophysics Data System (ADS)

    Zheng, Bin; Gong, Xiaoqun; Wang, Hanjie; Wang, Sheng; Wang, Huiquan; Li, Wei; Tan, Jian; Chang, Jin

    2015-10-01

    In living cells, due to the selective permeability and complicated cellular environment, the uptake efficiency and fluorescence decay of organic dyes during dye-labeling may be influenced, which may eventually result in poor fluorescent imaging. In this work, a protocol of UCNs@mSiO2-(FA and Azo) core-shell nanocarriers was designed and prepared successfully. The core-shell nanocarriers were assembled from two parts, including a mesoporous silica shell surface modified by folate (FA) and azobenzene (Azo), and an upconverting nanocrystal (UCN) core. The mesoporous silica shell is used for loading organic dyes and conjugating folate which helps to enhance the cellular uptake of nanocarriers. The UCN core works as a transducer to convert near infrared (NIR) light to local UV and visible light to activate a back-and-forth wagging motion of azobenzene molecules on the surface, while the azobenzene acts as a molecular impeller for propelling the release of organic dyes. The nanocarriers of loading organic dyes can maintain the stability of the fluorescent imaging effect better than free organic dyes. The experimental results show that with the help of the nanoparticle, cell uptake efficiency of the model dyes of rhodamine and 4‧, 6-diamidino-2-phenylindole (DAPI) was significantly improved. The release of dyes can only be triggered by NIR light exposure and their quantity is highly dependent on the duration of NIR light exposure, thus realizing NIR-regulated dye release spatiotemporally. Our work may open a novel avenue for precisely controlling UCN-based living cell imaging in biotechnology and diagnostics, as well as studying cell dynamics, cell-cell interactions, and tissue morphogenesis.

  1. 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

  2. Arsenic mineral dissolution and possible mobilization in mineral-microbe-groundwater environment.

    PubMed

    Islam, A B M R; Maity, Jyoti Prakash; Bundschuh, Jochen; Chen, Chien-Yen; Bhowmik, Bejon Kumar; Tazaki, Kazue

    2013-11-15

    Arsenic (As) is widely distributed in the nature as ores or minerals. It has been attracted much attention for the global public health issue, especially for groundwater As contamination. The aim of this study was to elucidate the characteristics of microbes in groundwater where As-minerals were dissolved. An ex situ experiment was conducted with 7 standard As-minerals in bacteria-free groundwater and stored in experimental vessels for 1 year without supplementary nutrients. The pH (6.7-8.4) and EhS.H.E. (24-548 mV) changed between initial (0 day) and final stages (365 days) of experiment. The dissolution of As was detected higher from arsenolite (4240 ± 8.69 mg/L) and native arsenic (4538 ± 9.02 mg/L), whereas moderately dissolved from orpiment (653 ± 3.56 mg/L) and realgar (319 ± 2.56 mg/L) in compare to arsenopyrite (85 ± 1.25mg/L) and tennantite (3 ± 0.06 mg/L). Optical microscopic, scanning electron microscopic observations and flurometric enumeration revealed the abundance of As-resistant bacillus, coccus and filamentous types of microorganisms on the surface of most of As-mineral. 4'-6-Diamidino-2-phenylindole (DAPI)-stained epifluorescence micrograph confirmed the presence of DNA and carboxyfluorescein diacetate (CFDA) staining method revealed the enzymatically active bacteria on the surface of As-minerals such as in realgar (As4S4). Therefore, the microbes enable to survive and mobilize the As in groundwater by dissolution/bioweathering of As-minerals. PMID:22954601

  3. Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

    PubMed Central

    Johnson, M. Brittany; Criss, Alison K.

    2013-01-01

    Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4',6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells. PMID:24056524

  4. Effects of nisin and reutericyclin on resistance of endospores of Clostridium spp. to heat and high pressure.

    PubMed

    Hofstetter, Simmon; Gebhardt, David; Ho, Linda; Gänzle, Michael; McMullen, Lynn M

    2013-05-01

    The effects of high pressure, temperature, and antimicrobial compounds on endospores of Clostridium spp. were examined. Minimal inhibitory concentrations (MIC) of nisin and reutericyclin were determined for vegetative cells and endospores of Clostridium sporogenes ATCC 7955, Clostridium beijerinckii ATCC 8260, and Clostridium difficile 3195. Endospores of C. sporogenes ATCC 7955 and C. beijerinckii ATCC 8260 were exposed to 90 °C and 90 °C/600 MPa in the presence of 16 mg L(-1) nisin or 6.4 mg L(-1) reutericyclin for 0-60 min in a 0.9% saline solution. Dipicolinic acid (DPA) release was measured using a terbium-DPA fluorescence assay, and endospore permeability was assessed using 4',6-diamidino-2-phenylindole (DAPI) fluorescence. Vegetative cells of C. sporogenes ATCC 7955 exhibited higher sensitivity to nisin relative to endospores, with MIC values 0.23 ± 0.084 mg L(-1) and 1.11 ± 0.48 mg L(-1), respectively. Nisin increased DPA release when endospores were treated at 90 °C; however, only C. sporogenes ATCC 7955 exhibited higher inactivation, suggesting strain or species specific effects. Reutericyclin did not enhance spore inactivation or DPA release. Use of nisin in combination with high pressure, thermal treatments enhanced inactivation of endospores of Clostridium spp. and may have application in foods. PMID:23498177

  5. Diversity and distribution of microbial eukaryotes in the deep tropical and subtropical North Atlantic Ocean

    NASA Astrophysics Data System (ADS)

    Morgan-Smith, Danielle; Clouse, Melissa A.; Herndl, Gerhard J.; Bochdansky, Alexander B.

