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

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

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

    1997-01-01

    Counting bacteria in drinking water samples by the epifluorescence technique after 4',6-diamidino-2-phenylindole (DAPI) staining is complicated by the fact that bacterial fluorescence varies with exposure of the cells to sodium hypochlorite. An Escherichia coli laboratory-grown suspension treated with sodium hypochlorite (5 to 15 mg of chlorine liter-1) for 90 min was highly fluorescent after DAPI staining probably due to cell membrane permeation and better and DAPI diffusion. At chlorine con...

  2. 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.

    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

  3. Binding of Hoechst 33258 and 4',6-diamidino-2-phenylindole to self-complementary decadeoxynucleotides with modified exocyclic base substituents

    Fluorescence titrations have been carried out to determine the association constants (Ka) for binding of the dyes Hoechst 33258 and DAPI to the self-complementary decamer d(CTGAATTCAG) and nine duplex derivatives with exocyclic substituent changes in the six central base pairs. Many Ka values are in the range (2-5) x 108 (duplex M)-1 at 5.5 degree C. Replacement of the leftmost adenine by 2-aminopurine in the sequence decreases Ka for Hoechst 33258 by a factor of 170. When the centermost adenine is replaced by 2-aminopurine, Ka for Hoechst 33258 and DAPI is too small to be evaluated. When the centermost adenine is replaced by purine, Ka for both dyes increases, but this very stable duplex-Hoechst 33258 complex is nonfluorescent. The measured affinities are compared to expectations derived from X-ray studies with dodecamer-dye complexes having an identical central binding sequence

  4. Presence of a Phytoplasma Associated with Witches’-Broom Disease in Ugni molinae Turcz. and Gaultheria phillyreifolia (Pers.) Sleumer Determined by DAPI, PCR, and DNA Sequencing Presencia de un Fitoplasma Asociado a la Enfermedad de "Escoba de Bruja" en Ugni molinae Turcz. y Gaultheria phillyreifolia (Pers.) Sleumer Determinado Mediante DAPI, PCR y Secuenciación de ADN

    Nolberto Arismendi S; Nancy Andrade S; Ricardo Riegel Sch; Roberto Carrillo Ll.

    2010-01-01

    Murta (Ugni molinae Turcz.) and common chaura (Gaultheria phillyreifolia (Pers.) Sleumer) are native species of Chile. Plants of both species have shown over-branching like witches' broom. The causal agents of these symptoms in many plants are phytoplasma. To verify the presence of these microorganisms, DAPI (4',6-diamidino-2-phenylindole) staining analysis and polymerase chain reaction (PCR) were performed in symptomatic and asymptomatic plants. Positive PCR samples were sequenced to identif...

  5. Mouse Karyotype Obtained by Combining DAPI Staining with Image Analysis

    2006-01-01

    In this study, mitotic metaphase chromosomes in mouse were identified by a new chromosome fluorescence banding technique combining DAPI staining with image analysis. Clear 4', 6-diamidino-2-phenylindole (DAPI) multiple bands like G-bands could be produced in mouse. The MetaMorph software was then used to generate linescans of pixel intensity for the banded chromosomes from short arm to long arm. These linescans were sufficient not only to identify each individual chromosome but also analyze the physical sites of bands in chromosome. Based on the results, the clear and accurate karyotype of mouse metaphase chromosomes was established. The technique is therefore considered to be a new method for cytological studies of mouse.

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

    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.

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

    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.

  9. Flow cytometry techniques in radiation biology

    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 postirradiation syndrome, we have 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 monoclonal 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 (DAPI) exclusion to identify viable cells, it was possible to follow regeneration of postirradiated rat marrow HSC

  10. Presence of a Phytoplasma Associated with Witches’-Broom Disease in Ugni molinae Turcz. and Gaultheria phillyreifolia (Pers. Sleumer Determined by DAPI, PCR, and DNA Sequencing Presencia de un Fitoplasma Asociado a la Enfermedad de "Escoba de Bruja" en Ugni molinae Turcz. y Gaultheria phillyreifolia (Pers. Sleumer Determinado Mediante DAPI, PCR y Secuenciación de ADN

    Nolberto Arismendi S

    2010-03-01

    Full Text Available Murta (Ugni molinae Turcz. and common chaura (Gaultheria phillyreifolia (Pers. Sleumer are native species of Chile. Plants of both species have shown over-branching like witches' broom. The causal agents of these symptoms in many plants are phytoplasma. To verify the presence of these microorganisms, DAPI (4',6-diamidino-2-phenylindole staining analysis and polymerase chain reaction (PCR were performed in symptomatic and asymptomatic plants. Positive PCR samples were sequenced to identify the pathogens involved. In individuals of both species with witches’ broom symptoms, DAPI staining showed fluorescent bodies in the phloem tissues, but not in asymptomatic plants. Verification by nested-PCR, phytoplasmatic DNA was amplified from diseased murta and chaura, but not in apparently healthy plants. Sequencing of amplified products allowed locating phytoplasma within the ash yellows group (16SrVII and related to Candidatus phytoplasma fraxini. This is the first report of phytoplasma in Chilean native species. Considering the diversity of plant species infected by the ash yellows group suggests that G. phillyreifolia and U. molinae could be a phytoplasma reservoir for other economically important agricultural crops.La murta (Ugni molinae Turcz. y la chaura común (Gaultheria phillyreifolia (Pers. Sleumer son especies nativas de Chile. En plantas de ambas especies se ha observado una sobre-ramificación de tipo "escoba de bruja". En muchas plantas los agentes causales de esta sintomatología son fitoplasmas. Para verificar la presencia de estos microorganismos se analizaron plantas con y sin síntomas mediante tinciones DAPI (4’,6-diamidino-2-fenilindol y reacción en cadena de la polimerasa (PCR. Muestras positivas en la PCR fueron secuenciadas para identificar al fitopatógeno implicado. En individuos de ambas especies con síntomas de escoba de bruja, la tinción DAPI permitió observar cuerpos fluorescentes en los tejidos del floema, situaci

  11. Karyological observation on Saccharina japonica chromosomes stained with DAPI%海带染色体的DAPI染色及核型初步分析

    刘宇; 毕燕会; 周志刚

    2012-01-01

    Saccharina japonica( Aresch. )C. E. Lane,C. Mayes et G. W. Saunders( = Laminaria japonica Aresch. ) (Phaeophyta) is an important economic seaweed in China. There is a distinct outcome about the chromosome number in this kelp due to the tiny size of these chromosomes. The karyotypes and chromosomes of S. Japonica were observed after a series of treatments including pretreatment with 0. 2% colchicines for about 10 h, Carnoy' s fixative, and mixture of enzymes prior to dropping from 30 cm height overhead glass slides for spreading the surface coat. The prepared chromosomes were stained with 4',6-diamidino-2-phenylindole ( DAPI), a fluorescent probe sensitive and specific to DNA. The chromosome numbers of the haploid male and female gametophytes were 31 respectively,and there were 62 in diploid sporophytes. Most of the chromosomes were either droplet or short bacilliform. In the meantime, the female gametophyte chromosomes were between 0. 78 μm and 2. 61 μm in size, larger than the males that were between 0.57 |xm and 2.17 μm. Based upon the relative size of chromosome, the karyotypes of the female or male gametophyte chromosomes were primarily analyzed. All the results laid a solid foundation for a basic technique for localization of molecular markers on kelp chromosomes.%鉴于海带染色体比较小且数目存在分歧等原因,利用0.2%秋水仙素对海带配子体及孢子体处理10 h左右,经过卡诺试剂固定、多种酶组合处理及30 cm的高位滴片,可以获得质量比较高的海带染色体;使用灵敏度高、特异性强的DNA荧光染料DAPI进行染色,结果显示,海带雌、雄配子体的染色体各为31条,孢子体染色体为62条,大多为短杆状或者点状;雌配子体染色体的大小为0.78~2.61 μm,稍大于雄配子体(大小为0.57~2.17 μm).根据染色体的大小,对海带配子体的染色体核型进行了初步分析.这些结果为分子标记的染色体定位等细胞学研究奠定了技术基础.

  12. New Method for Counting Bacteria Associated with Coral Mucus ▿ †

    Garren, Melissa; Azam, Farooq

    2010-01-01

    The ability to count bacteria associated with reef-building corals in a rapid, reliable, and cost-effective manner has been hindered by the viscous and highly autofluorescent nature of the coral mucus layer (CML) in which they live. We present a new method that disperses bacterial cells by trypsinization prior to 4′,6-diamidino-2-phenylindole (DAPI) staining and quantification by epifluorescence microscopy. We sampled seawater and coral mucus from Porites lobata from 6 reef sites influenced b...

  13. Molecular cytogenetic characterization of some representatives of the subgenera Artemisia and Absinthium (genus Artemisia, Asteraceae)

    Vallès, J.; Siljak-Yakovlev, S.; Hidalgo, O.; Garnatje, T.; Garcia, S.; Pellicer, J.

    2008-01-01

    A molecular cytogenetic study has been performed in three species of the genus Artemisia, complementing previous works on two subgenera that had been scarcely studied from this standpoint, Artemisia ( A. chamaemelifolia, A. vulgaris) and Absinthium ( A. absinthium). Chromomycin A3 and 4',6-diamidino-2-phenylindole (DAPI) banding have been carried out, as well as fluorescent in situ hybridization (...

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

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

    2015-01-01

    The aim of this study was to observe the effects of bone marrow mesenchymal stem-cell transplantation (BMSCs) in repairing acute spinal cord damage in rats and to examine the potential beneficial effects. 192 Wistar rats were randomized into 8 groups. Spinal cord injury was created. Behavior and limb functions were scored. Repairing effects of BMSCs transplantation was evaluated and compared. In vitro 4’,6-diamidino-2-phenylindole (DAPI)-tagged BMSCs were observed, and whether they migrated t...

  15. Significance of Size and Nucleic Acid Content Heterogeneity as Measured by Flow Cytometry in Natural Planktonic Bacteria

    Gasol, Josep M.; Zweifel, Ulla Li; Peters, Francesc; Fuhrman, Jed A.; Hagström, Åke

    1999-01-01

    Total bacterial abundances estimated with different epifluorescence microscopy methods (4′,6-diamidino-2-phenylindole [DAPI], SYBR Green, and Live/Dead) and with flow cytometry (Syto13) showed good correspondence throughout two microcosm experiments with coastal Mediterranean water. In the Syto13-stained samples we could differentiate bacteria with apparent high DNA (HDNA) content and bacteria with apparent low DNA (LDNA) content. HDNA bacteria, “live” bacteria (determined as such with the Mo...

  16. Naringin Protects against Rotenone-induced Apoptosis in Human Neuroblastoma SH-SY5Y Cells

    KIM, HAK-JAE; Song, Jeong Yoon; PARK, HAE JEONG; Park, Hyun-Kyung; Yun, Dong Hwan; CHUNG, JOO-HO

    2009-01-01

    Rotenone, a mitochondrial complex I inhibitor, can induce the pathological features of Parkinson's disease (PD). In the present study, naringin, a grapefruit flavonoid, inhibited rotenone-induced cell death in human neuroblastoma SH-SY5Y cells. We assessed cell death and apoptosis by measuring mitogen-activated protein kinase (MAPKs) and caspase (CASPs) activities and by performing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 4,6-diamidino-2-phenylindole (DAPI) st...

  17. Ligand-induced formation of nucleic acid triple helices.

    Pilch, D S; Breslauer, K J

    1994-01-01

    We demonstrate that ligand binding can be used to induce the formation of triplex structures that would not otherwise form. Specifically, we show that binding of berenil or 4',6-diamidino-2-phenylindole DAPI) induces formation of the poly(rA).poly(rA).poly(dT) triplex, providing an example of an RNA(purine).RNA(purine).DNA(pyrimidine) triplex. We also show that binding of berenil, DAPI, ethidium, or netropsin can induce formation of the poly(dT).poly(rA).poly(dT) triplex, thereby overcoming a...

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

    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

  19. Effects of Ultrasound on the Survival and Characteristics of Cryptosporidium Oocysts and Giardia Cysts

    Al-Sabi, Mohammad Nafi Solaiman; Gad, J.; Klinting, M.;

    2011-01-01

    power and time durations on two of the most common waterborne protozoa Cryptosporidium and Giardia, and examined its effects on parasite characteristics and survival rate using immunofluorescence dyes; DAPI (4’,6-diamidino-2-phenylindol) staining/PI (propidium iodide), and analyzed by flow cytometry....... Results: The results showed that exposure of Cryptosporidium oocysts and Giardia cysts to ultrasound could reduce their survival rate even if the exposure time was limited to a few seconds. When sonication time was extended, changes in parasite characteristics became clearly visible. Several other factors...

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

    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 bre...

  1. Cationic Antimicrobial Peptide Cytotoxicity

    Laverty, Garry; Gilmore, Brendan

    2014-01-01

    Fluorescence microscopy serves as a valuable tool for assessing the structural integrity and viability of eukaryotic cells. Through the use of calcein AM and the DNA stain 4,6-diamidino-2 phenylindole (DAPI), cell viability and membrane integrity can be qualified. Our group has previously shown the ultra-short cationic antimicrobial peptide H-OOWW-NH2; the amphibian derived 27-mer peptide Maximin-4and the ultra-short lipopeptide C12-OOWW-NH2 to be effective against a range of bacterial biofil...