    2013-08-01

    Employing a combination of 4',6-diamidino-2-phenylindole and fluorescein isothiocyanate (DAPI-FITC) staining and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH), we distinguished a variety of taxonomic and morphological types of eukaryotic microbes in the central and deep water masses of the tropical and subtropical North Atlantic Ocean. Samples were taken along a transect across the tropical Atlantic, along the equatorial upwelling and into the West-African upwelling region. Samples were collected as deep as 7000 m in the Romanche Fracture Zone within the Mid-Atlantic Ridge. Approximately 50-70% of FISH-identified eukaryotes in deep water masses belong to one of seven groups: kinetoplastids, labyrinthulomycetes, fungi, diplonemids, group II alveolates, MAST 4 (stramenopiles), and an unidentified organism with a peculiar nuclear morphology. A smaller percentage of total eukaryotes was identified in the Central Water, especially in the oxygen minimum zone, than in deep water masses. CARD-FISH probes designed to identify broad taxonomic groups revealed kinetoplastids and fungi were more abundant than noted in previous studies employing 18S rRNA gene clone libraries. Group II alveolates, in contrast, were much less prevalent than previously reported. On a second survey, eukaryotic microbes were enumerated in the deep-sea basins below the North Atlantic subtropical gyre including the Vema Fracture Zone, which is another prominent trench in the Mid-Atlantic Ridge. The abundance of eukaryotes and chlorophyll concentrations were significantly different between the two cruises, which covered very different hydrographic regimes with associated high and low levels of primary production, respectively.

  6. Protein Phosphatase 2A (PP2A) Regulatory Subunits ParA and PabA Orchestrate Septation and Conidiation and Are Essential for PP2A Activity in Aspergillus nidulans

    PubMed Central

    Zhong, Guo-wei; Jiang, Ping; Qiao, Wei-ran; Zhang, Yuan-wei; Wei, Wen-fan

    2014-01-01

    Protein phosphatase 2A (PP2A) is a major intracellular protein phosphatase that regulates multiple aspects of cell growth and metabolism. Different activities of PP2A and subcellular localization are determined by its regulatory subunits. Here we identified and characterized the functions of two protein phosphatase regulatory subunit homologs, ParA and PabA, in Aspergillus nidulans. Our results demonstrate that ParA localizes to the septum site and that deletion of parA causes hyperseptation, while overexpression of parA abolishes septum formation; this suggests that ParA may function as a negative regulator of septation. In comparison, PabA displays a clear colocalization pattern with 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei, and deletion of pabA induces a remarkable delayed-septation phenotype. Both parA and pabA are required for hyphal growth, conidiation, and self-fertilization, likely to maintain normal levels of PP2A activity. Most interestingly, parA deletion is capable of suppressing septation defects in pabA mutants, suggesting that ParA counteracts PabA during the septation process. In contrast, double mutants of parA and pabA led to synthetic defects in colony growth, indicating that ParA functions synthetically with PabA during hyphal growth. Moreover, unlike the case for PP2A-Par1 and PP2A-Pab1 in yeast (which are negative regulators that inactivate the septation initiation network [SIN]), loss of ParA or PabA fails to suppress defects of temperature-sensitive mutants of the SEPH kinase of the SIN. Thus, our findings support the previously unrealized evidence that the B-family subunits of PP2A have comprehensive functions as partners of heterotrimeric enzyme complexes of PP2A, both spatially and temporally, in A. nidulans. PMID:25280816

  7. 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.

  8. 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. PMID:27315046

  9. 40 CFR 721.9750 - 2-Chloro-4,6-bis(substituted)-1,3,5-triazine, dihydrochloride.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false 2-Chloro-4,6-bis(substituted)-1,3,5-triazine, dihydrochloride. 721.9750 Section 721.9750 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances...

  10. 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. PMID:26530993

  11. 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.

  12. 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

  13. 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. PMID:19714475

  14. 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

  15. 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

  16. 40 CFR 721.9750 - 2-Chloro-4,6-bis(substituted)-1,3,5-triazine, dihydrochloride.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false 2-Chloro-4,6-bis(substituted)-1,3,5... New Uses for Specific Chemical Substances § 721.9750 2-Chloro-4,6-bis(substituted)-1,3,5-triazine... identified generically as 2-chloro-4,6-bis(substituted)-1,3,5-triazine, dihydrochloride (PMN P-91-659)...

  17. Antibacterial Effects and Biocompatibility of Titania Nanotubes with Octenidine Dihydrochloride/Poly(lactic-co-glycolic acid).

    PubMed

    Xu, Zhiqiang; Lai, Yingzhen; Wu, Dong; Huang, Wenxiu; Huang, Sijia; Zhou, Lin; Chen, Jiang

    2015-01-01

    Titanium (Ti) implants with long-term antibacterial ability and good biocompatibility are highly desirable materials that can be used to prevent implant-associated infections. In this study, titania nanotubes (TNTs) were synthesized on Ti surfaces through electrochemical anodization. Octenidine dihydrochloride (OCT)/poly(lactic-co-glycolic acid) (PLGA) was infiltrated into TNTs using a simple solvent-casting technique. OCT/PLGA-TNTs demonstrated sustained drug release and maintained the characteristic hollow structures of TNTs. TNTs (200 nm in diameter) alone exhibited slight antibacterial effect and good osteogenic activity but also evidently impaired adhesion and proliferation of bone marrow mesenchymal stem cells (BMSCs). OCT/PLGA-TNTs (100 nm in diameter) supported BMSC adhesion and proliferation and showed good osteogenesis-inducing ability. OCT/PLGA-TNTs also exhibited good long-term antibacterial ability within the observation period of 7 d. The synthesized drug carrier with relatively long-term antibacterial ability and enhanced excellent biocompatibility demonstrated significant potential in bone implant applications. PMID:26090449

  18. Residual antimicrobial effect of chlorhexidine digluconate and octenidine dihydrochloride on reconstructed human epidermis.