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

    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

  3. The Antiapoptic Effects of Hominis Placenta Extract

    Jung-Chul Seo

    2001-02-01

    Full Text Available Purpose. Free radicals are implicated in the pathophysiology of aging, ischemic injury and neurodegenerative disorders. To deform]no whether Hominis Placenta extract prevents H2O2-induced apoptosis, we have performed morphological and biochemical analyses for the detection of apoptotic phenomena in the pineal tumor cell line PGT-beta We have also peformed cytochemical and immunocytochemical analyses for the detection of changes in nitric oxide synthase (NOS activity and estimated the expression . of apoptotic genes using reverse transcription-polymerase chain reaction (RT-PCR Methods. PGT-beta cells were pretreated with Hominis Placenta extracts (0,10-2/ug/ml for 2 hours and then exposed to H2O2 (0, 50uM for 3 hours. Appearance of apoptotic characteristics were monitored using 4, 6-diamidino-2-phenylindole dihydrochloride (DAPI staining assay, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL assay and flow cytometric analysis. NOS activity was measured by NADPH-diaphorase cytochemistry. Expression of inducible NOS (iNOS and nuclear factor kappa B (NF k B was assessed via immunocytochemistry. The expression of apoptotic genes was examined by RT-PCR. Results. After 3 flours of exposure to H2O2, it was shown that PGT-beta cells treated with H2O2(50 uM exhibit classical apoptotic features and increases in NOS activity and caspase-3 expression. Treatment with Hominis Placenta extract resulted in a reduced occurrence of apoptotic features. DAPI staining, TUNEL and flow cytometric assays revealed decreases in the occurrence of nuclear fragmentation and in the sub-Gl fraction in the PGT-beta cells treated with Hominis Placenta extract. Cells treated with Hominis Placenta extract also showed lower activity of NADPH-diaphorase and immunoreactivities of both iNOS and NF k B than those of H2O2-treated cells which were not treated with Hominis Placenta extract. By RT-PCR, it was shown that the level of caspase-3 m

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

    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

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

    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

  6. Influence of in vitro supplementation with lipids from conventional and Alpine milk on fatty acid distribution and cell growth of HT-29 cells

    Dänicke Sven

    2011-08-01

    Full Text Available Abstract Background To date, the influence of milk and dairy products on carcinogenesis remains controversial. However, lipids of ruminant origin such as conjugated linoleic acids (CLA are known to exhibit beneficial effects in vitro and in vivo. The aim of the present study was to determine the influence of milk lipids of different origin and varying quality presenting as free fatty acid (FFA solutions on cellular fatty acid distribution, cellular viability, and growth of human colon adenocarcinoma cells (HT-29. Methods FAME of conventional and Alpine milk lipids (MLcon, MLalp and cells treated with FFA derivatives of milk lipids were analyzed by means of GC-FID and Ag+-HPLC. Cellular viability and growth of the cells were determined by means of CellTiter-Blue®-assay and DAPI-assay (4',6-diamidino-2-phenylindole dihydrochloride, respectively. Results Supplementation with milk lipids significantly decreased viability and growth of HT-29 cells in a dose- and time-dependent manner. MLalp showed a lower SFA/MUFA ratio, a 8 fold increased CLA content, and different CLA profile compared to MLcon but did not demonstrate additional growth-inhibitory effects. In addition, total concentration and fatty acid distribution of cellular lipids were altered. In particular, treatment of the cells yielded highest amounts of two types of milk specific major fatty acids (μg FA/mg cellular protein after 8 h of incubation compared to 24 h; 200 μM of MLcon (C16:0, 206 ± 43, 200 μM of MLalp (C18:1 c9, (223 ± 19. Vaccenic acid (C18:1 t11 contained in milk lipids was converted to c9,t11-CLA in HT-29 cells. Notably, the ratio of t11,c13-CLA/t7,c9-CLA, a criterion for pasture feeding of the cows, was significantly changed after incubation for 8 h with lipids from MLalp (3.6 - 4.8, compared to lipids from MLcon (0.3 - 0.6. Conclusions Natural lipids from conventional and Alpine milk showed similar growth inhibitory effects. However, different changes in cellular

  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.

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

    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

  9. The microflora of rainbow trout intestine : a comparison of traditional and molecular identification

    Spanggaard, Bettina; Huber, I.; Nielsen, J.;

    2000-01-01

    The culturability of the intestinal microflora of 48 rainbow trout was detected by comparing direct microscopic counts (4',6-diamidino-2-phenylindole, DAPI) with plate counts (tryptone soya agar, TSA). In general, a high percentage (average 50%) of the microflora could be cultured. The counts...... of the intestinal microflora varied 3-5 log units between fish within the same sampling point. A total of 504 bacteria were identified by physiologic criteria and 153 strains also by partial sequencing of the 16S rRNA gene. High agreement was found between classical and molecular identification. The dominant...... intestinal microflora was identified as bacteria belonging to the gamma subclass of Proteobacteria (of the genera Citrobacter, Aeromonas and Pseudomonas), to the Gram- positive bacteria with low G + C-content (of the genus Carnobacterium) and as bacteria belonging to the beta subclass of Proteobacteria...

  10. ANTICANCER ACTIVITY OF OSCILLATORIA TEREBRIFORMIS CYANOBACTERIA IN HUMAN LUNG CANCER CELL LINE A549

    S.Mukund

    2014-04-01

    Full Text Available Purpose: To evaluate the anti-cancer properties of the cyanobacterial extract of Oscillatoria terebriformis Methods: The extract was tested in Human lung cancer cell lines and examined for its effect on cell viability, nuclear morphology and sub-G1 formation. Cell viability was determined by micro culture tetrazolium technique (MTT, nuclear morphology investigated using 4’-6-diamidino-2-phenylindole (DAPI staining technique, and apoptosis assay using DNA fragmentation. Results: The results showed decreasing cell viability in a concentration-dependent manner. Altered cell morphology after treatment with the extract demonstrated that cells experienced apoptosis. Conclusion: The data demonstrate that Oscillatoria Terebriformis extract induced apoptosis in Human lung cancer A549 cells, and therefore, has a potential as an anti-cancer agent.

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

    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. Active bacteria (CTC+) in temperate lakes: temporal and cross-system variations

    Søndergaard, Morten; Danielsen, M.

    2001-01-01

    The temporal variation in the abundance and proportion of highly respiration-active bacteria in the eutrophic lakes Esrum and Frederiksborg Slotssø was determined with the redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC). In addition, a comparative late summer study was undertaken across a...... gradient of nutrient enrichment in Danish lakes. The purpose was to investigate the importance of substrate (chlorophyll) and temperature for the control of CTC-active cells (CTC+). The abundance of CTC+ cells was much lower and more variable than the total number of cells counted after 4',6-diamidino-2......-phenylindole (DAPI) staining. The proportion of CTC+ cells in Lake Esrum and Frederiksborg Slotssø was normally <5%, and between 2.5 and 20% in 14 other lakes. The abundance as well as the proportion of CTC+ cells increased with chlorophyll in Lake Esrum and Frederiksborg Slotssø, and chlorophyll explained 43...

  13. The microflora of rainbow trout intestine : a comparison of traditional and molecular identification

    Spanggaard, Bettina; Huber, I.; Nielsen, J.; Nielsen, T.; Appel, K.F.; Gram, Lone

    The culturability of the intestinal microflora of 48 rainbow trout was detected by comparing direct microscopic counts (4',6-diamidino-2-phenylindole, DAPI) with plate counts (tryptone soya agar, TSA). In general, a high percentage (average 50%) of the microflora could be cultured. The counts of...... the intestinal microflora varied 3-5 log units between fish within the same sampling point. A total of 504 bacteria were identified by physiologic criteria and 153 strains also by partial sequencing of the 16S rRNA gene. High agreement was found between classical and molecular identification. The...... dominant intestinal microflora was identified as bacteria belonging to the gamma subclass of Proteobacteria (of the genera Citrobacter, Aeromonas and Pseudomonas), to the Gram- positive bacteria with low G + C-content (of the genus Carnobacterium) and as bacteria belonging to the beta subclass of...

  14. Octenidine dihydrochloride: chemical characteristics and antimicrobial properties.

    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

  15. 21 CFR 522.1362 - Melarsomine dihydrochloride for injection.

    2010-04-01

    ... deep intramuscular injection in the lumbar muscles (L3-L5). Use a 23 gauge 1 inch needle for dogs less... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Melarsomine dihydrochloride for injection. 522... ANIMAL DRUGS § 522.1362 Melarsomine dihydrochloride for injection. (a) Specifications. The drug...

  16. Ternary mutual diffusion in aqueous (ethambutol dihydrochloride + hydrochloric acid) solutions

    Highlights: • Ternary diffusion coefficients for aqueous system ethambutol dihydrochloride and hydrochloric acid. • Diffusion of ethambutol dihydrochloride driven by hydrochloric acid gradients. • Coupled diffusion as indicated by cross-diffusion coefficients. - Abstract: Ternary mutual diffusion coefficients measured by the Taylor dispersion method are reported for aqueous solutions of {ethambutol dihydrochloride (1) + HCl (2)} at 25 °C and various carrier solution compositions. Mutual diffusion coefficients estimated from limiting ionic conductivities using Nernst equations are used to discuss the composition dependence of the measured diffusion coefficients. 1H NMR studies, combined with DFT calculations, confirm a fully extended conformation for the diprotonated form of the drug present under these conditions, and are consistent with an electrostatic mechanism for the strongly coupled diffusion of diprotonated ethambutol and HCl

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

    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

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

    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

  19. Simulation Analysis of Dilution Crystallization of Spectinomycin Dihydrochloride Pentahydrate

    鲍颖; 王静康; 王永莉

    2005-01-01

    A mathematical model for dilution crystallization of spectinomycin dihydrochloride pentahydrate was established on the basis of the population and mass balance. Three operating modes, i.e., constant mass rate addition of diluent, constant mass fraction of diluent and constant size-independent growth rate, were investigated over a wide range of controlled parameters. The intrinsic characteristics of the crystallization process and the influence of operation parameters were analyzed in detail. Suitable strategy for better performance was suggested.

  20. Chromatin and Cell Wall Staining of Schizosaccharomyces pombe.

    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

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

    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

  2. Effect of post-traumatic mild hypothermia on hippocampal cell death after traumatic brain injury in rats.

    Jia, Feng; Mao, Qing; Liang, Yu-Min; Jiang, Ji-Yao

    2009-02-11

    In this investigation, we evaluated the effect of post-traumatic mild hypothermia on cell death in the hippocampus after fluid percussion traumatic brain injury (TBI) in rats. Adult male Sprague-Dawley rats were randomly divided into three groups (n = 40/group): TBI with hypothermia treatment (32 degrees C), TBI with normothermia (37 degrees C), and sham injury. The TBI model was induced by a fluid percussion TBI device. Mild hypothermia (32 degrees C) was achieved by partial immersion in a water bath (0 degrees C) under general anesthesia for 4h. All rats were killed at 24 or 72h after TBI. The ipsilateral hippocampal CA1 in all rats were analyzed by hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling (TUNEL), and 4',6-diamidino-2-phenylindole (DAPI) staining for determining cell death. Caspase-3 expression was examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. At 24h, based on TUNEL and DAPI results, the cell death index was 28.80 +/- 2.60% and 32.10 +/- 1.40% in the normothermia TBI group, while reaching only 14.30 +/- 2.70% and 18.40 +/- 2.10% in the hypothermic TBI group (p percussion injury. Taken together with other studies, these observations support the premise that post-traumatic mild hypothermia can provide cerebral protection for patients with TBI. PMID:19236165

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

    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

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

    Oldenburg Delene J

    2007-03-01

    Full Text Available Abstract 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.

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

    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

  6. Karyotype characterization of Trigona fulviventris Guérin, 1835 (Hymenoptera, Meliponini by C banding and fluorochrome staining: report of a new chromosome number in the genus

    Domingues Alayne Magalhães Trindade

    2005-01-01

    Full Text Available Although many species of the genus Trigona have been taxonomically described, cytogenetic studies of these species are still rare. The aim of the present study was to obtain cytogenetic data by conventional staining, C banding and fluorochrome staining for the karyotype characterization of the species Trigona fulviventris. Cytogenetic analysis revealed that this species possesses a diploid chromosome number of 2n = 32, different from most other species of this genus studied so far. This variation was probably due to the centric fusion in a higher numbered ancestral karyotype, this fusion producing the large metacentric chromosome pair and the lower chromosome number observed in Trigona fulviventris. Heterochromatin was detected in the pericentromeric region of the first chromosome pair and in one of the arms of the remaining pairs. Base-specific fluorochrome staining with 4'-6-diamidino-2-phenylindole (DAPI showed that the heterochromatin was rich in AT base pairs (DAPI+ except for pair 13, which was chromomycin A3 (CMA3 positive indicating an excess of GC base pairs. Our data also suggests that there was variation in heterochromatin base composition.

  7. Human Bone Marrow Mesenchymal Stem Cell Behaviors on PCL/Gelatin Nanofibrous Scaffolds Modified with A Collagen IV-Derived RGD-Containing Peptide

    Ali Mota

    2014-03-01

    Full Text Available Objective: We introduce an RGD (Arg-Gly-Asp-containing peptide of collagen IV origin that possesses potent cell adhesion and proliferation properties. Materials and Methods: In this experimental study, the peptide was immobilized on an electrospun nanofibrous polycaprolactone/gelatin (PCL/Gel hybrid scaffold by a chemical bonding approach to improve cell adhesion properties of the scaffold. An iodine-modified phenylalanine was introduced in the peptide to track the immobilization process. Native and modified scaffolds were characterized with scanning electron microscopy (SEM and fourier transform infrared spectroscopy (FTIR. We studied the osteogenic and adipogenic differentiation potential of human bone marrow-derived mesenchymal stem cells (hBMSCs. In addition, cell adhesion and proliferation behaviors of hBMSCs on native and peptide modified scaffolds were evaluated by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay and 4',6-diamidino-2-phenylindole (DAPI staining, and the results compared with tissue culture plate, as the control. Results: FTIR results showed that the peptide successfully immobilized on the scaffold. MTT assay and DAPI staining results indicated that peptide immobilization had a dramatic effect on cell adhesion and proliferation. Conclusion: This peptide modified nanofibrous scaffold can be a promising biomaterial for tissue engineering and regenerative medicine with the use of hBMSCs.

  8. Detection and Treatment of Mycoplasma Contamination in Cultured Cells

    Hsuan Jung

    2003-04-01

    Full Text Available Background: Mycoplasmas, the smallest and simplest prokaryotes that reside in endosomesof mammalian cells, are widespread contaminants found in cell cultures.About 30% of all cell cultures, varying from 15 to 80%, are reportedlycontaminated with mycoplasmas. Here, we present our experience in successfullydetecting and treating mycoplasmal infection in various cell lines.Methods: The nested polymerase chain reaction (PCR detection and microscopicexamination, including phase-contrast, fluorescent, as well as differentialinterference contrast, were used for detecting potential mycoplasma contaminationof cell lines used in our laboratory. As soon as mycoplasma was identified,antibiotic treatment was initiated.Results: Mycoplasmal contamination was detected in six of 15 cell lines using thenested PCR amplification of mycoplasma DNA, which was further demonstratedusing 4, 6-Diamidino-2-phenylindole (DAPI staining and fluorescentmicroscopy. Alternate treatment with two antibiotics, macrolide (tiamulinand tetracycline (minocycline, effectively eliminated mycoplasma, whichwas validated by both PCR and microscopic studies.Conclusions: The nested PCR using genomic DNA extracted from cultured cells as templatesis a rapid and sensitive method for detecting mycoplasma contamination.Treatment with combined antibiotics can completely eradicatemycoplasmal infection from cultured cells. For the ease of use, PCR and/orDAPI staining appear suitable for detecting potential mycoplasmal contaminationin laboratories that rely heavily on the cell culture system.

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

    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.

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

    Kyoung-Hee Bae

    2013-01-01

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

  11. Feasibility of Bone Marrow Stromal Cells Autologous Transplantation for Dilated Cardiomyopathy

    ZHOU Cheng; YANG Chenyuan; XIAO Shiliang; FEI Hongwen

    2007-01-01

    The feasibility of bone marrow stromal cells autologous transplantation for rabbit model of dilated cardiomyopathy induced by adriamycin was studied. Twenty rabbits received 2 mg/kg of adriamycin intravenously once a week for 8 weeks (total dose, 16 mg/kg) to induce the cardiomyopathy model with the monitoring of cardiac function by transthoracic echocardiography. Marrow stromal cells were isolated from cell-transplanted group rabbits and were culture-expanded on the 8th week. On the 10th week, cells were labeled with 4,6-diamidino-2-phenylindole (DAPI), and then injected into the myocardium of the same rabbits. The results showed that viable cells labeled with DAPI could be identified in myocardium at 2nd week after transplantation. Histological findings showed the injury of the myocardium around the injection site was relieved with less apoptosis and more expression of bcl-2. The echocardiography found the improvement of local tissue movement from (2.12±0.51) cm/s to (3.81±0.47) cm/s (P<0.05) around the inject site, but no improvement of heart function as whole. It was concluded bone marrow stromal cells transplantation for dilated cardiomyopathy was feasibe. The management of cells in vitro, the quantity and the pattern of the cells transplantation and the action mechanism still need further research.

  12. Drug uptake (DAPI) of trypanosomes (T. brucei) and antitrypanosomal activity in vitro, in culture and in vivo studied by microscope fluorometry, chromatogram spectrophotometry and radiotracer techniques

    The present study had the following objectives: 1) Investigation of the specific binding and location of the diamidine DAPI within trypanosomes by fluorescence microscopy. 2) Development and standardization of a microscope fluorometry technique for measuring DAPI uptake of single trypanosomes. 3) Determination of the effect of incubation media, exposure time, and drug concentration on DAPI uptake of single trypanosomes. 4) Development of a technique applicable for quantitative fluorescence chemical analysis of DAPI uptake of trypanosomes. 5) Determination of drug uptake of trypanosomes using 14C labelled DAPI. 6) Comparison of the values obtained by the three methods. (orig./MG)

  13. Formulation and Evaluation of Trimetazidine Dihydrochloride Extended Release Tablets by Melt Congealing Method

    Javeer, S. D.; Pandit, Reshma; Jain, S. P.; Amin, Purnima

    2010-01-01

    Trimetazidine dihydrochloride, a cellular antiischemic agent indicated in the management and prophylaxis of angina pectoris is given as 20 mg thrice daily in the conventional dosage regimen. The purpose of the present study was to formulate and evaluate twice a day extended release tablets containing 30 mg trimetazidine dihydrochloride. The method developed to formulate these extended release tablets was melt congealing followed by wet granulation which exhibited uniform sustained release act...

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

    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

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

    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.

  16. Flow-cytometry techniques in radiation biology

    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.

  17. Synthesis and Biological Evaluation of Curcumin Derivatives with Water-Soluble Groups as Potential Antitumor Agents: An in Vitro Investigation Using Tumor Cell Lines

    Luyang Ding

    2015-12-01

    Full Text Available Three series of curcumin derivatives including phosphorylated, etherified, and esterified products of curcumin were synthesized, and their anti-tumor activities were assessed against human breast cancer MCF-7, hepatocellular carcinoma Hep-G2, and human cervical carcinoma HeLa cells. Compared with curcumin, compounds 3, 8, and 9 exhibited stronger antitumor cell line growth activities against HeLa cells. Compound 12 also showed higher antitumor cell line growth activities on MCF-7 cells than curcumin. Among them, 4-((1E,6E-7-(4-Hydroxy-3-methoxyphenyl-3,5-dioxohepta-1,6-dienyl-2-methoxyphenyl dihydrogen phosphate(3 showed the strongest activity with an half maximal inhibitory concentration (IC50 of 6.78 µM against HeLa cells compared with curcumin with an IC50 of 17.67 µM. Stabilities of representatives of the three series were tested in rabbit plasma in vitro, and compounds 3 and 4 slowly released curcumin in plasma. The effect of compound 3 on HeLa cell apoptosis was determined by examining morphological changes by DAPI (4′,6-diamidino-2-phenylindole staining as well as Annexin V-FITC/ Propidium Iodide (PI double staining and flow cytometry. The results showed that 3 induced cellular apoptosis in a dose-dependent manner. Together our findings show that 3 merits further investigation as a new potential antitumor drug candidate.

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

    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

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

    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 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

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

    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

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

    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

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

    Tu Qiufen [Key Laboratory Advanced Technologies of Material, Ministry of Education, Southwest Jiaotong University, Chengdu 610031 (China); Zhang Yi; Ge Dongxia; Wu Jiang [Institute of Biomedical Engineering, West China Center of Medical Sciences, Sichuan University, Chengdu 610041 (China); Chen Huaiqing [Institute of Biomedical Engineering, West China Center of Medical Sciences, Sichuan University, Chengdu 610041 (China)], E-mail: chq@scu.edu.cn

    2008-11-15

    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.

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

    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.

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

    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

  5. Programmed cell death during terminal bud senescence in a sympodial branching tree,Eucommia ulmoides

    XU Wenjie; Kalima-N'Koma MWANGE; CUI Keming

    2004-01-01

    Eucommia ulmoides Oliv. is a typical sympodial branching tree. The apical bud of the branch ages and dies every year, replaced by the nearby axillary bud in the second year. Structural assays and a series of biochemical analyses were performed to analyze the senescence mechanism in the apical bud. It was revealed that most cells of the apical bud underwent the programmed cell death (PCD) during the senescence: the chromosomes were congregated and the nuclear contents were condensed, as shown by 4′,6-diamidino-2-phenylindole (DAPI) fluorescence. DNA fragmentation was detected during senescence using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end in situ labeling (TUNEL) method, coincident with the appearance of a DNA ladder. Moreover, a 20 kD DNase related to fragmentation was found. PCD was initiated first in the young leaves, leaf primordia and peripheral zone cells, then in the central mother cells and initial layer cells in the apical meristem. The terminal buds remain in vegetative growth during senescence, in contrast to buds of many annual plants.

  6. Inhibition of Dual Specific Oncolytic Adenovirus on Esophageal Cancer via Activation of Caspases by a Mitochondrial-dependent Pathway

    SU Jia-qiang; CHI Bao-rong; LI Xiao; LIU Lei; LIU Li-ming; QI Yan-xin; WANG Zhuo-yue; JIN Ning-yi

    2012-01-01

    We investigated the anti-tumor effects of dual cancer specific-oncolytic adenovirus Ad-VP on esophageal cancer(EC).The anti-tumor activity of Ad-VP was compared with that of the control recombinant adenoviruses (Ad-GP,Ad-Apoptin,Ad-EGFP) in human esophageal cancer cell EC-109 and human normal liver cell L02 in vitro.In 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assays,the growth of EC-109 cells was slightly inhibited by Ad-GP.Ad-Apoptin and Ad-EGFP.However,Ad-VP induced a significant cytotoxic effect.Infection of EC-109 cells with Ad-VP resulted in a significant induction of apoptosis of them in vitro,detected by 4′,6-diamidino-2-phenylindole(DAPI) or acridine orange and ethidium bromide staining.The results of Western blot and flow cytometric assay indicate the loss of mitochondrial membrane potential(△ψm),the release of cytochrome c and the activation of caspase-3,6 and 7 in Ad-VP infiected EC-109 cells.In contrast,all these assays show almost no effects of the recombinant adenoviruses on L02 cells.These results demonstrate that the treatment of tumors with Ad-VP selectively inhibits tumor growth and induces apoptosis of esophageal cancer cells.Ad-VP may provide a novel and powerful strategy for cancer gene therapy.

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

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

    2005-01-01

    AIM: Oligomeric proanthocyanidins (OPC), natural polyphenolic compounds found in plants, are known to have antioxidant and anti-cancer effects. We investigated whether the anti-cancer effects of the OPC are induced by apoptosis on human colorectal cancer cell line, SNU-C4.METHODS: Colorectal cancer cell line, SNU-C4 was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. The cytotoxic effect of OPC was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenylt-etrazolium bromide (MTT) assay. To find out the apoptotic cell death, 4, 6-diamidino-2-phenylindole (DAPI) staining,terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, reverse transcriptionpolymerase chain reaction (RT-PCR), and caspase-3 enzyme assay were performed.RESULTS: In this study, cytotoxic effect of OPC on SNUC4 cells appeared in a dose-dependent manner. OPC treatment (100 μg/mL) revealed typical morphological apoptotic features. Additionally OPC treatment (100 μg/mL)increased level of BAX and CASPASE-3, and decreased level of BCL-2 mRNA expression. Caspase-3 enzyme activity was also significantly increased by treatment of OPC (100 μg/mL) compared with control.CONCLUSION: These data indicate that OPC caused cell death by apoptosis through caspase pathways on human colorectal cancer cell line, SNU-C4.

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

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

    2016-01-01

    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

  9. Signaling pathways involved in apoptosis induced by novel angucycline antibiotic landomycin E in Jurkat T leukemia cells

    Panchuk R. R.

    2011-04-01

    Full Text Available Aim. To study the molecular mechanisms of action of novel anticancer antibiotic landomycin E (LE. Methods. Annexin V/propidium iodide, DAPI (4',6-diamidino-2-phenylindole staining, Western-blot analysis. Results. LE applied in 2 µg/ml dose (IC50, induced reactive oxygen species (ROS-dependent splitting of poly [ADP-ribose] polymerase 1 (PARP-1 and DNA Fragmentation Factor 45 (DFF45 proteins involved in DNA reparation. This effect was observed 6 h after the start of treatment and it positively correlated with phosphatidyl serine externalization (early morphological marker of apoptosis. We suggest that cleavage of PARP-1 and DFF45 was mediated by active caspase-7 which is a key effector caspase in the LE-induced apoptosis in leukemia cells. We found that activation of initiator procaspase-10 (involved in receptor- mediated apoptosis was the earliest detected event in LE-induced apoptotic signaling pathways; however, this activation was shown to be ROS-independent. We also demonstrated that the induction of apoptosis by LE is accompanied by activation of apoptosis-inducing factor (AIF in mitochondria. Conclusions. Our data suggest that LE-induced cascade of apoptotic events is started by the initiator caspase-10 which leads to activation of the effector caspase-7 and AIF that is known to induce caspase-independent apoptosis involving ROS generation.

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

    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

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

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

    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

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

    Kuan-Han Lee

    2014-01-01

    Full Text Available 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-trimethoxyphenoxybenzoic 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.

  14. A multiple-method approach reveals a declining amount of chloroplast DNA during development in Arabidopsis

    Oldenburg Delene J

    2009-01-01

    Full Text Available Abstract Background A decline in chloroplast DNA (cpDNA during leaf maturity has been reported previously for eight plant species, including Arabidopsis thaliana. Recent studies, however, concluded that the amount of cpDNA during leaf development in Arabidopsis remained constant. Results To evaluate alternative hypotheses for these two contradictory observations, we examined cpDNA in Arabidopsis shoot tissues at different times during development using several methods: staining leaf sections as well as individual isolated chloroplasts with 4',6-diamidino-2-phenylindole (DAPI, real-time quantitative PCR with DNA prepared from total tissue as well as from isolated chloroplasts, fluorescence microscopy of ethidium-stained DNA molecules prepared in gel from isolated plastids, and blot-hybridization of restriction-digested total tissue DNA. We observed a developmental decline of about two- to three-fold in mean DNA per chloroplast and two- to five-fold in the fraction of cellular DNA represented by chloroplast DNA. Conclusion Since the two- to five-fold reduction in cpDNA content could not be attributed to an artifact of chloroplast isolation, we conclude that DNA within Arabidopsis chloroplasts is degraded in vivo as leaves mature.

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

    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.

  16. 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

    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.

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

    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

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

    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.

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

    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.

  20. Mapping small DNA sequences by fluorescence in situ hybridization directly on banded metaphase chromosomes

    A procedure for mapping small DNA probes directly on banded human chromosomes by fluorescence in situ hybridization has been developed. This procedure allows for the simultaneous visualization of banded chromosomes and hybridization signal without overlaying two separate photographic images. This method is simple and rapid, requires only a typical fluorescence microscope, has proven successful with DNA probes as small as 1 kilobase, is applicable for larger probes, and will greatly facilitate mapping the vast number of probes being generated to study genetic disease and define the human genome. Human metaphase chromosomes were prepared from phytohemagglutinin-stimulated lymphocyte cultures synchronized with bromodeoxyuridine and thymidine. Probes were labeled with biotin-dUTP, and the hybridization signal was amplified by immunofluorescence. Chromosomes were stained with both propidium iodide and 4',6-diamidino-2-phenylindole (DAPI), producing R- and Q-banding patterns, respectively, allowing unambiguous chromosome and band identification while simultaneously visualizing the hybridization signal. Thirteen unique DNA segments have been localized to the long arm of chromosome 11 by using this technique, and localization of 10 additional probes by using radioactive in situ hybridization provides a comparison between the two procedures. These DNA segments have been mapped to all long-arm bands on chromosome 11 and in regions associated with neoplasias and inherited disorders

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

    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

  2. Solid state stability and solubility of triethylenetetramine dihydrochloride.

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

    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)...

  4. Feasibility of optimizing trimetazidine dihydrochloride release from controlled porosity

    Basant A. Habib

    2014-05-01

    Full Text Available The aim of this study was to develop and optimize Trimetazidine dihydrochloride (TM controlled porosity osmotic pump (CPOP tablets of directly compressed cores. A 23 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 Y1, Y2, Y3, and Y4, 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% ⩽ Y1 ⩽ 20%, 40% ⩽ Y2 ⩽ 60%, 80% ⩽ Y3 ⩽ 100%, and Y4 > 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.

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

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

    1987-01-01

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

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

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

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

    M. M. Gupta; Niraj Gupta; Chauhan, Bhupendra S.; Shweta Pandey

    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 p...

  8. ESTIMATION OF MANIDIPINE DIHYDROCHLORIDE BY A NEW RP-HPLC METHOD

    Raja Sundararajan et al

    2012-09-01

    Full Text Available A rapid reverse phase high performance liquid chromatographic method was developed for the estimation of Manidipine dihydrochloride in its pure form as well as in tablet dosage forms. Chromatography was carried out on an phenomenex C18 column (2504.6mm, 5 µm, using a mixture of acetonitrile and water (80: 20 v/v with pH adjusted to 3.5 with ortho phosphoric acid (0.1 % v/v as the mobile phase at a flow rate of 1.3 mL/min The detection wavelength is 230 nm. The retention time of the drug was 3.54 min. The method produced linear responses within concentration range of 25 to 125 µg/mL of Manidipine dihydrochloride. The method was found to be reproducible for analysis of the drug in tablet dosage forms.

  9. DESIGN, DEVLOPMENT AND EVALUATION OF TRIMETAZIDINE DIHYDROCHLORIDE MODIFIED RELEASE TABLET USING 32 FACTORIAL DESIGN

    Biswal Biswajit; Patel M.B

    2011-01-01

    The present investigation was undertaken to fabricate modified release tablet of Trimetazidine dihydrochloride using hydroxypropyl methylcellulose (HPMC) and Ethyl cellulose as a matrixing agent. A 32 full factorial design was employed for the optimization of formulation. The percentage drug released at a given time (Q1, Q2 , Q4,Q8 ,Q 16 , Q24) and the time required for a given percentage of drug to be released (t50%) were selected as dependent variables. The in vitro drug dissolution study w...

  10. Floating Tablet of Trimetazidine Dihydrochloride: An Approach for Extended Release with Zero-Order Kinetics

    Abdelbary, Ahmed; El-Gazayerly, Omaima N.; El-Gendy, Nashwa A.; Ali, Adel A.

    2010-01-01

    Trimetazidine dihydrochloride is an effective anti-anginal agent; however, it is freely soluble in water and suffers from a relatively short half-life. To solve this encumbrance, it is a prospective candidate for fabricating trimetazidine extended-release formulations. Trimetazidine extended-release floating tablets were prepared using different hydrophilic matrix forming polymers including HPMC 4000 cps, carbopol 971P, polycarbophil, and guar gum. The tablets were fabricated by dry coating t...