    PubMed

    Müller, G; Langer, J; Siebert, J; Kramer, A

    2014-01-01

    The objective of the present investigation was to examine the residual antimicrobial activity after a topical exposure of reconstructed human epidermis (RHE) to equimolar solutions of either chlorhexidine digluconate (CHG, 0.144% w/v) or octenidine dihydrochloride (OCT, 0.1% w/v) for 15 min. RHE-associated antiseptic agents were more effective on Staphylococcus aureus than on Pseudomonas aeruginosa. S. aureus was not detected after 24 h of contact, which demonstrated a microbicidal efficacy of greater than 5-log10 reduction. In contrast, P. aeruginosa was reduced by approximately 2 log10 at the same incubation time, which parallels the growth of the initial inoculum. This result could be interpreted either as a microbiostatic effect or as an adherence of P. aeruginosa to a low positively charged surface. Small amounts of CHG and OCT can penetrate the stratum corneum. Using these antiseptic agents, the viability of keratinocytes was reduced to 65-75% of that of the untreated RHE control following 24 h incubation in the presence of test microorganisms. With consideration of antimicrobial activity and cytotoxic effect, OCT corresponds better to a biocompatible antiseptic agent than CHG. PMID:23887383

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

    PubMed

    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

  20. Antibacterial Effects and Biocompatibility of Titania Nanotubes with Octenidine Dihydrochloride/Poly(lactic-co-glycolic acid)

    PubMed Central

    Xu, Zhiqiang; Lai, Yingzhen; Wu, Dong; Huang, Wenxiu; Huang, Sijia; Zhou, Lin; Chen, Jiang

    2015-01-01

    Titanium (Ti) implants with long-term antibacterial ability and good biocompatibility are highly desirable materials that can be used to prevent implant-associated infections. In this study, titania nanotubes (TNTs) were synthesized on Ti surfaces through electrochemical anodization. Octenidine dihydrochloride (OCT)/poly(lactic-co-glycolic acid) (PLGA) was infiltrated into TNTs using a simple solvent-casting technique. OCT/PLGA-TNTs demonstrated sustained drug release and maintained the characteristic hollow structures of TNTs. TNTs (200 nm in diameter) alone exhibited slight antibacterial effect and good osteogenic activity but also evidently impaired adhesion and proliferation of bone marrow mesenchymal stem cells (BMSCs). OCT/PLGA-TNTs (100 nm in diameter) supported BMSC adhesion and proliferation and showed good osteogenesis-inducing ability. OCT/PLGA-TNTs also exhibited good long-term antibacterial ability within the observation period of 7 d. The synthesized drug carrier with relatively long-term antibacterial ability and enhanced excellent biocompatibility demonstrated significant potential in bone implant applications. PMID:26090449

  1. 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

  2. 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

  3. 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

  4. Alpha- and beta-Proteobacteria control the consumption and release of amino acids on lake snow aggregates.

    PubMed

    Schweitzer, B; Huber, I; Amann, R; Ludwig, W; Simon, M

    2001-02-01

    We analyzed the composition of aggregate (lake snow)-associated bacterial communities in Lake Constance from 1994 until 1996 between a depth of 25 m and the sediment surface at 110 m by fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes of various specificity. In addition, we experimentally examined the turnover of dissolved amino acids and carbohydrates together with the microbial colonization of aggregates formed in rolling tanks in the lab. Generally, between 40 and more than 80% of the microbes enumerated by DAPI staining (4',6'-diamidino-2-phenylindole) were detected as Bacteria by the probe EUB338. At a depth of 25 m, 10.5% +/- 7.9% and 14.2% +/- 10.2% of the DAPI cell counts were detected by probes specific for alpha- and beta-Proteobacteria. These proportions increased to 12.0% +/- 3.3% and 54.0% +/- 5.9% at a depth of 50 m but decreased again at the sediment surface at 110 m to 2.7% +/- 1.4% and 41.1% +/- 8.4%, indicating a clear dominance of beta-Proteobacteria at depths of 50 and 110 m, where aggregates have an age of 3 to 5 and 8 to 11 days, respectively. From 50 m to the sediment surface, cells detected by a Cytophaga/Flavobacteria-specific probe (CF319a) comprised increasing proportions up to 18% of the DAPI cell counts. gamma-Proteobacteria always comprised minor proportions of the aggregate-associated bacterial community. Using only two probes highly specific for clusters of bacteria closely related to Sphingomonas species and Brevundimonas diminuta, we identified between 16 and 60% of the alpha-Proteobacteria. In addition, with three probes highly specific for close relatives of the beta-Proteobacteria Duganella zoogloeoides (formerly Zoogloea ramigera), Acidovorax facilis, and Hydrogenophaga palleroni, bacteria common in activated sludge, 42 to 70% of the beta-Proteobacteria were identified. In the early phase (<20 h) of 11 of the 15 experimental incubations of aggregates, dissolved amino acids were consumed by the

  5. 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

  6. 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. PMID:26653793

  7. Safety and tolerability of levocetirizine dihydrochloride in infants and children with allergic rhinitis or chronic urticaria.