  11. Efficacy of octenidine dihydrochloride and 2-phenoxyethanol in the topical treatment of inflammatory acne:

    Aberer, W.; Kränke, Birger; Mayr-Kanhäuser, Sigrid

    2008-01-01

    Background: With the increase in antibiotic-resistant strains of microorganisms in acne lesions, the search for alternative treatment methods has become important. We studied the efficacy of a combination of the antiseptic substances octenidine dihydrochloride and 2-phenoxyethanol (O/P) in mild to moderate inflammatory acne vulgaris. Methods: Thirty patients were instructed to apply O/P once or twice daily for a 6-week treatment period. Determination of efficacy included the numerical documen...

  12. Binary diffusion coefficients of L-histidine methyl ester dihydrochloride in aqueous solutions

    Highlights: • Mutual diffusion coefficients of L-histidine methyl ester dihydrochloride in aqueous solutions. • Influence of the thermodynamic and kinetic factors on the variation of the mutual diffusion coefficients. • Estimation of the hydrodynamic radius of L-histidine methyl ester dihydrochloride. - Abstract: The Taylor dispersion technique has been used for measuring mutual diffusion coefficients of L-histidine methyl ester as its dihydrochloride at T = 298.15 K and finite concentrations from (0.001 to 0.100) mol · dm−3. On the basis of experimental mutual diffusion coefficients, the hydrodynamic radii, Rh, the diffusion coefficient at infinite dilution D0 and the dependence of thermodynamic factors, FT, on the concentration, have been estimated using the Onsager–Fuoss equation. Further insight on the diffusion has been obtained from 1H and 13C NMR spectroscopy and DFT calculations, which suggest that the L-histidine methyl ester is present as its dication in acidic solution in a fully extended conformation, with considerable charge delocalization over the imidazolium ring. These experimental and computational results allow us to have a better understanding of the thermodynamic and kinetic behavior of this amino acid derivative in aqueous solutions

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

    Wei Zhao; Jun-Jie Li; Da-Yong Cao; Xiao Li; Lin-Ying Zhang; Yong He; Shu-Qiang Yue

    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-diamidino2-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 di-ferences 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.

  14. Genetic fidelity assessment in micropropagated plants using cytogenetical analysis and heterochromatin distribution: a case study with Nepenthes khasiana Hook f.

    Devi, Soibam Purnima; Kumaria, Suman; Rao, Satyawada Rama; Tandon, Pramod

    2015-09-01

    Rapid clonal propagation of selected genotypes has been one of the most extensively exploited approaches of biotechnology. However, inclusion of somaclonal variations in tissue-culture-derived plants results in the production of undesirable plant off-types which limits its applications in tissue culture industry. Therefore, the most critical concern has been the maintenance of genetic uniformity of micropropagated plants. Assessment of genetic fidelity in tissue-culture-raised plants of three consecutive regenerations of Nepenthes khasiana has been successfully carried out using chromosome counts and heterochromatin distribution pattern wherein changes in the number of chromosomes and the distribution of AT and GC base pairs were recorded. The cells studied in the plantlets of the first regeneration (23.33 %) showed deviant number of chromosome which was increased to 33.33 % and 40 % in the plantlets of the second and the third regenerations, respectively. Also, 4',6-diamidino-2-phenylindole (DAPI)(+) and chromomycin A3 (CMA)(+) binding sites, on an average of 5.74 ± 0.47 and 5.00 ± 0.30, were observed in the plantlets of the first regeneration. Subsequently, DAPI(+) binding sites were increased to 6.61 ± 0.39 and 6.74 ± 0.57 in the plantlets of the second and the third regenerations, respectively, with a corresponding decrease in the CMA(+) binding sites (4.63 ± 0.45 and 4.16 ± 0.47 CMA(+) sites in the plantlets of the second and the third regenerations, respectively). The study reveals an increase in cytological variations in the morphologically similar micropropagated plants of N. khasiana with the subsequent regenerations which further necessitate the determination of genetic integrity of micropropagated plants. PMID:25616932

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

    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.

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

    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

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

    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

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

    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

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

    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

  20. Ocorrência de infecção Cryptosporidium spp. em peixe-boi marinho (Trichechus manatus Occurrence of Cryptosporidium spp. infection in antillean manatee (Trichechus manatus

    João Carlos Gomes Borges

    2009-03-01

    Full Text Available A criptosporidiose constitui-se como uma zoonose que pode afetar o homem e uma ampla variedade de animais domésticos e silvestres, principalmente indivíduos imunodeficientes. O objetivo desse trabalho foi registrar a ocorrência de infecção por Cryptosporidium em peixe-boi marinho. Após ser constatada a mudança de comportamento de um peixe-boi marinho mantido nos oceanários do Centro Mamíferos Aquáticos, ICMBio - FMA, animal foi submetido à exame clínico e, posteriormente, à coleta de amostra fecal. As amostras fecais foram analisadas pela técnica de Kinyoun, teste de imunofluorescência direta e pelo corante 4'.6'-Diamidino-2-Phenilindole (DAPI. No exame clínico, o animal apresentou sinais de desconforto abdominal. Os resultados obtidos nas análises de microscopia de luz e fluorescente revelaram a presença de oocistos de Cryptosporidium nas fezes desse peixe-boi.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.

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

    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

  2. Molecular characterization of constitutive heterochromatin in three species of Trypoxylon (Hymenoptera, Crabronidae, Trypoxylini by CMA3/DAPI staining

    Rodolpho Menezes

    2011-07-01

    Full Text Available 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, 1856 the molecular composition of the heterochromatin was different 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, 1873 which has the lowest chromosome number in the genus and other characteristics of the likely ancestral Trypoxylon karyotype.

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

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

    2013-01-01

    The aim of this study was to develop and optimize Trimetazidine dihydrochloride (TM) controlled porosity osmotic pump (CPOP) tablets of directly compressed cores. A 23 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%) i...

  4. In vitro antiplaque activity of octenidine dihydrochloride (WIN 41464-2) against preformed plaques of selected oral plaque-forming microorganisms.

    Slee, A M; O'Connor, J R

    1983-01-01

    The antibacterial activity of octenidine dihydrochloride (WIN 41464-2) against intact preformed in vitro plaques of four indigenous oral plaque-forming microorganisms, Streptococcus mutans, Streptococcus sanguis, Actinomyces viscosus, and Actinomyces naeslundii, was studied. Both absolute (plaque bactericidal index) and relative (chlorhexidine coefficient) indices of antiplaque efficacy were established. Octenidine dihydrochloride compared favorably with chlorhexidine digluconate with respect...

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

    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

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

    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

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

    Zuo, Chaohui, E-mail: zuochaohui@vip.sina.com [Department of Gastroduodenal and Pancreatic Surgery, Translation Medicine Research Center of Liver Cancer, Hunan Province Tumor Hospital & Affiliated Tumor Hospital of Xiangya Medical School, Central South University, Changsha, Hunan Province (China); Department of Pathology, Immunology and Laboratory Medicine and Shands Cancer Center, University of Florida, Gainesville, FL (United States); Qiu, Xiaoxin [Department of Gastroduodenal and Pancreatic Surgery, Translation Medicine Research Center of Liver Cancer, Hunan Province Tumor Hospital & Affiliated Tumor Hospital of Xiangya Medical School, Central South University, Changsha, Hunan Province (China); Cancer Research Institute, University of South China, Hengyang, Hunan Province (China); Liu, Nianli; Yang, Darong [Cancer Research Institute, University of South China, Hengyang, Hunan Province (China); Xia, Man [Department of Gastroduodenal and Pancreatic Surgery, Translation Medicine Research Center of Liver Cancer, Hunan Province Tumor Hospital & Affiliated Tumor Hospital of Xiangya Medical School, Central South University, Changsha, Hunan Province (China); Department of Pathology, Immunology and Laboratory Medicine and Shands Cancer Center, University of Florida, Gainesville, FL (United States); Liu, Jingshi [Department of Gastroduodenal and Pancreatic Surgery, Translation Medicine Research Center of Liver Cancer, Hunan Province Tumor Hospital & Affiliated Tumor Hospital of Xiangya Medical School, Central South University, Changsha, Hunan Province (China); Wang, Xiaohong [Cancer Research Institute, University of South China, Hengyang, Hunan Province (China); 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.

  8. Homeopathic mother tincture of Phytolacca decandra induces apoptosis in skin melanoma cells by activating caspase-mediated signaling via reactive oxygen species elevation

    Samrat Ghosh; Kausik Bishayee; Avijit Paul; Avinaba Mukherjee; Sourav Sikdar; Debrup Chakraborty; Naoual Boujedaini

    2013-01-01

    OBJECTIVE:Preventive measures against skin melanoma like chemotherapy are useful but suffer from chronic side effects and drug resistance.Ethanolic extract of Phytolacca decandra (PD),used in homeopathy for the treatment of various ailments like chronic rheumatism,regular conjunctivitis,psoriasis,and in some skin diseases was tested for its possible anticancer potential.METHODS:Cytotoxicity of the drug was tested by conducting 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay on both normal (peripheral blood mononuclear cells) and A375 cells.Fluorescence microscopic study of 4',6-diamidino-2-phenylindole dihydrochloride-stained cells was conducted for DNA fragmentation assay,and changes in cellular morphology,if any,were also recorded.Lactate dehydrogenase activity assay was done to evaluate the percentages of apoptosis and necrosis.Reactive oxygen species (ROS) accumulation,if any,and expression study of apoptotic genes also were evaluated to pin-point the actual events of apoptosis.RESULTS:Results showed that PD administration caused a remarkable reduction in proliferation of A375 cells,without showing much cytotoxicity on peripheral blood mononuclear cells.Generation of ROS and DNA damage,which made the cancer cells prone to apoptosis,were found to be enhanced in PD-treated cells.These results were duly supported by the analytical data on expression of different cellular and nuclear proteins,as for example,by downregulation of Akt and Bcl-2,up-regulation of p53,Bax and caspase 3,and an increase in number of cell deaths by apoptosis in A375 cells.CONCLUSION:Overall results demonstrate anticancer potentials of PD on A375 cells through activation of caspase-mediated signaling and ROS generation.

  9. Synthesis and high in vitro cytotoxicity of some (S,S)-ethylenediamine-N,N’-di-2-propanoate dihydrochloride esters

    Pantelić Nebojša; Zmejkovski Bojana B.; Stanojković Tatjana P.; Jeftić Verica V.; Radić Gordana P.; Trifunović Srećko R.; Kaluđerović Goran N.; Sabo Tibor J.

    2014-01-01

    Novel (S,S)-R2eddip ester, O,O’-diisoamyl-(S,S)-ethylenediamine-N,N’-di-2-propanoate dihydrochloride, 1, was synthesized and characterized by IR, 1H and 13C NMR spectroscopy, mass spectroscopy and elemental analysis.In vitro antitumor action of 1, and two more R2eddip esters, O,O’-dialkyl-(S,S)-ethylenediamine-N,N’-di-2-propanoate dihydrochlorides, obtained before, (alkyl = n-Bu, n-Pe; 2 and 3, respectively), was determined against cervix adenocarcino...

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

    Mylien T. Hoang

    2015-09-01

    Full Text Available 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.

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

    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

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

    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

  13. Differentiation of smooth muscle progenitor cells in peripheral blood and its application in tissue engineered blood vessels

    Shang-zhe XIE; Ning-tao FANG; Shui LIU; Ping ZHOU; Yi ZHANG; Song-mei WANG; Hong-yang GAO; Luan-feng PAN

    2008-01-01

    Background: A major shortcoming in tissue engineered blood vessels (TEBVs) is the lack of healthy and easily attainable smooth muscle cells (SMCs). Smooth muscle progenitor cells (SPCs), especially from peripheral blood, may offer an alternative cell source for tissue engineering involving a less invasive harvesting technique. Methods: SPCs were isolated from 5-ml fresh rat peripheral blood by density-gradient centrifugation and cultured for 3 weeks in endothelial growth medium-2-MV (EGM-2-MV) medium containing platelet-derived growth factor-BB (PDGF BB). Before seeded on the synthesized scaffold, SPC-derived smooth muscle outgrowth cell (SOC) phenotypes were assessed by immuno-fluorescent staining, Western blot analysis, and reverse transcription polymerase chain reaction (RT-PCR). The cells were seeded onto the silk fibroin-modified poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (SF-PHBHHx) scaffolds by 6×104 cells/cm'2 and cultured under the static con-dition for 3 weeks. The growth and proliferation of the seeded cells on the scaffold were analyzed by 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT) assay, scanning electron microscope (SEM), and 4,6-diamidino-2-phenylindole (DAPI) staining. Results: SOCs displayed specific "hill and valley" morphology, expressed the specific markers of the SMC lineage: protein, and extracellular matrix components elastin and matrix Gla protein (MGP), as well as vascular endothelial growth factor (VEGF). After seeded on the SF-PHBHHx scaffold, the cells showed excellent metabolic activity and proliferation. Conclusion: SPCs isolated from peripheral blood can be differentiated into the SMCs in vitro and have an impressive growth potential in the biodegradable synthesized scaffold. Thus, SPCs may be a promising cell source for constructing TEBVs.

  14. Localization of a female-specific marker on the chromosomes of the brown seaweed Saccharina japonica using fluorescence in situ hybridization.

    Yu Liu

    Full Text Available BACKGROUND: There is a heteromorphic alternative life in the brown seaweed, Saccharina japonica (Aresch. C. E. Lane, C. Mayes et G. W. Saunders ( = Laminaria japonica Aresch., with macroscopic monoecious sporophytes and microscopic diecious gametophytes. Female gametophytes are genetically different from males. It is very difficult to identify the parent of a sporophyte using only routine cytological techniques due to homomorphic chromosomes. A sex-specific marker is one of the best ways to make this determination. METHODOLOGY/PRINCIPAL FINDINGS: To obtain clear images, chromosome preparation was improved using maceration enzymes and fluorochrome 4', 6-diamidino-2-phenylindole (DAPI. The chromosome number of both male and female haploid gametophytes was 31, and there were 62 chromosomes in diploid sporophytes. Although the female chromosomes ranged from 0.77 µm to 2.61 µm in size and were larger than the corresponding ones in the males (from 0.57 µm to 2.16 µm, there was not a very large X chromosome in the females. Based on the known female-related FRML-494 marker, co-electrophoresis and Southern blot profiles demonstrated that it was inheritable and specific to female gametophytes. Using modified fluorescence in situ hybridization (FISH, this marker could be localized on one unique chromosome of the female gametophytes as well as the sporophytes, whereas no hybridization signal was detected in the male gametophytes. CONCLUSIONS/SIGNIFICANCE: Our data suggest that this marker was a female chromosome-specific DNA sequence. This is the first report of molecular marker localization on algal chromosomes. This research provides evidence for the benefit of using FISH for identifying molecular markers for sex identification, isolation of specific genes linked to this marker in the females, and sex determination of S. japonica gametophytes in the future.