    PubMed

    Hampel, Frank; Ratner, Paul; Haeusler, Jean-Marc C

    2010-01-01

    Allergic rhinitis (AR) and chronic idiopathic urticaria (CIU) are common causes of substantial illness and disability in preschool children. Antihistamines are commonly used to treat preschool children with these conditions, but their use is based mostly on extrapolated efficacy from adult populations; it is thus important to characterize the safety of antihistamines in the pediatric population. This study was designed to assess the safety of levocetirizine dihydrochloride oral liquid drops in infants and children with AR or CIU. Two multicenter, double-blind, randomized, parallel-group studies randomized infants aged 6-11 months (study 1, n = 69) and children aged 1-5 years (study 2, n = 173) to levocetirizine, 1.25 mg (q.d. or b.i.d., respectively), or placebo for 2 weeks, using a 2:1 ratio. Safety evaluations included treatment-emergent adverse events (TEAEs), vital signs, electrocardiographic (ECG) assessments, and laboratory tests. The overall incidence of TEAEs was similar between levocetirizine and placebo in both studies. Most TEAEs were mild or moderate in intensity. TEAEs prompted discontinuation of therapy in three patients receiving levocetirizine in study 1. No clinically relevant changes from baseline in vital signs or laboratory parameters were apparent in either study; changes from baseline in these evaluations were similar between groups. No significant changes were observed in ECG parameters, including corrected QT interval. Levocetirizine, 1.25 and 2.5 mg/day, was well tolerated in infants aged 6-11 months and in children aged 1-5 years, respectively, with AR or CIU. PMID:20819318

  8. Safety and efficacy of 22 weeks of treatment with sapropterin dihydrochloride in patients with phenylketonuria.

    PubMed

    Lee, Phillip; Treacy, Eileen P; Crombez, Eric; Wasserstein, Melissa; Waber, Lewis; Wolff, Jon; Wendel, Udo; Dorenbaum, Alex; Bebchuk, Judith; Christ-Schmidt, Heidi; Seashore, Margretta; Giovannini, Marcello; Burton, Barbara K; Morris, Andrew A

    2008-11-15

    Phenylketonuria (PKU) is an inherited metabolic disease characterized by phenylalanine (Phe) accumulation, which can lead to neurocognitive and neuromotor impairment. Sapropterin dihydrochloride, an FDA-approved synthetic formulation of tetrahydrobiopterin (6R-BH4, herein referred to as sapropterin) is effective in reducing plasma Phe concentrations in patients with hyperphenylalaninemia due to tetrahydrobiopterin (BH4)-responsive PKU, offering potential for improved metabolic control. Eighty patients, > or =8 years old, who had participated in a 6-week, randomized, placebo-controlled study of sapropterin, were enrolled in this 22-week, multicenter, open-label extension study comprising a 6-week forced dose-titration phase (5, 20, and 10 mg/kg/day of study drug consecutively for 2 weeks each), a 4-week dose-analysis phase (10 mg/kg/day), and a 12-week fixed-dose phase (patients received doses of 5, 10, or 20 mg/kg/day based on their plasma Phe concentrations during the dose titration). Dose-dependent reductions in plasma Phe concentrations were observed in the forced dose-titration phase. Mean (SD) plasma Phe concentration decreased from 844.0 (398.0) micromol/L (week 0) to 645.2 (393.4) micromol/L (week 10); the mean was maintained at this level during the study's final 12 weeks (652.2 [382.5] micromol/L at week 22). Sixty-eight (85%) patients had at least one adverse event (AE). All AEs, except one, were mild or moderate in severity. Neither the severe AE nor any of the three serious AEs was considered related to sapropterin. No AE led to treatment discontinuation. Sapropterin is effective in reducing plasma Phe concentrations in a dose-dependent manner and is well tolerated at doses of 5-20 mg/kg/day over 22 weeks in BH4-responsive patients with PKU. PMID:18932221

  9. Feasibility of optimizing trimetazidine dihydrochloride release from controlled porosity osmotic pump tablets of directly compressed cores.

    PubMed

    Habib, Basant A; Rehim, Randa T Abd El; Nour, Samia A

    2014-05-01

    The aim of this study was to develop and optimize Trimetazidine dihydrochloride (TM) controlled porosity osmotic pump (CPOP) tablets of directly compressed cores. A 2(3) full factorial design was used to study the influence of three factors namely: PEG400 (10% and 25% based on coating polymer weight), coating level (10% and 20% of tablet core weight) and hole diameter (0 "no hole" and 1 mm). Other variables such as tablet cores, coating mixture of ethylcellulose (4%) and dibutylphthalate (2%) in 95% ethanol and pan coating conditions were kept constant. The responses studied (Yi ) were cumulative percentage released after 2 h (Q%2h), 6 h (Q%6h), 12 h (Q%12h) and regression coefficient of release data fitted to zero order equation (RSQzero), for Y 1, Y 2, Y 3, and Y 4, respectively. Polynomial equations were used to study the influence of different factors on each response individually. Response surface methodology and multiple response optimization were used to search for an optimized formula. Response variables for the optimized formula were restricted to 10% ⩽ Y 1 ⩽ 20%, 40% ⩽ Y 2 ⩽ 60%, 80% ⩽ Y 3 ⩽ 100%, and Y 4 > 0.9. The statistical analysis of the results revealed that PEG400 had positive effects on Q%2h, Q%6h and Q%12h, hole diameter had positive effects on all responses and coating level had positive effect on Q%6h, Q%12h and negative effect on RSQzero. Full three factor interaction (3FI) equations were used for representation of all responses except Q%2h which was represented by reduced (3FI) equation. Upon exploring the experimental space, no formula in the tested range could satisfy the required constraints. Thus, direct compression of TM cores was not suitable for formation of CPOP tablets. Preliminary trials of CPOP tablets with wet granulated cores were promising with an intact membrane for 12 h and high RSQzero. Further improvement of these formulations to optimize TM release will be done in further studies. PMID

  10. Fast Disintegrating Combination Tablet of Taste Masked Levocetrizine Dihydrochloride and Montelukast Sodium: Formulation Design, Development, and Characterization.