  15. Synergistic effects of prostaglandin E1 and lithium in a rat model of cerebral ischemia

    Rong RAN; Bo GAO; Rui SHENG; Li-sha ZHANG; Hui-lin ZHANG; Zhen-lun GU; Zheng-hong QIN

    2008-01-01

    Aim:Heat shock proteins (HSPs) are important regulators of cellular survival and exert neuroprotective effects against cerebral ischemia.Both prostaglandin El (PGEI) and lithium have been reported to protect neurons against ischemic injury.The present study was undertaken to examine if lithium could potentiate the neuroprotection of PGE 1 against cerebral ischemia,and if the synergetic effects take place at the level of HSPs.Methods:Brain ischemia was induced by a permanent middle cerebral artery occlusion (pMCAO) in rats.Rats were pretreated with subcutaneous injection of lithium for 2 d and a single intravenous administration of PGEI immediately after ischemic insult.Cerebrocortical blood flow of each group was closely monitored prior to onset of ischemia,5 min,15 rain,30 min and 60 min after surgical operation.Body temperature was measured before,5 min,2 h and 24 h after the onset of pMCAO.The infarct volume,brain edema and motor behavior deficits were analyzed 24 h after ischemic insult.Cytoprotective HSP70 and heme oxygenase-1 (HO-1) in the striatum of the ipsilateral hemisphere were detected by immunoblotting.Brain sections from the striatum of the ipsilateral hemisphere were double-labeled with the anti-HSP70 antibody and 4,6-diamidino-2-phenylindole (DAPI).Results:Treatment with PGEI (8 and 16 ~tg/kg,iv) or lithium (0.5 mEq/kg,sc) alone reduced infarct volume,neurological deficits and brain edema induced by focal cerebral ischemia in rats.Moreover,a greater neuroprotection was observed when PGEI and lithium were given together.Co-administration of PGE1 and lithium significantly upregulated cytoprotective HSP70 and HO-1 protein levels.Conclusion:Lithium and PGEI may exert synergistic effects in treatment of cerebral ischemia and thus may have potential clinical value for the treatment of stroke.

  16. Karyotype characterization, constitutive heterochromatin and nucleolus organizer regions of Paranaita opima (Coleoptera, Chrysomelidae, Alticinae

    Mara Cristina de Almeida

    2006-01-01

    Full Text Available Species of the subtribe Oedionychina not only have a highly uniform diploid number of 2n = 22 (20+X+y but have the karyotypic peculiarity of possessing extremely large sex chromosomes. We analyzed Paranaita opima embryos and gonadal cells to determine their diploid number, chromosomal morphology, type of sex determination system, constitutive heterochromatin pattern and which chromosomes bear nucleolus organizer regions (NORs. The diploid number of P. opima was 2n = 22 (20+XY/XX with all chromosomes being metacentric. Chromosome pair 6 showed an interstitial secondary constriction on the short arm. The C-banding technique revealed centromeric constitutive heterochromatin in all chromosomes, which, in pair 6, extended up to the secondary constriction of the short arm, additional C-bands also being present on the Y chromosome. Silver nitrate nucleolar organizer region (Ag-NOR staining showed NORs on the secondary constriction of pair 6. Fluorochrome analysis with chromomycin A3 (CMA3, 4'-6-diamidino-2-phenylindole (DAPI and the distamycin A (DA counterstain showed that the short arm of chromosome pair 6 exhibited a GC-rich block extending from the proximal to the median region, including part of the secondary constriction. The same techniques also showed AT-rich blocks at the centromeric region of all chromosomes and at the terminal region of the short arm of pair 6. The basic karyotype characteristics and C band pattern of P. opima are similar to those described for other species in the subtribe Oedionychina. The pattern of autosomal NORs observed in P. opima corresponds to that registered in the majority of the Chrysomelidae species.

  17. Helicobacter pylori antigens, acetylsalicylic acid, LDL and 7-ketocholesterol - their potential role in destabilizing the gastric epithelial cell barrier. An in vitro model of Kato III cells.

    Gajewski, Adrian; Mnich, Eliza; Szymański, Karol; Hinc, Krzysztof; Obuchowski, Michał; Moran, Anthony P; Chmiela, Magdalena

    2016-01-01

    Colonization of gastric tissue in humans by H. pylori Gram-negative bacteria initiates gastric and duodenal ulcers and even gastric cancers. Infections promote inflammation and damage to gastric epithelium which might be followed by the impairment of its barrier function. The role of H. pylori components in these processes has not been specified. H. pylori cytotoxicity may potentially increase in the milieu of anti-inflammatory drugs including acetylsalicylic acid (ASA). The lipid transport-associated molecule such as low density lipoprotein (LDL), which is a classic risk factor of coronary heart disease (CHD) and 7-ketocholesterol (7-kCh) a product of cholesterol oxidation, which may occur during the oxidative stress in LDL could also be considered as pro-inflammatory. The aim of this study was to evaluate the cytotoxicity of H. pylori antigens, ASA, LDL and 7-kCh towards Kato III gastric epithelial cells, on the basis of the cell ability to reduce tetrazolium salt (MTT) and morphology of cell nuclei assessed by 4',6-diamidino-2-phenylindole (DAPI) staining. Kato III cells were stimulated for 24 h, at 37°C and 5% CO2, with H. pylori antigens: cytotoxin associated gene A (CagA) protein, the urease A subunit (UreA), lipopolysaccharide (LPS) and ASA, LDL or 7-kCh. H. pylori LPS, ASA, LDL and 7-kCh, but not H. pylori glycine acid extract (GE), demonstrated cytotoxicity against Kato III cells, which was related to a diminished percentage of MTT reducing cells and to an increased cell population with the signs of DNA damage. The results suggest that damage to gastric epithelial cells can be induced independently by H. pylori antigens, ASA and endogenous lipid transport-associated molecules. During H. pylori infection in vivo, especially in CHD patients, synergistic or antagonistic interactions between these factors might possibly influence the disease course. Further study is necessary to explain these potential effects. PMID:26619253

  18. The effects of strontium ranelate on osteoblast apoptosis in simulated microgravity%雷奈酸锶在模拟微重力环境对成骨细胞凋亡的影响

    宋淑军; 司少艳; 刘俊丽; 周金莲; 牛忠英; 谭小清; 张建中

    2012-01-01

    Objective To investigate the role of strontium ranelate, the novel antiosteoporotic agent, in regulating the apoptosis of osteoblasts under simulated microgravity. Methods Apoptotic cells were identified on the basis of nuclear morphology with 4,6-diamidino-2 phenylindole ( DAPI) staining and rotating cell culture system was used to simulate microgravitational effect. Results Simulated microgravity increased apoptosis of osteoblasts cells and this could be reversed by strontium ranelate treatment. Conclusion Here we demonstrate that strontium ranelate plays a protective role in osteoblast survival in simulated microgravity environment. This provides a theoretical and experimental evidence for the treatment of microgravity-induced bone loss with strontium ranelate.%观察雷奈酸锶这种新型的抗骨质疏松药,在模拟微重力环境对成骨细胞凋亡的影响.方法 细胞凋亡是利用4,6-二氨基-2-苯基吲哚所染细胞核形态的改变来辨认,利用旋转细胞培养系统模拟微重力环境.结果 模拟微重力环境可以促进成骨细胞的凋亡,雷奈酸锶可以纠正模拟微重力环境对成骨细胞凋亡的影响.结论 在模拟微重力环境雷奈酸锶对成骨细胞生存具有保护作用,这为雷奈酸锶治疗微重力环境骨丢失提供了理论和实验证据.

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

    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

  20. High resolution DNA content measurements of mammalian sperm

    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.

  1. Induction of actin disruption and downregulation of P-glycoprotein expression by solamargine in multidrug-resistant K562/A02 cells

    LI Xia; ZHAO Ying; JI Mei; LIU Shan-shan; CUI Min; LOU Hong-xiang

    2011-01-01

    Background Solamargine (SM), a steroidal glycoalkaloid isolated from the Chinese herb Solarium incanum, has been shown to inhibit the growth of some cancer cell lines and induce significant apoptosis. However, the effects of SM on multidrug-resistant (MDR) cells and the molecular mechanisms involved are poorly understood. The purpose of this study was to evaluate the anti-MDR effects of SM and the associated mechanisms in MDR K562/A02 cells.Methods The cytotoxicity of SM was measured by 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) assay. The 14',6-diamidino-2-phenylindole (DAPI) nuclear staining and flow cytometry were used to detect SM-induced apoptosis. The mRNA expression of P-glycoprotein (P-gp) was investigated by real-time PCR (RT-PCR). Western blotting was used to determine the expression of Bcl-2, Bax, and actin. The changes in the morphology of actin were examined with immunofluorescence staining.Results MTT results showed that SM effectively killed the MDR sublines K562/A02, KB/VCR, and H460/paclitaxel (Taxol), and their parental cell lines K562, KB, and H460 to an equivalent or more sensitive degree. Based on the results by flow cytometry and immunostaining, the pro-apoptotic effects of SM were observed in MDR K562/A02 cells. Furthermore, the RT-PCR results showed that SM induced the downregulation of MDR1 mRNA. In addition, the expression of P-gp and actin was decreased in the SM-treated cells, as measured by western blotting and immunostaining.Conclusions These results demonstrate that SM effectively triggers apoptosis in MDR tumor cells, which is associated with actin disruption and downregulation of MDR1 expression. This compound may merit further investigation as a potential therapeutic agent that bypasses the MDR mechanism for the treatment of MDR tumors.

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

    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.

  3. Cold-shock eliminates female nucleus in fertilized eggs to induce androgenesis in the loach (Misgurnus anguillicaudatus, a teleost fish

    Morishima Kagayaki

    2011-11-01

    Full Text Available Abstract Background Androgenesis (all-male inheritance is generally induced by means of irradiating the eggs to inactivate the maternal genome, followed by fertilization with normal sperm. In fish, the conventional technique for induced androgenesis has been applied for rapid fixation to traits, recovery of cryopreserved genotypes, sex-control, etc. A new method of androgenesis that eliminates the need to irradiate the egg was proposed using the loach, Misgurnus anguillicaudatus (a teleost fish. Results When the eggs of wild-type females were fertilized with sperm of albino or orange phenotype males and cold-shocked at 0 to 3°C for 60 min duration just after fertilization, generally more than 30% (with a peak of 100% of the hatched progeny were androgenotes. While a few of them were the normal diploid, most of them turned out to be abnormal haploid. All-male inheritance was verified by the expression of the recessive color trait (albino or orange and microsatellite genotypes comprising only paternally derived alleles. Nuclear behavior after the cold-shock treatment was traced by microscopic observation of DAPI (4'6-diamidino-2-phenylindole-stained samples and hematoxylin-eosin stained histological sections, and the extrusion of egg (maternal nucleus was observed in eggs treated in the optimum timing. Conclusion In this paper, we demonstrate that cold-shock treatment (at 0 and 3°C of loach eggs for 60 min just after fertilization successfully induces androgenetic haploid development. The most likely mechanism of cold-shock induced androgenesis is an elimination of the egg nucleus together along with the second polar body and subsequent development of a decondensed sperm nucleus or male pronucleus.

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

    Ehsan Yazdanparast

    2014-05-01

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

  5. 13-Acetoxysarcocrassolide Induces Apoptosis on Human Gastric Carcinoma Cells Through Mitochondria-Related Apoptotic Pathways: p38/JNK Activation and PI3K/AKT Suppression

    Ching-Chyuan Su

    2014-10-01

    Full Text Available 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.

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

    Newton Ruiz

    Full Text Available 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.

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

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

    2008-11-01

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

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

    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

  9. Detailed interrogation of trypanosome cell biology via differential organelle staining and automated image analysis

    Wheeler Richard J

    2012-01-01

    Full Text Available Abstract Background Many trypanosomatid protozoa are important human or animal pathogens. The well defined morphology and precisely choreographed division of trypanosomatid cells makes morphological analysis a powerful tool for analyzing the effect of mutations, chemical insults and changes between lifecycle stages. High-throughput image analysis of micrographs has the potential to accelerate collection of quantitative morphological data. Trypanosomatid cells have two large DNA-containing organelles, the kinetoplast (mitochondrial DNA and nucleus, which provide useful markers for morphometric analysis; however they need to be accurately identified and often lie in close proximity. This presents a technical challenge. Accurate identification and quantitation of the DNA content of these organelles is a central requirement of any automated analysis method. Results We have developed a technique based on double staining of the DNA with a minor groove binding (4'', 6-diamidino-2-phenylindole (DAPI and a base pair intercalating (propidium iodide (PI or SYBR green fluorescent stain and color deconvolution. This allows the identification of kinetoplast and nuclear DNA in the micrograph based on whether the organelle has DNA with a more A-T or G-C rich composition. Following unambiguous identification of the kinetoplasts and nuclei the resulting images are amenable to quantitative automated analysis of kinetoplast and nucleus number and DNA content. On this foundation we have developed a demonstrative analysis tool capable of measuring kinetoplast and nucleus DNA content, size and position and cell body shape, length and width automatically. Conclusions Our approach to DNA staining and automated quantitative analysis of trypanosomatid morphology accelerated analysis of trypanosomatid protozoa. We have validated this approach using Leishmania mexicana, Crithidia fasciculata and wild-type and mutant Trypanosoma brucei. Automated analysis of T. brucei

  10. A study of embryonic development in eriophyoid mites (Acariformes, Eriophyoidea) with the use of the fluorochrome DAPI and confocal microscopy.

    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. FORMULATION AND EVALUATION OF EXTENDED RELEASE MATRIX TABLETS OF TRIMETAZIDINE DIHYDROCHLORIDE

    Mogili Dinesh

    2013-01-01

    Full Text Available Oral ingestion has long been the most convenient and commonly employed route of drug delivery. Indeed, for Extended release systems, the oral route of administration has by far received the most attention with respect to research on physiological and drug constraints as well as design and testing of products. The primary objective of the extended release (Matrix drug delivery system is to ensure safety and to improve efficacy of drug as well as patient compliance. The present invention provides a novel sustained release composition comprising Trimetazidine Dihydrochloride. The objective of the present study was to formulate and evaluate once daily extended release matrix tablets of Trimetazidine Dihydrochloride using hydrophilic polymers Hydroxypropylmethylcellulose, Polyox, and natural polymer Xanthan gum. Trimetazidine has a half life 6 hrs and usual oral dosage regimen 0.5 mg and 60 mg daily. To reduce the frequency of administration and to improve patient compliance, a once-daily extended release formulation of Trimetazidine is desirable. The most commonly used method of modulating the drug release is to include it in a matrix system. Hydrophilic polymer matrix systems were widely used in oral controlled drug delivery because they make it easier to achieve a desirable drug-release profile, they are cost effective and they have broad US Food and Drug Administration acceptance. Hence, in present work, an attempt has been made to develop once daily sustained release matrix tablets of Trimetazidine using putative hydrophilic matrix materials. The drug release for extended duration using a hydrophilic matrix system is restricted because of rapid diffusion of dissolved drug through the hydrophilic gel network.

  12. Combined FISH, anti-γ-Hb and DAPI for detection of fetal nucleated RBCs in maternal blood

    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.

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

    Kietzmann Manfred; Siebert Joerg; Braun Michael; Stahl Jessica

    2011-01-01

    Abstract 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 ...