    PubMed

    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

  11. 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

  12. Flow cytometric determination of genome size for eight commercially important fish species in China.

    PubMed

    Zhu, Dongmei; Song, Wen; Yang, Kun; Cao, Xiaojuan; Gul, Yasmeen; Wang, Weiming

    2012-09-01

    The genome size (C value) of eight commercially important fish species in China was measured using flow cytometry. Chicken (Gallus domesticus) erythrocytes were used as reference cells. When using propidium iodide (PI) as the fluorescent dye, genome sizes were 1.09 ± 0.08, 2.75 ± 0.12, 1.05 ± 0.05, 1.35 ± 0.11, 0.99 ± 0.05, 0.90 ± 0.08, 0.90 ± 0.07, and 0.88 ± 0.07 pg for Japanese eel (Anguilla japonica), mullet (Myxocyprinus asiaticus), yellowcheek carp (Elopichthys bambusa), blunt snout bream (Megalobrama amblycephala), yellow catfish (Pelteobagrus fulvidraco), ricefield eel (Monopterus albus), mandarin fish (Siniperca chuatsi), and snakehead (Ophicephalus argus), respectively. However, genome sizes were 1.25 ± 0.00, 3.08 ± 0.02, 1.25 ± 0.00, 1.57 ± 0.01, 0.96 ± 0.01, 1.00 ± 0.01, 0.91 ± 0.01, and 0.89 ± 0.01 pg for these fishes, respectively, when 4', 6-diamidino-2-phenylindole (DAPI) was used as the fluorescent dye. Regardless of the dye used, the more evolutionarily advanced species had a smaller genome size than those with a lower evolutionary status. For each species, we also measured the size of erythrocytes and their nucleus and evaluated the relationships between erythrocyte size, nucleus size, chromosome number, and genome size. Genome size was positively correlated with erythrocyte nucleus size and chromosome number when using PI as the fluorescent dye, but it was only correlated with erythrocyte nucleus size when DAPI was used. PMID:22956044

  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. Subnuclear distribution of the entire complement of linker histone variants in Arabidopsis thaliana.

    PubMed

    Ascenzi, R; Gantt, J S

    1999-11-01

    Linker histones (e.g. H1, H5, H1 degrees ) are thought to exert control on chromatin function by restricting nucleosomal dynamics. All higher eukaryotes possess a diverse family of linker histones, which may exhibit functional specialization. Arabidopsis thaliana apparently contains a minimal complement of linker histone structural variants and therefore is an ideal model for investigating functional differentiation among linker histones. Histones H1-1 and H1-2 are relatively similar proteins that are expressed in a wide variety of tissues and make up the majority of linker histone while H1-3 is a highly divergent minor variant protein that is induced by drought stress. We are interested in determining whether the in vivo distribution of each of these proteins also differs. To this end, we have produced subtype-specific antibodies and have localized each of the three proteins at the intranuclear and DNA sequence levels by indirect immunofluorescence and immunoprecipitation, respectively. Antibodies against linker histones H1-1 and H1-2 decorate nuclei in patterns very similar to 4',6-diamidino-2-phenylindole (DAPI) staining, but different than the staining pattern of total histones. In contrast, antibodies made against two regions of H1-3 bind to chromatin in a diffuse pattern distinct from the DAPI-staining pattern. We also describe a technique to determine the localization of plant linker histone variants along regions of chromatin, employing in vivo chemical DNA-protein cross-linking to preserve native associations followed by immunoprecipitation with subtype-specific antibodies. We use this technique to demonstrate that, in contrast to the major linker histones, H1-3 does not bind the repetitive sequences pAL1 and 5S rDNA. In addition, we show that linker histones are bound to the compacted nucleosomal arrays at the telomere but with reduced stoichiometry. Taken together, our results suggest that plants, as has been shown for animals, possess a variant linker

  15. 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

  16. Biogeochemical controls on the bacterial populations in the eastern Atlantic Ocean

    NASA Astrophysics Data System (ADS)

    Neogi, S. B.; Koch, B. P.; Schmitt-Kopplin, P.; Pohl, C.; Kattner, G.; Yamasaki, S.; Lara, R. J.

    2011-12-01

    Little is known about bacterial dynamics in the oligotrophic ocean, particularly about cultivable bacteria. We examined the abundance of total and cultivable bacteria in relation to changes in biogeochemical conditions in the eastern Atlantic Ocean with special regard to Vibrio spp., a group of bacteria that can cause diseases in human and aquatic organisms. Surface, deep water and plankton (<20 μm, 20-55 μm and >55 μm) samples were collected between 50° N and 24° S. Chlorophyll-a was very low (<0.3 μg l-1) in most areas of the nutrient-poor Atlantic, except at a few locations near upwelling regions. In surface water, dissolved organic carbon (DOC) and nitrogen (DON) concentrations were 64-95 μM C and 2-10 μM N accounting for ≥90 % and ≥76 % of total organic C and N, respectively. DOC and DON gradually decreased to ~45 μM C and <5 μM N in the bottom water. In the surface layer, culture independent total bacteria and other prokaryotes represented by 4´-6-diamidino-2-phenylindole (DAPI) counts, ranged mostly between 107 and 108 cells l-1, while cultivable bacterial counts (CBC) and Vibrio spp. were found at concentrations of 104-107 and 102-105 colony forming units (CFU) l-1, respectively. Most bacteria (>99 %) were found in the nanoplankton fraction (<20 μm), however, bacterial abundance did not correlate with suspended particulates (chlorophyll-a, particulate organic C [POC] and N [PON]). Instead, we found a highly significant correlation between bacterial abundance and temperature (p < 0.001) and a significant correlation with DOC and DON (p < 0.005 and <0.01, respectively). In comparison to CBC and DAPI-stained prokaryotes, cultivable Vibrio showed a stronger and highly significant correlation with DOC and DON (p < 0.0005 and p < 0.005, respectively). In cold waters of the mesopelagic and abyssal zones, CBC was 50 to 100-times lower than in the surface layer; however, cultivable Vibrio spp. could be isolated from the bathypelagic zone and even

  17. Biogeochemical controls on the bacterial population in the eastern Atlantic Ocean

    NASA Astrophysics Data System (ADS)

    Neogi, S. B.; Koch, B. P.; Schmitt-Kopplin, P.; Pohl, C.; Kattner, G.; Yamasaki, S.; Lara, R. J.