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

    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. DESIGN, DEVLOPMENT AND EVALUATION OF TRIMETAZIDINE DIHYDROCHLORIDE MODIFIED RELEASE TABLET USING 32 FACTORIAL DESIGN

    Biswal Biswajit

    2011-10-01

    Full Text Available The present investigation was undertaken to fabricate modified release tablet of Trimetazidine dihydrochloride using hydroxypropyl methylcellulose (HPMC and Ethyl cellulose as a matrixing agent. A 32 full factorial design was employed for the optimization of formulation. The percentage drug released at a given time (Q1, Q2 , Q4,Q8 ,Q 16 , Q24 and the time required for a given percentage of drug to be released (t50% were selected as dependent variables. The in vitro drug dissolution study was carried out in 1.2 pH , 4.5 pH acetate buffer and 6.8 pH phosphate buffer employing USP Type -1(basket at 75 rpm. The similarity factor (f2 was calculated for selection of best batch considering mean in vitro dissolution data of marketed product as a reference profile. It is concluded that the desired drug release pattern can be obtained by using a proper combination of HPMC (high gelling ability and ethyl cellulose (retard drug for longer period of time. The retardation capacity of ethyl cellulose and faster hydration rate of HPMC favors its use in modified release tablets. The matrix integrity during dissolution testing was maintained by using hydroxypropyl methylcellulose2.

  16. Acid–base titrimetric assay of hydroxyzine dihydrochloride in pharmaceutical samples

    Kanaka¬pura Basavaiah Vinay

    2010-07-01

    Full Text Available Two simple titrimetric methods have been developed for the determination of hydroxyzine dihydrochloride (HDH in pure form and in tablets. The principle of the methods are simple acid–base reactions in which the hydrochloride content of the drug was determined by titrating with an aqueous standardized NaOH solution either visually using phenolphthalein as indicator (method A or potentiometrically using glass-calomel electrode system (method B. The methods were applicable over the range of 2-20 mg HDH. The procedures were also applied for the determination of HDH in its dosage forms and the results were found to be in good agreement with those obtained by the reference method. The precision, expressed by intra-day and inter-day relative standard deviation values, was satisfactory (RSD ≤ 2.76%. The accuracy was satisfactory as well (RE ≤ 2.67%. Excipients used as additives in pharmaceutical formulations did not interfere in the proposed procedures as shown by the recovery study via a standard addition technique with recovery percentage in the range 97.48–106.3% with a standard deviation of 1.76–3.42 %.

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

    An S

    2013-02-01

    Full Text Available Songhie An,* Kihoon Nam,* Sunghyun Choi, Cheng Z Bai, Yan Lee, Jong-Sang ParkDepartment of Chemistry, Seoul National University, Seoul, Republic of Korea*These authors contributed equally to this workBackground: 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

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

    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: LIM0.5 and LID0.5. 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. LIM0.5 and LID0.5 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 potentially be a valuable

  19. Development of LC Method for the Simultaneous Determination of Antidepressant Drug Combination Melitracen Hydrochloride and Flupentixol Dihydrochloride in their Combined Dosage Form

    Usmangani K. Chhalotiya

    2011-01-01

    Full Text Available A simple, specific and stability-indicating reversed-phase high-performance liquid chromatographic method was developed for the simultaneous determination of melitracen hydrochloride and flupentixol dihydrochloride in tablet dosage form. A Brownlee C-18, 5 μm column having 250×4.6 mm i.d. in isocratic mode, with mobile phase containing 0.025 M potassium dihydrogen phosphate: methanol (10 : 90, v/v; pH 7.3 was used. The flow rate was 1.0 mL/min, and effluents were monitored at 230 nm. The retention times of melitracen hydrochloride and flupentixol dihydrochloride were 7.75 min and 5.50 min, respectively. The linearity for melitracen hydrochloride and flupentixol dihydrochloride were in the range of 0.5–60 μg/mL. The recoveries obtained for melitracen hydrochloride and flupenthixol dihydrochloride was 99.81–100.77% and 99.42–100.12%, respectively. Both the drugs were subjected to acid and alkali hydrolysis, chemical oxidation, and dry heat degradation and photodegradation. The proposed method was validated and successfully applied to the estimation of melitracen hydrochloride and flupentixol dihydrochloride in combined tablet dosage form.

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

    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

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

    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

  2. FORMULATION AND EVALUATION OF TASTE MASKED RAPIDLY DISINTEGRATING TABLET CONTAINING FLUPENTIXOL DIHYDROCHLORIDE

    Ahmed A. Elbary

    2011-09-01

    Full Text Available The aim of the present study was to develop rapid disintegrating tablets of Flupentixol dihydrochloride, a slightly bitter antipsychatric drug. An attempt has been made to prepare bitterless rapid disintegrating tablet using Eudragit E100 as a taste masking agent. The tablet was prepared with three superdisintegrants e.g. sodium starch glycolate, crosscarmellose sodium and crospovidone , each one was added in three different concentration 2%, 3% and 4% ; mass extrusion was the technique used for the preparation of these tablets. The blend was examined for angle of repose, bulk density, tapped density, compressibility index and Hausner’s ratio. The compressed tablets were evaluated for hardness, drug content, friability, disintegration time in-vitro and in-vivo, wetting time and dissolution rate. The contents of the prepared tablets were characterized by X-ray diffraction and Fourier transform infrared spectroscopy (FTIR. Different nine formulas showed in-vitro disintegration times ranges from 11.8 sec to 61 sec , but it was 150 sec for F1 ( formula without any superdisintegrant. These results were nearly correlated with in vivo disintegration times for the ten formulas. In vitro dissolution studies showed the release in the following descending order of superdisintegrants: Crospovidone > Croscarmellose sodium > Sodium Starch Glycolate. Maximum in vitro dissolution rate was found to be with formulation F10 which contains crospovidone (4%. Thus, F10 was considered the best among the other formulations. The stability study was conducted as the International Conference on Harmonization (ICH guidelines and the formulations subjected again for changes in hardness, friability, drug content, wetting time and disintegration time. Crospovidone at a concentration of 4% w/w is suitable for preparing rapid disintegrating tablet of Flupentixol dihydrocloride.

  3. Formulation and Evaluation of Modified Release Matrix Tablets of Trimetazidine Dihydrochloride

    Vinod J

    2013-02-01

    Full Text Available The purpose of the present study was to formulate the oral modified release tablets of Trimetazidine dihydrochloride by using Polyethylene oxide (Polyox WSR 303 LEO (35-55% as a rate controlling polymer. The tablets were prepared by direct compression method and coated by using film coating polymers. The powder mixtures were evaluated for angle of repose, loose bulk density, tapped bulk density and compressibility index and showed satisfactory results. All the ingredients were lubricated and compressed using 8.5mm circular shaped standard concave plain punches. The tablets were evaluated for uniformity of weight, content of active ingredient, thickness, friability, hardness and In-vitro dissolution studies. Drug content in the formulation was determined by UV- Visible Spectrophotometric method. All the formulations showed compliance with Pharmacopoeial standards. The in vitro release study of matrix tablets were carried out in pH 6.8 phosphate buffer for 12 hours. The prepared matrix tablets showed 100.00% release over a period of 12 hours. The dissolution profile of Formulation, F5 was similar to Innovator product in three different media such as pH 1.2, pH 4.5 acetate buffer and pH 6.8 phosphate buffer. It was observed that the amount of polymer in the tablets influences the drug release. In vitro release study results revealed that the release of the drug was retarded with the proportional increase in polymer concentration. It was indicated that the using a hydrophilic non-cellulose polymer in an appropriate concentration in tablet could control the rate of drug release.

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

    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

  5. Synthesis and high in vitro cytotoxicity of some (S,S-ethylenediamine-N,N’-di-2-propanoate dihydrochloride esters

    Pantelić Nebojša

    2014-01-01

    Full Text Available Novel (S,S-R2eddip ester, O,O’-diisoamyl-(S,S-ethylenediamine-N,N’-di-2-propanoate dihydrochloride, 1, was synthesized and characterized by IR, 1H and 13C NMR spectroscopy, mass spectroscopy and elemental analysis.In vitro antitumor action of 1, and two more R2eddip esters, O,O’-dialkyl-(S,S-ethylenediamine-N,N’-di-2-propanoate dihydrochlorides, obtained before, (alkyl = n-Bu, n-Pe; 2 and 3, respectively, was determined against cervix adenocarcinoma (HeLa, human melanoma (Fem-x, human chronic myelogenous leukemia (K562 cells, and a non-cancerous cell line human embryonic lung fibroblast (MRC-5, using MTT assay. Esters 1-3 showed higher cytotoxicity and better selectivity in comparison to cisplatin, used as reference compound. The highest activityis expressed by1,with IC50(Fem-xvalue1.51 ± 0.09 µM. [Projekat Ministarstva nauke republike Srbije, br. 172035 i br. 175011

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

    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

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

    M. M. Gupta

    2014-01-01

    Full Text Available 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.

  8. Biological Phosphorus Removal During High-Rate, Low-Temperature, Anaerobic Digestion of Wastewater.

    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

  9. Bacterial abundance, activity, and viability in the eutrophic River Warnow, northeast Germany.

    Freese, H M; Karsten, U; Schumann, R

    2006-01-01

    The River Warnow is the drinking water source for the city of Rostock. Its eutrophic status is accompanied by high amounts of bacteria, which may reach up to 24 x 10(6) cells mL(-1) as recorded during a seasonal study in 2002. Because the river is eutrophic and also heavily loaded with organic matter, this burden is a problem for drinking water purification, as it must be removed completely to not trigger new bacterial growth in the pipeline network. Therefore, restoration measures in the river have to be planned, and bacteria have to be favored as decomposers. That includes the investigation of the physiological state of bacteria in situ. Viable and active cells in the lower reaches of River Warnow were estimated using a broad set of methods. Intact bacteria were investigated by the LIVE/DEAD BacLight bacterial viability kit, containing a mixture of permeant and impermeant nucleic acid stains. Cells with ribosomes were visualized by fluorescence in situ hybridization with the EUB338 oligonucleotide probe. Intact cells and ribosome-containing bacteria represented 24% of total numbers stained by 4'6,-diamidino-2-phenylindole (DAPI) or 66 and 62%, respectively, in relation to all bacteria visualized by the LIVE/DEAD kit. Both fractions were considered as viable, although the fraction of RIB + bacteria is most likely underestimated by the protocol applied. 5-Cyano-2,3-ditolyltetrazolium chloride (CTC) was applied to mark respiring bacteria. The esterase substrate CellTracker Green 5-chloromethylfluorescein diacetate showed cells with intracellular hydrolytic activity. Whereas 1.5% of DAPI-stained bacteria were observed as respiring, 3.8% exhibited intracellular hydrolytic activity on average. If these active fractions were calculated as the percentages of intact cells, much higher fractions of 5.4% were respiring and 16% hydrolytic. Temperature was a main factor influencing total and viable cell numbers simultaneously. The results confirm that there are different

  10. Biological phosphorus removal during high-rate, low-temperature, anaerobic digestion of wastewater

    Ciara eKeating

    2016-03-01

    Full Text Available 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 0.4-1.5 kg COD m-3 d-1 and hydraulic retention times of 8-24 hours, while phosphate removal efficiency ranged from 28-78% 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 characterised polyphosphate accumulating organisms (PAOs such as Rhodocyclus, Chromatiales, Actinobacter and Acinetobacter was

  11. Analysis of a valuable chromosome rearrangement induced by ionizing radiations in a cultivated chili pepper line (Capsicum baccatum var. Pendulum - solanaceae)

    Capsicum (chili peppers) is an important genus including five crop species consumed by man as spice and food. Most contributions about induced mutagenesis in Capsicum refer to gene mutations while induced changes at chromosome level are scarce. We started a program to achieve chromosome rearrangements by ionizing radiations in C. baccatum variety pendulum cultivar 'cayenne', which has a karyotype with 2n=2x=24, 11 metacentrics + 1 subtelocentric pairs, 4 pairs (1, 3, 10, 12) carrying nucleolar organizing regions (NORs) and associated satellites in short arm. Seeds were treated with different acute doses of X-rays developing M1-4 generations. A rearranged chromosome carrying NORs in both arms was found in M2 seedlings from the only surviving M1 plant after a 300 Gy treatment. The structural change was analyzed by: 1) Feulgen's staining to observe chromosome number, size and shape; 2) silver impregnation to detect active NORs; 3) fluorescent chromosome banding to reveal type and position of constitutive heterochromatic regions [triple staining with chromomycin/distamycin/4-6-diamidino-2-phenylindole (CMA/DA/DAPI)]; 4) fluorescent in situ hybridization with 18S- 25S rDNA repeated sequence as probe. A reciprocal translocation between two NOR-bearing chromosomes in the M1 plant has occurred, which gave viable progeny carrying the chromosomal interchange without any deviating phenotype after four generations, nor in heterozygous neither in homozygous condition. The lack of chromosome instability suggests a small reciprocal interchange between a member of pair 1 and of 3, both carrying active NORs in shorts arms. The results of this rearrangement were two chromosomes with little change in size, one of them easily recognized by the presence of NORs and associated CMA+/DAPI- heterochromatin in both arms. As the translocation here reported produced a conspicuous rearranged marker chromosome, the obtained plant line is considered very valuable for studies on chromosome

  12. EFFECTS OF BETAHISTINE DIHYDROCHLORIDE AS ADJUVANT TO ENALAPRIL THERAPY OF PATIENTS WITH CHRONIC HEART FAILURE CLASS II-II (NYHA SUFFERING FROM GIDDINESS

    S. Y. Martsevich

    2008-01-01

    Full Text Available Aim. To study adjuvant effect of betahistine dihydrochloride to ACE inhibitors in patients with chronic heart failure (CHF class II-III suffering from giddiness.Material and methods. 61 patients with CHF class II-III, ejection fraction ≤45% (Simpson suffering from giddiness were involved into randomized open parallel study. Patients were randomized to Betahistine dihydrochloride plus basic CHF therapy or only basic therapy groups. Enalapril dose titration was performed in all patients. Quality of life and giddiness severity evaluation, electrocardiogram was performed initially and after treatment. Clinical examination results, drug therapy and adverse event were registered at each visit.Results. The target ACE inhibitor dose (≥20 mg daily was reached in 97 % of patients. It led to significant reduction of dyspnea, edemas, CHF class reduction and life quality increase. Significant differences between investigated groups were not found. Reduction of giddiness severity was shown in both groups. There was a trend to more prominent improvement of life quality (р=0,08 and more frequent achievement of target ACE inhibitor dose in patients treated with betahistine dihydrochloride.Conclusion. The target ACE inhibitor dose can be achieved more than in 90% of patients with CHF class II-III without hypotension symptoms. Adjuvant usage of betahistine dihydrochloride is necessary in patients with CHF still suffering from giddiness after achievement of target ACE inhibitor dose.

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

    Kietzmann Manfred

    2011-08-01

    Full Text Available Abstract 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.

  14. Application of well-defined indium tin oxide nanorods as Raman active platforms

    Zhao, Songqing; Guo, Yi; Song, Sheng; Choi, Daniel; Hahm, Jong-In

    2012-01-01

    We determine the surface enhanced Raman (SER) capability of indium tin oxide nanorods (ITO NRs) whose physical, chemical, and optical properties are precisely and uniformly controlled during synthesis. We demonstrate that the Raman intensities observed from varying concentrations of the pure and mixed molecules of rhodamine 6G and 4′,6-diamidino-2-phenylindole are much larger on ITO NRs relative to those measured on commercially available ITO-coated glass or Si. Our efforts signify the first ...