    2011-08-01

    Little is known about bacterial dynamics in the oligotrophic ocean, particularly about its cultivable population. We examined the abundance of total and cultivable bacteria in relation to changes in biogeochemical conditions in the eastern Atlantic Ocean with special regard to Vibrio spp., a group of bacteria that can cause diseases in human and aquatic organisms. Surface, deep water and plankton samples (<20 μm, 20-55 μm and >55 μm) were collected between 50° N and 24° S. Chlorophyll-a was very low (<0.3 μg l-1) in most areas of the nutrient-poor Atlantic, except at a few locations near upwelling regions. In surface water, dissolved organic carbon (DOC) and nitrogen (DON) concentrations were 64-95 μM C and 2-10 μM N accounting for ≥90 % and ≥76 % of total organic C and N, respectively. DOC and DON gradually decreased to ~45 μM C and <5 μM N in the bottom water while dissolved inorganic nutrients (Si, P, N) increased with depth. In the surface layer, culture independent total bacteria, represented by 4´-6-diamidino-2-phenylindole (DAPI) counts, ranged mostly between 107 and 108 cells l-1, while cultivable bacterial counts (CBC) and Vibrio spp. were found at concentrations of 104-107 and 102-105 colony forming units (CFU) l-1, respectively. Most bacteria (>99 %) were found in the nanoplankton fraction (<20 μm), however, bacterial abundance did not correlate with suspended particulates (chlorophyll-a, particulate organic C and N). Instead, we found a highly significant correlation between bacterial abundance and temperature (p < 0.001) and a significant correlation with DOC and DON. Among the cultivable bacteria, the abundance of Vibrio was also highly significantly correlated with DOC and DON (p < 0.0005 and p < 0.005, respectively). In cold waters of the mid-pelagic and abyssal zones, CBC was 50 to 100-times lower than in the surface layer; however, cultivable Vibrio spp. could be isolated from the bathypelagic zone and even near the seafloor

  18. 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

  19. 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

  20. Apoptosis of U937 Cells Induced by Hematoporphyrin Monomethyl Ether-Mediated Sonodynamic Action

    PubMed Central

    Su, Xiaomin; Wang, Pan; Wang, Xiaobing; Cao, Bing; Li, Long

    2013-01-01

    Abstract Purpose The present study aims to investigate apoptosis of U937 cells induced by hematoporphyrin monomethyl ether (HMME)-mediated sonodynamic therapy (SDT). Materials HMME concentration was kept constant at 10 μg/mL. Tumor cells suspended in serum-free RPM1640 were exposed to ultrasound at 1.1 MHz for up to 60 seconds with an intensity of 1 W/cm2 in the presence and absence of HMME. The viability of cells was determined by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltertrazolium bromide tetrazolium (MTT) test. Apoptosis was analyzed using a flow cytometer with Annexin V-PE/7-ADD staining as well as fluorescence microscopy with 4′-6-diamidino-2-phenylindole (DAPI) staining. The DNA damage of U937 cells, intracellular reactive oxygen species (ROS), and mitochondria membrane potential (MMP) were also analyzed by a flow cytometer after exposures. Western blotting and reverse transcriptase–polymerase chain reaction were used to analyze the protein and mRNA expression level of caspase-3 and poly(ADP-ribose) polymerase (PARP). Results Fluorescent imaging revealed that HMME mainly localized in the mitochondria. MTT assay showed 55.6% of cell survival at 4 hours post-SDT. Flow cytometric analysis displayed a significant increase in the early- and late-apoptotic cell populations (35.6%) of U937 cells by HMME-mediated SDT. Compared with the control, ultrasound-alone, and HMME-alone groups, the intracellular ROS and the MMP loss were greatly increased in the combined SDT group. Obvious nuclear condensation was also found with DAPI staining, and the DNA fragment increased to 33.9% at 2 hours post-SDT treatment. Immunofluorescent staining indicated obvious Bax translocation after SDT. Western blot showed visible enhancement of caspase-3 and PARP cleavage. In addition, caspase-3 and PARP mRNA expression of U937 cells increased remarkably after SDT treatment. Conclusions The findings demonstrated that HMME-mediated sonodynamic action (HMME-SDT) significantly

  1. The percutaneous permeation of a combination of 0.1% octenidine dihydrochloride and 2% 2-phenoxyethanol (octenisept®) through skin of different species in vitro