  15. Dioscin-induced autophagy mitigates cell apoptosis through modulation of PI3K/Akt and ERK and JNK signaling pathways in human lung cancer cell lines

    Hsieh, Ming-Ju; Tsai, Te-Lung; Hsieh, Yih-Shou; Wang, Chau-Jong; Chiou, Hui-Ling

    2013-01-01

    Our previous study has revealed that dioscin, a compound with anti-inflammatory, lipid-lowering, anticancer and hepatoprotective effects, may induce autophagy in hepatoma cells. Autophagy is a lysosomal degradation pathway that is essential for cell survival and tissue homeostasis. In this study, the role of autophagy and related signaling pathways during dioscin-induced apoptosis in human lung cancer cells was investigated. Results from 4′-6-diamidino-2-phenylindole and annexin-V/PI double-s...

  16. Tracking Chemotactic Migration of a Genetically Engineered Bacterium in the Presence of Constructed Nutrient Gradients Within a Sandy Aquifer in Cape Cod, Massachusetts

    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.

  17. Whole-body gene expression pattern registration in Platynereis larvae

    Asadulina Albina

    2012-12-01

    Full Text Available Abstract Background Digital anatomical atlases are increasingly used in order to depict different gene expression patterns and neuronal morphologies within a standardized reference template. In evo-devo, a discipline in which the comparison of gene expression patterns is a widely used approach, such standardized anatomical atlases would allow a more rigorous assessment of the conservation of and changes in gene expression patterns during micro- and macroevolutionary time scales. Due to its small size and invariant early development, the annelid Platynereis dumerilii is particularly well suited for such studies. Recently a reference template with registered gene expression patterns has been generated for the anterior part (episphere of the Platynereis trochophore larva and used for the detailed study of neuronal development. Results Here we introduce and evaluate a method for whole-body gene expression pattern registration for Platynereis trochophore and nectochaete larvae based on whole-mount in situ hybridization, confocal microscopy, and image registration. We achieved high-resolution whole-body scanning using the mounting medium 2,2’-thiodiethanol (TDE, which allows the matching of the refractive index of the sample to that of glass and immersion oil thereby reducing spherical aberration and improving depth penetration. This approach allowed us to scan entire whole-mount larvae stained with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP in situ hybridization and counterstained fluorescently with an acetylated-tubulin antibody and the nuclear stain 4’6-diamidino-2-phenylindole (DAPI. Due to the submicron isotropic voxel size whole-mount larvae could be scanned in any orientation. Based on the whole-body scans, we generated four different reference templates by the iterative registration and averaging of 40 individual image stacks using either the acetylated-tubulin or the nuclear-stain signal for each developmental

  18. Arsenic mineral dissolution and possible mobilization in mineral–microbe–groundwater environment

    Highlights: ► Bacteria use arsenic minerals for their growth without supplementary nutrient. ► Enzymatically active bacteria survive in the arsenic contaminated environment. ► Mostly bacillus, coccus and filamentous dissolves the arsenic mineral. ► Except enargite, soluble-As was detected with respect to other arsenic mineral. ► Dissolution: native-As > arsenolite > orpiment > realgar > arsenopyrite > tennantite. -- Abstract: 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 ± 1.25 mg/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

  19. Quantitative Expression and Co-Localization of Wnt Signalling Related Proteins in Feline Squamous Cell Carcinoma.

    Giuliano, Antonio; Swift, Rebecca; Arthurs, Callum; Marote, Georgina; Abramo, Francesca; McKay, Jenny; Thomson, Calum; Beltran, Mariana; Millar, Michael; Priestnall, Simon; Dobson, Jane; Costantino-Casas, Fernando; Petrou, Terry; McGonnell, Imelda M; Davies, Anthony J; Weetman, Malcolm; Garden, Oliver A; Masters, John R; Thrasivoulou, Christopher; Ahmed, Aamir

    2016-01-01

    Feline oral squamous cell carcinoma (FOSCC) is an aggressive neoplasm in cats. Little is known about the possible molecular mechanisms that may be involved in the initiation, maintenance and progression of FOSCC. Wnt signalling is critical in development and disease, including many mammalian cancers. In this study, we have investigated the expression of Wnt signalling related proteins using quantitative immunohistochemical techniques on tissue arrays. We constructed tissue arrays with 58 individual replicate tissue samples. We tested for the expression of four key Wnt/ß-catenin transcription targets, namely Cyclin D1 (CCND1 or CD1), FRA1, c-Myc and MMP7. All antibodies showed cross reactivity in feline tissue except MMP7. Quantitative immunohistochemical analysis of single proteins (expressed as area fraction / amount of tissue for normal vs tumor, mean ± SE) showed that the expression of CD1 (3.9 ± 0.5 vs 12.2 ± 0.9), FRA1 (5.5 ± 0.6 vs 16.8 ± 1.1) and c-Myc (5.4 ± 0.5 vs 12.5 ± 0.9) was increased in FOSCC tissue by 2.3 to 3 fold compared to normal controls (p<0.0001). By using a multilabel, quantitative fluorophore technique we further investigated if the co-localization of these proteins (all transcription factors) with each other and in the nucleus (stained with 4',6-diamidino-2-phenylindole, DAPI) was altered in FOSCC compared to normal tissue. The global intersection coefficients, a measure of the proximity of two fluorophore labeled entities, showed that there was a significant change (p < 0.01) in the co-localization for all permutations (e.g. CD1/FRA1 etc), except for the nuclear localization of CD1. Our results show that putative targets of Wnt signalling transcription are up-regulated in FOSCC with alterations in the co-localization of these proteins and could serve as a useful marker for the disease. PMID:27559731

  20. The effect of simvastatin on proliferation and apoptosis of the osteoblast in simulated microgravity environment%辛伐他汀在模拟微重力环境对成骨细胞增殖和凋亡的影响

    宋淑军; 司少艳; 刘俊丽; 周金莲; 史亮; 贾桂玥; 谭小清; 张建中

    2011-01-01

    目的 观察辛伐他汀在模拟微重力环境下对成骨细胞增殖和凋亡的影响.方法 利用沿水平轴连续回转(30r/min)细胞培养系统模拟微重力环境,使用MTT比色法观察小鼠成骨样细胞MC3T3-E1的增殖情况;细胞凋亡是根据4,6-二氨基-2-苯基吲哚所染细胞核形态的改变来辨认.结果 模拟微重力环境可以降低MC3T3-E1增殖功能,辛伐他汀在模拟微重力环境可以增强MC3T3-E1增殖功能,但对细胞的凋亡并无显著影响.结论 在模拟微重力环境下辛伐他汀对成骨细胞增殖功能具有保护作用,为辛伐他汀治疗微重力环境骨丢失提供了理论和实验证据.%Objective To observe the effect of simvastatin on proliferation and apoptosis of the osteoblast in simulated microgravity environment. Methods The microgravity environment was simulated using a horizontally rotated (30 r/min) cell culture system. The proliferation of mouse MC3T3-E1 osteoblast-like cells was observed using MTT assay. Cell apoptosis was identified according to the variation of nuclear morphology using 4, 6-diamidino-2 phenylindole ( DAPI) staining. Results Simulated microgravity environment could decrease the proliferation of MC3T3-E1 cells. Simvastatin could increase the proliferation of MC3T3-E1 in simulated microgravity environment. However, simvastatin had no effect on apoptosis of the osteoblast. Conclusion Simvastatin plays a protective role for osteoblast proliferation in simulated microgravity environment. This provides theoretical and experimental evidences of simvastatin for the treatment of microgravity-induced bone loss.

  1. HIV antiretroviral drug combination induces endothelial mitochondrial dysfunction and reactive oxygen species production, but not apoptosis

    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

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

    Islam, A.B.M.R., E-mail: uttambangla@yahoo.com [Department of Human Ecology, School of International Health, Graduate School of Medicine, The University of Tokyo,7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); ITO Public Nuisance Research Institute, 1-26-8, Omori Kita, Otaku, Tokyo 142-0016 (Japan); Maity, Jyoti Prakash, E-mail: jyoti_maity@yahoo.com [Department of Earth and Environmental Sciences, National Chung Cheng University, 168 University Road, Ming-Shung, Chiayi County 62102, Taiwan (China); Bundschuh, Jochen [Faculty of Engineering and Surveying, University of Southern Queensland, Toowoomba, Queensland 435 (Australia); KTH-International Groundwater Arsenic Research Group, Department of Land and Water Resources Engineering, Royal Institute of Technology (KTH) Teknikringen 76,SE-10044 Stockholm (Sweden); Department of Earth Sciences, National Cheng Kung University, University Road, Tainan, 70101, Taiwan (China); Chen, Chien-Yen [Department of Earth and Environmental Sciences, National Chung Cheng University, 168 University Road, Ming-Shung, Chiayi County 62102, Taiwan (China); Bhowmik, Bejon Kumar [Department of Biotechnology, Graduate School of Agriculture and Life science, The University of Tokyo, 1-1-1,Yayoi, Bunkyo-ku, Tokyo 113-0033 (Japan); Tazaki, Kazue [Department of Earth Sciences, Faculty of Science, Kanazawa University, Kakuma, Kanazawa, Ishikawa 920-1192 (Japan)

    2013-11-15

    Highlights: ► Bacteria use arsenic minerals for their growth without supplementary nutrient. ► Enzymatically active bacteria survive in the arsenic contaminated environment. ► Mostly bacillus, coccus and filamentous dissolves the arsenic mineral. ► Except enargite, soluble-As was detected with respect to other arsenic mineral. ► Dissolution: native-As > arsenolite > orpiment > realgar > arsenopyrite > tennantite. -- Abstract: 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 Eh{sub S.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 ± 1.25 mg/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 (As{sub 4}S{sub 4}). Therefore, the microbes enable to survive and mobilize the As in groundwater by dissolution/bioweathering of As-minerals.

  3. Biological effects of positron emitters in thyroid cell cultures

    Full text of publication follows. Aim: Today, the use of 124I- (β+, half-life 4.2 d) is an increasing field in positron emission tomography (PET). In principle, positrons deposit their energy in the surrounding material like electrons. Therefore, we investigated the biological effects of positron emitters in comparison to electrons in vitro. Materials and Methods: two different thyroid cell lines (Fischer Rat Thyroid Cell Line No. 5 (FRTL5) and human papillary thyroid cancer cell line BCPAP) were investigated in vitro. While FRTL5 has been described to express a high level of sodium iodine transporter (NIS), the NIS expression of BCPAP is known to be low. Parallel cultures were incubated with either 50 -400 kBq/ml 124I- (IBA) or 50-400 kBq/ml 131I- (GE Health care). Cell count and radioiodine uptake were determined 24 h to 144 h after isotope application. Additionally, 4',6-diamidino-2-phenylindole (DAPI) staining of ethanol fixed cells was performed and induced apoptosis was determined by morphological analysis of the cell nucleus (condensation, fragmentation) by fluorescence microscopy. Results: BCPAP showed no significant uptake (<0.1 % per one million cells). The proliferation of BCPAP cells was not significantly influenced by radioiodine incubation. The uptake of NIS-expressing FRTL5 cells ranged between 0.6 % and 4 % per one million cells, independently of the isotope. In FRTL5 cells the incubation with 131I- induced a significant dose-dependent inhibition of proliferation (p<0.05). The positron emitter 124I- induced analogue effects on proliferation compared to the electron emitter 131I- (30-40 % inhibition, 144 h incubation with 400 kBq/ml). In parallel, in FRTL5 cell lines an isotope-independent increase of morphological changes in the cell nuclei (up to 5 fold) could be determined. In contrast, no significant changes could be verified in BCPAP cell nuclei. Conclusions: As expected from the physical point of view, the biological effects of positrons

  4. 盐酸大观霉素溶析结晶过程模拟与分析%Simulation Analysis of Dilution Crystallization of Spectinomycin Dihydrochloride Pentahydrate

    鲍颖; 王静康; 王永莉

    2005-01-01

    A mathematical model for dilution crystallization of spectinomycin dihydrochloride pentahydrate was established on the basis of the population and mass balance. Three operating modes, i.e., constant mass rate addition of diluent, constant mass fraction of diluent and constant size-independent growth rate, were investigated over a wide range of controlled parameters. The intrinsic characteristics of the crystallization process and the influence of operation parameters were analyzed in detail. Suitable strategy for better performance was suggested.

  5. Transplantation of adipose-derived stem cells with fibrin glue for treatment of acute myocardial infarction in rat%纤维蛋白胶联合干细胞移植治疗大鼠急性心肌梗死

    张雪莲; 马依彤; 王常勇; 马翔; 阿迪拉·阿扎提; 刘芬; 陈邦党; 王宝珠

    2012-01-01

    Objective To investigate the cell survival of the combination of fibrin glue and adiposederived stem cells (ADSCs) in rats when implanted into ischemic myocardium and the improvement of heart function.Methods The rat ADSCs were isolated from the subcutaneous adipose tissues.The surface phenotype of these cells was analyzed by flow cytometry.Myocardial infarction was induced in female rats using coronary artery ligation.One week after MI,surviving rats were randomized (random nuber) into 4 groups,control group (n =10),fibrin group (n =10),cell group (n =10) and combination group (n =10).100 μl of PBS was injected into the ischemic myocardium in control group.100 μl of Fibrin glue were injected into ischemic myocardium in fibrin group.100 μl of ADSCs labeled with DAPI were injected into the infract along the border zone in cell group.ADSCs in 100 μl of fibrin glue were injected into the infract in combination group.Four weeks after the injection the surviving rats underwent examination of heart functions by the Hemodynamics.The rats were killed and their hearts were taken out to undergo immunohistochemistry with 4,6-diamidino-2-phenylindole (DAPI) and actin and factor Ⅶ to measure the area of cardiac infarction and the capillary density.The heart infarcted size was calculated by masson trichrome staining.All data was analyzed by software SPSS 15.0,ANOVA comparison tests and the student t test were used,and P < 0.05 was considered as statistically significant.Results Four weeks after the cells were transplanted,LVSP and + dp/dtmax of combination group were highest among all groups.The heart infarcted size of the combination group was (28.5 ± 3.6) %,significantly less than those of the cell group (33.33 ± 2.3) % and fibrin group (35.96 ± 2.11) %,both P < 0.05.The capillary density of the combination group was (108.7 ± 11.38) /mm2,significantly greater than those of the cell group and that of the fibrin group,and greater than that of the control

  6. The Early Embryonic Development of Red Crucian Carp Stained With DAPI%用DAPI染色法观察红鲫胚胎发育

    张纯; 刘少军

    2011-01-01

    Using fluorescence stered microscope, the early embryonic development of red crucian carp stained with DA-PI is described in the present study. The results show that this method is convenient and can make the figures of embryo clearer. It is worth mentioning that using this method, the positioning of DNA in nuclei can be observed clearly. For example, the blastocysts cells showing mitosis frequently was observed by this method. The method provides a meaningful way for the study of early embryonic development of fish and relative feature of cell division.%以红鲫为研究材料,采用DAPI荧光染料对脱膜的红鲫胚胎进行染色,在荧光体视显微镜下观察红鲫胚胎早期卵裂到囊胚发育的过程.结果表明,该染色方法操作便捷,能更清晰的观察到胚胎发育的外形.值得一提的是:该方法较光学显微镜观察能清晰的观察到核DNA在细胞中的定位,如囊胚期能观察到早期卵裂细胞正进行频繁的有丝分裂等.该方法的获得为研究鱼类早期胚胎发育及相关细胞分裂特征提供了有意义的途径.