    PubMed Central

    2011-01-01

    Background A water based combination of 0.1% octenidine dihydrochloride and 2% 2 - phenoxyethanol is registered in many European countries as an antiseptic solution (octenisept®) for topical treatment with high antimicrobial activity for human use, but octenidine based products have not been registered for veterinary use yet. The aim of the present study was to investigate whether octenidine dihydrochloride or 2 -phenoxyethanol, the two main components of this disinfectant, permeate through animal skin in vitro. Therefore, permeation studies were conducted using Franz-type diffusion cells. 2 ml of the test compound were applied onto 1.77 cm2 split skin of cats, dogs, cows and horses. To simulate wounded skin, cattle skin was treated with adhesive tapes 100 times, as well. Up to an incubation time of 28 hours samples of the acceptor chamber were taken and were analysed by UV-HPLC. Using the method of the external standard, the apparent permeability coefficient, the flux Jmax, and the recovery were calculated. Furthermore, the residues of both components in the skin samples were determined after completion of the diffusion experiment. Results After 28 hours no octenidine dihydrochloride was found in the receptor chamber of intact skin samples, while 2.7% of the topical applied octenidine dihydrochloride permeated through barrier disrupted cattle skin. 2 - phenoxyethanol permeated through all skin samples with the highest permeability in equine, followed by bovine, canine to feline skin. Furthermore, both components were found in the stratum corneum and the dermis of all split skin samples with different amounts in the examined species. Conclusion For 2-phenoxyethanol the systemic impact of the high absorption rate and a potential toxicological risk have to be investigated in further studies. Due to its low absorption rates through the skin, octenidine dihydrochloride is suitable for superficial skin treatment in the examined species. PMID:21835019

  2. Chromatographic analysis of 1,3-bis(dimethylamino)isopropyl 4-chlorophenoxyacetate dihydrochloride, a new CNS stimulant.

    PubMed

    Kiss, I T; Kovács-Hadady, K

    1992-01-01

    A reversed-phase liquid chromatographic method is developed for the assay of the new CNS stimulant, 1,3-bis(dimethylamino)isopropyl 4-chlorophenoxyacetate dihydrochloride (BCE-001), and its main contaminant, 4-chlorophenoxyacetic acid (PCPA). In the first place the possible reactions of BCE-001 with mobile phase components are investigated and it is found that BCE-001 is readily hydrolysed in water and undergoes transesterification in methanol or ethanol. The methyl ester is formed rapidly and quantitatively so that BCE-001 can be assayed as methyl 4-chlorophenoxyacetate. PCPA is formed as a result of the hydrolysis of BCE-001 during the sample pretreatment and chromatographic separation and this causes an overestimate of the PCPA impurity in the bulk drug. An effective method is developed to prevent the hydrolysis of BCE-001 during sample pretreatment. PMID:1391102

  3. NTP Toxicology and Carcinogenesis Studies of 3,3'-Dimethoxybenzidine Dihydrochloride (CAS No. 20325-40-0) in F344/N Rats (Drinking Water Studies).

    PubMed

    1990-01-01

    3,3'-Dimethoxybenzidine dihydrochloride is an off-white powder with a melting point of 274 degrees C. 3,3'-Dimethoxybenzidine is used principally as an intermediate in the production of commercial bisazobiphenyl dyes for coloring textiles, paper, plastic, rubber, and leather. In the synthesis of the bisazobiphenyl dyes, the amine groups of 3,3'-dimethoxybenzidine are chemically linked with other aromatic amines. A small quantity of 3,3'-dimethoxybenzidine is also used as an intermediate in the production of o-dianisidine diisocyanate, which is used in isocyanate-based adhesive systems and as a component of polyurethane elastomers. 3,3'-Dimethoxybenzidine dihydrochloride was evaluated in toxicity and carcinogenicity studies as part of the National Toxicology Program's Benzidine Dye Initiative. This Initiative was designed to evaluate the representative benzidine congeners and benzidine congener-derived and benzidine-derived dyes. 3,3'-Dimethoxybenzidine dihydrochloride was nominated for study because of the potential for human exposure during production of bisazobiphenyl dyes and because benzidine, a structurally related chemical, is a known human carcinogen. NTP Toxicology and Carcinogenesis studies were conducted by administering 3,3'-dimethoxybenzidine dihydrochloride (greater than 97.5% pure) in drinking water to groups of F344/N rats of each sex for 14 days, 13 weeks, 9 months, or 21-months. The 21-month studies were intended to last 24 months but were terminated early because of rapidly declining survival due to neoplasia. Studies were performed only in rats because similar studies are being performed in mice at the National Center for Toxicology Research. Genetic toxicology studies were conducted with Salmonella typhimurium, Chinese hamster over (CHO) cells, and Drosophila melanogaster. Fourteen-Day Studies: All rats receiving drinking water concentrations up to 4,500 ppm lived to the end of the studies. Rats that received water containing 4,500 ppm 3

  4. Combination of adhesive-tape-based sampling and fluorescence in situ hybridization for rapid detection of Salmonella on fresh produce.

    PubMed

    Bisha, Bledar; Brehm-Stecher, Byron F

    2010-01-01

    This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55°C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (≤ 500 μL) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37°C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports

  5. Inducing cell cycle arrest and apoptosis by dimercaptosuccinic acid modified Fe3O4 magnetic nanoparticles combined with nontoxic concentration of bortezomib and gambogic acid in RPMI-8226 cells

    PubMed Central

    Zhang, Wei; Qiao, Lixing; Wang, Xinchao; Senthilkumar, Ravichandran; Wang, Fei; Chen, Baoan

    2015-01-01

    The purpose of this study was to determine the potential benefits of combination therapy using dimercaptosuccinic acid modified iron oxide (DMSA-Fe3O4) magnetic nanoparticles (MNPs) combined with nontoxic concentration of bortezomib (BTZ) and gambogic acid (GA) on multiple myeloma (MM) RPMI-8226 cells and possible underlying mechanisms. The effects of BTZ-GA-loaded MNP-Fe3O4 (BTZ-GA/MNPs) on cell proliferation were assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,4,-diphenyltetrazolium bromide (MTT) method. Cell cycle and apoptosis were detected using the terminal deoxyribonucleotidyl transferase (TdT)-mediated biotin-16-dUTP nick-end labeling (TUNEL) assay and flow cytometry (FCM). Furthermore, DMSA-Fe3O4 MNPs were characterized in terms of distribution, apoptotic morphology, and cellular uptake by transmission electron microscopy (TEM) and 4,6-diamidino-2-phenylindole (DAPI) staining. Subsequently, the effect of BTZ-GA/MNPs combination on PI3K/Akt activation and apoptotic-related protein were appraised by Western blotting. MTT assay and hematoxylin and eosin (HE) staining were applied to elevate the functions of BTZ-GA/MNPs combination on the tumor xenograft model and tumor necrosis. The results of this study revealed that the majority of MNPs were quasi-spherical and the MNPs taken up by cells were located in the endosome vesicles of cytoplasm. Nontoxic concentration of BTZ-GA/MNPs increased G2/M phase cell cycle arrest and induced apoptosis in RPMI-8226 cells. Furthermore, the combination of BTZ-GA/MNPs activated phosphorylated Akt levels, Caspase-3, and Bax expression, and down-regulated the PI3K and Bcl-2 levels significantly. Meanwhile, the in vivo tumor xenograft model indicated that the treatment of BTZ-GA/MNPs decreased the tumor growth and volume and induced cell apoptosis and necrosis. These findings suggest that chemotherapeutic agents polymerized MNPs-Fe3O4 with GA could serve as a better alternative for targeted therapeutic approaches to treat multiple

  6. 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.

  7. Isolation of the MAT1-1 mating type idiomorph and evidence for selfing in the Chinese medicinal fungus Ophiocordyceps sinensis.

    PubMed

    Bushley, Kathryn E; Li, Yi; Wang, Wen-Jing; Wang, Xiao-Liang; Jiao, Lei; Spatafora, Joseph W; Yao, Yi-Jian

    2013-09-01

    Ophiocordyceps sinensis is one of the most valued medicinal fungi in China. Research on the mating system and sexual development is vitally important to this endangered species. Previous efforts devoted to investigate the mating type (MAT) locus of O. sinensis, however, resulted in an incomplete understanding. In this study, the MAT1-1 locus of O. sinensis was investigated. The conserved α-box and HMG-box regions of the MAT1-1-1 and MAT1-1-3 genes, respectively, and a conserved region of the DNA lyase gene were successfully amplified using degenerate PCR. A combination of TAIL-PCR and long-range PCR were used to connect these genes and obtain the sequence of the MAT1-1 locus. Screening of 22 single spore isolates by PCR demonstrated that both the MAT1-1-1 and MAT1-2-1 genes cooccurred within the same isolate. Additionally, both MAT1-1-1 and MAT1-2-1 are expressed in vegetative mycelia, providing evidence that O. sinensis is likely capable of selfing. DAPI (4,6-diamidino-2-phenylindole) staining of ascospores and hyphae showed that a majority of hyphal compartments are binucleate, suggesting that O. sinensis may be pseudohomothallic. Analyses of sequence diversity showed lower levels of genetic diversity in MAT1-1-1 compared to MAT1-2-1, indicating the possibility that different selective pressures act on the two MAT idiomorphs. The MAT1-1-1 sequences of O. sinensis and Tolypocladium inflatum cluster as a monophyletic group consistent with phylogenetic classification of Ophiocordycipitaceae. Comparison of the structure of the MAT1-1 locus across hypocrealean taxa showed that O. sinensis contains all three mating type genes (MAT1-1-1, MAT1-1-2, and MAT1-1-3) and supported previous observations that of the four families in Hypocreales, MAT1-1-3 has undergone a lineage specific loss only in some members of the Cordycipitaceae. PMID:24012300

  8. 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

  9. Repositioning: the fast track to new anti-malarial medicines?

    PubMed Central

    2014-01-01

    Background Repositioning of existing drugs has been suggested as a fast track for developing new anti-malarial agents. The compound libraries of GlaxoSmithKline (GSK), Pfizer and AstraZeneca (AZ) comprising drugs that have undergone clinical studies in other therapeutic areas, but not achieved approval, and a set of US Food and Drug Administration (FDA)-approved drugs and other bio-actives were tested against Plasmodium falciparum blood stages. Methods Molecules were tested initially against erythrocytic co-cultures of P. falciparum to measure proliferation inhibition using one of the following methods: SYBR®I dye DNA staining assay (3D7, K1 or NF54 strains); [3H] hypoxanthine radioisotope incorporation assay (3D7 and 3D7A strain); or 4’,6-diamidino-2-phenylindole (DAPI) DNA imaging assay (3D7 and Dd2 strains). After review of the available clinical pharmacokinetic and safety data, selected compounds with low μM activity and a suitable clinical profile were tested in vivo either in a Plasmodium berghei four-day test or in the P. falciparum Pf3D70087/N9 huSCID ‘humanized’ mouse model. Results Of the compounds included in the GSK and Pfizer sets, 3.8% (9/238) had relevant in vitro anti-malarial activity while 6/100 compounds from the AZ candidate drug library were active. In comparison, around 0.6% (24/3,800) of the FDA-approved drugs and other bio-actives were active. After evaluation of available clinical data, four investigational drugs, active in vitro were tested in the P. falciparum humanized mouse model: UK-112,214 (PAF-H1 inhibitor), CEP-701 (protein kinase inhibitor), CEP-1347 (protein kinase inhibitor), and PSC-833 (p-glycoprotein inhibitor). Only UK-112,214 showed significant efficacy against P. falciparum in vivo, although at high doses (ED90 131.3 mg/kg [95% CI 112.3, 156.7]), and parasitaemia was still present 96 hours after treatment commencement. Of the six actives from the AZ library, two compounds (AZ-1 and AZ-3) were marginally

  10. 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.