  7. Ultrasonic microdialysis coupled with capillary electrophoresis electrochemiluminescence study the interaction between trimetazidine dihydrochloride and human serum albumin.

    Sun, Shuangjiao; Long, Chanjuan; Tao, Chunyao; Meng, Sa; Deng, Biyang

    2014-12-01

    The paper describes a homemade ultrasonic microdialysis device coupled with capillary electrophoresis electrochemiluminescence (CE-ECL) for studying the interaction between human serum albumin (HSA) and trimetazidine dihydrochloride (TMZ). The time required for equilibrium by ultrasonic microdialysis was 45min, which was far less than that by traditional dialysis (240min). It took 80min to achieve the required combination equilibrium by normal incubation and only 20min by ultrasonic. Compared with traditional dialysis, the use of ultrasonic microdialysis simplified experimental procedures, shortened experimental time and saved consumption of sample. A simple, sensitive and selective determination of TMZ was developed using CE-ECL and the parameters that affected ECL intensity were optimized. Under the optimized conditions, the linear range of TMZ was from 0.075 to 80μmol/L (r(2)=0.9974). The detection limit was 26nmol/L with RSD of 2.8%. The number of binding sites and binding constant were 1.54 and 15.17L/mol, respectively. PMID:25440662

  8. Inactivation of Listeria monocytogenes, Salmonella spp. and Escherichia coli O157:H7 on cantaloupes by octenidine dihydrochloride.

    Upadhyay, Abhinav; Chen, Chi-Hung; Yin, Hsinbai; Upadhyaya, Indu; Fancher, Samantha; Liu, Yanyan; Nair, Meera Surendran; Jankelunas, Leanne; Patel, Jitendra R; Venkitanarayanan, Kumar

    2016-09-01

    The efficacy of a new generation disinfectant, octenidine dihydrochloride (OH), as wash and coating treatments for reducing Listeria monocytogenes (LM), Salmonella spp. (SAL), and Escherichia coli O157:H7 (EC) on cantaloupe was investigated. Cantaloupe rind plugs inoculated separately with the three bacterial species (∼8 log CFU/cm(2)) were washed for 1, 3, 5 min at 25 °C in water, or chlorine (200 ppm), ethanol (1%), OH (0.01, 0.05, 0.1%) and surviving populations were measured after treatment. Additionally, inoculated cantaloupe rind plugs were coated with 2% chitosan or chitosan containing OH (0.01, 0.05, 0.1%) and sampled for surviving pathogens. Subsequently, the antimicrobial efficacy of OH wash and coating (0.1, 0.2%) on whole cantaloupes was determined. All OH wash reduced LM, SAL, and EC on cantaloupe rinds by > 5 log CFU/cm(2) by 2 min, and reduced populations to undetectable levels (below 2 log CFU/cm(2)) by 5 min (P Washing and coating whole cantaloupes with OH reduced the three pathogens by at least 5 log and 2 log CFU/cm(2), respectively (P wash and coating to reduce LM, SAL, and EC on cantaloupes. PMID:27217367

  9. The antimicrobial effect of Octenidine-dihydrochloride coated polymer tracheotomy tubes on Staphylococcus aureus and Pseudomonas aeruginosa colonisation

    Leonhard Matthias

    2009-07-01

    Full Text Available Abstract Background The surface of polymeric tracheotomy tubes is a favourable environment for biofilm formation and therefore represents a potential risk factor for the development of pneumonia after tracheotomy. The aim of this in-vitro study was to develop octenidine-dihydrochloride (OCT coated polymer tracheotomy tubes and investigate any effects on Staphylococcus (S. aureus and Pseudomonas (P. aeruginosa colonization. Additionally the resistance of the OCT coating was tested using reprocessing procedures like brushing, rinsing and disinfection with glutaraldehyde Results Contamination with S. aureus: Before any reprocessing, OCT coated tracheotomy tubes were colonized with 103 cfu/ml and uncoated tracheotomy tubes with 105 cfu/ml (P = 0.045. After reprocessing, no differences in bacterial concentration between modified and conventional tubes were observed. Contamination with P. aeruginosa: Before reprocessing, OCT coated tubes were colonized with 106 cfu/ml and uncoated tubes with 107 cfu/ml (P = 0.006. After reprocessing, no significant differences were observed. Conclusion OCT coating initially inhibits S. aureus and P. aeruginosa colonisation on tracheotomy tubes. This effect, however, vanishes quickly after reprocessing of the tubes due to poor adhesive properties of the antimicrobial compound. Despite the known antimicrobial effect of OCT, its use for antimicrobial coating of tracheotomy tubes is limited unless methods are developed to allow sustained attachment to the tube.

  10. 干细胞因子促进骨髓间充质干细胞向心肌细胞的分化%Differentiation of mesenchymal stem cells into myocardial cells promoted by stem cell factors

    鲍翠玉; 郭军; 马业新; 郑敏

    2006-01-01

    BACKGROUND: Mesenchymal stem cells (MSCs) can differentiate into myocardial cells in vitro and in vivo, but the amount is small and directed differentiation rate is low.OBJECTIVE: To explore the effect of stem cell factor (SCF) on promotion of MSCs differentiating into myocardial cells.DESIGN: Opening experiment.SETTING: Institute of Cardiovascular Disease, Xianning College and Department of Cardiology, Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: The experiment was performed at the Experimental Center of Institute of Cardiovascular Disease, Xianning College from October 2003to August 2004. A total of 20 infant SD rats aged 1-2 days were selected for culture of myocardial cells. Another 25 clean adult SD rats were selected and randomly divided into SCF group, blank control group with 12 rats in each group. The left one rat was used for MSCs extraction, culture and purification.METHODS: ①MSCs were labeled with 4', 6-diamidino-2-phenylindole,dihydrochloride (DAPI) before co-culture. SCF was given into rats of the SCF group by subcutaneous injection successively for 5 days, 20 μg/kg per day. Isolated MSCs were co-cultured with myocardial cells that had been cultured for 3 days. Saline of the same volume was given in the blank control group by subcutaneous injection. MSCs without any intervention were co-cultured with myocardial cells that had been cultured for 3 days.②Expressions of MHC α/β and troponin T were recorded and measured with digital micro-camera shot and immunofluorescence technique, respectively. Percentage of DAPI labeled MSCs differentiating into myocardial cells was measured.MAIN OUTCOME MEASURES: ①Growth of MSCs, ②detection of labeling rate of DAPI on MSCs, ③analysis of activity and purity of co-cultured myocardial cells, and ④differentiation of MSCs into myocardial cells after co-culture.RESULTS: ①Bone marrow cell suspension was inoculated in plastic petri dish. Round

  11. 家蚕中肠上皮细胞增殖和分化的初步研究%A Preliminary Study on Proliferation and Differentiation of Intestinal Epithelial Cells of the Silkworm, Bombyx mori

    陈毅彪; 李婷; 郑春琴; 郝向伟; 韦转琴; 向仲怀; 鲁成; 崔红娟

    2013-01-01

    In this study,we indentified the potential location of intestinal stem cells in the silkworm (Bombyx mori)through analysis of proliferation and differentiation of midgut epithelial cells during different developmental stages of larva to pupa.And we observed the morphological structure and cellular component of silkworm midgut during metamorphosis and development of silkworm larvae by Hematoxylin and Eosin (H&E) staining and 4',6-diamidino-2-phenylindole (DAPI) staining.The results showed that the morphological structure and cellular component of silkworm midgut had remarkable changes in the process of molting and metamorphosis of silkworm larvae.The intestinal wall was thin at full appetite stage of each instar,became thicker before molting,and reached peak value at molting stage.There were three types of cells,namely columnar cells (CC),goblet cells (GC) and regenerative cells (RC),in the midgut epithelium.These three types of cells increased gradually with the advance of larval instar.Among them,the goblet cells increased continuously in all instars and reached peak value at molting stage,while the small cells near basal lamina increased at pre-molting stage.Observation by 5-Bromo-2-deoxyUridine (BrdU) and Phospho-histone H3 (PHH3)immunofluorescence staining revealed that midgut epithelial cells,especially the small cells near basal lamina of midgut epithelium,had the highest proliferation rate at premolting stage of each instar.Meanwhile,BrdU label retention assay disp.layed positive signal of BrdU retention in the midgut epithelium near basal lamina.These results demonstrated rapid proliferation of small cells near basal lamina of midgut epithelium during molting of silkworm larvae,suggesting the existence of potential intestinal stem cells in these small cells.%通过分析家蚕自幼虫期到蛹期发育过程中肠上皮细胞的增殖与分化情况,鉴定家蚕中肠干细胞的潜在定位.采用苏木精和伊红(Hematoxylin and Eosin,H&E)染色与4

  12. Extraction-Free Ion-Pair Methods for the Assay of Trifluoperazine Dihydrochloride in Bulk Drug, Tablets, and Spiked Human Urine Using Three Sulfonphthalein Dyes

    Prashanth, K. N.; Swamy, N.; Basavaiah, K.

    2014-11-01

    Three simple and sensitive extraction-free spectrophotometric methods are described for the determination of trifluoperazine dihydrochloride (TFH). The methods are based on ion pair complex formation between the nitrogenous compound trifluoperazine (TFP) converted from trifluoperazine dihydrochloride and sulfonphthalein dyes, namely, bromocresol green (BCG), bromothymol blue (BTB), and bromophenol blue (BPB) in dichloromethane medium in which all the above experimental variables were circumvented. The colored products are measured at 425 nm in the BCG method, 415 nm in the BTB method, and 420 nm in the BPB method. The stoichiometry of the ion-pair complexes formed between the drug and dye (1:1) was determined by Job's continuous variations method, and the stability constants of the complexes were also calculated. These methods quantify TFP over the concentration ranges of 1.25-20.0 μg/ml in the BCG method, 1.5-21.0 μg/ml in the BTB method, and 1.5-18.0 μg/ml in the BPB method. The molar absorptivity (l·mol-1·cm-1) and Sandell sensitivity (ng/cm2) were calculated to be 2.06·104 and 0.0197; 1.82·104 and 0.0224; and 2.22·104 and 0.0183 for the BCG, BTB, and BPB methods, respectively. The methods were successfully applied to the determination of TFP in pure drug, pharmaceuticals, and in spiked human urine with good accuracy and precision.

  13. Utility of positron annihilation lifetime technique for the assessment of spectroscopic data of some charge-transfer complexes derived from N-(1-Naphthyl)ethylenediamine dihydrochloride.

    Refat, Moamen S; Adam, Abdel Majid A; Sharshar, T; Saad, Hosam A; Eldaroti, Hala H

    2014-03-25

    In this work, structural, thermal, morphological, pharmacological screening and positron annihilation lifetime measurements were performed on the interactions between a N-(1-Naphthyl)ethylenediamine dihydrochloride (NEDA·2HCl) donor and three types of acceptors to characterize these CT complexes. The three types of acceptors include π-acceptors (quinol and picric acid), σ-acceptors (iodine) and vacant orbital acceptors (tin(IV) tetrachloride and zinc chloride). The positron annihilation lifetime parameters were found to be dependent on the structure, electronic configuration, the power of acceptors and molecular weight of the CT complexes. The positron annihilation lifetime spectroscopy can be used as a probe for the formation of charge-transfer (CT) complexes. PMID:24291622

  14. Molecular cytogenetic characterization of some representatives of the subgenera Artemisia and Absinthium (genus Artemisia, Asteraceae

    Vallès, J.

    2008-12-01

    Full Text Available A molecular cytogenetic study has been performed in three species of the genus Artemisia, complementing previous works on two subgenera that had been scarcely studied from this standpoint, Artemisia ( A. chamaemelifolia, A. vulgaris and Absinthium ( A. absinthium. Chromomycin A3 and 4',6-diamidino-2-phenylindole (DAPI banding have been carried out, as well as fluorescent in situ hybridization (FISH of 5S and 18S-5.8S-26S ribosomal DNA. Morphometrical data of karyotype characters were calculated and idiograms with the position of the AT- and GC-rich regions as well as rDNA loci were constructed. Colocalization of most of these regions has been observed, confirming previous findings in this genus. Both ribosomal DNA appear always colocalized, which is a distinct feature with respect to most angiosperms surveyed. Regarding the differential characteristics of each species, a symmetrical karyotype has been found in the species studied. Artemisia absinthium shows long chromosomes and absence of centromeric banding signals that, conversely, are absent in A. vulgaris andA. chamaemelifolia. The last species also presents B-chromosomes in which ribosomal DNA and heterochromatin have been detected. Despite these differences, karyotype morphology and signal pattern of the three species are quite coincidental. This might reflect a close phylogenetic relationship between both subgenera, which is consistent with the available molecular phylogenies presenting species of the subgenera Artemisia and Absinthium intermixed.

    Se ha llevado a cabo un estudio citogenético molecular en tres especies del género Artemisia, que complementa trabajos previos sobre dos subgéneros que han sido poco estudiados desde este punto de vista, Artemisia (A. chamaemelifolia, A. vulgaris y Absinthium (A. absinthium. Se han efectuado tinciones de bandeo con cromomicina A3

  15. Formulation development of fast releasing oral thin films of levocetrizine dihydrochloride with Eudragit® Epo and optimization through Taguchi orthogonal experimental design

    P K Lakshmi

    2011-01-01

    Full Text Available The aim of this study was to develop a fast releasing oral polymeric film, prepared using the solvent casting method, with good mechanical properties, instant disintegration and dissolution, an acceptable taste in the oral cavity. Levocetirizine dihydrochloride, an antihistamine, was incorporated to relieve the symptoms of allergic rhinitis. Four batches of films with drug were prepared using different combinations of polymers and plasticizers Eudragit; EPO, HPMC E 5 LV, and PVA were the selected polymers. Glycerin, dibutyl phthalate, propylene glycol, and PEG 400 were the selected plasticizers. The resultant films were evaluated for weight variation, assay, content uniformity, folding endurance, thickness, tensile strength, percent elongation, surface pH, in vitro disintegration and in vitro dissolution. The formulations from the preliminary trial were analyzed using Taguchi OA experimental design, which was applied to optimize the type of polymers, concentration of polymers, plasticizer, and sweetener based on their disintegration data at three different levels. The optimized films disintegrated in less than 30s, releasing 70-90% of drug within 2 mins. The percentage release varied with the type of polymer and concentration of polymer. The films made with EPO released 96% of drug in 2 mins, which was the best release among all.

  16. Stability-indicating determination of trimetazidine dihydrochloride inthe presence of two of its related substances using a direct GC/MS method.

    Belal, Tarek S; Awad, Tamer; Clark, C Randall

    2014-01-01

    A novel, simple, direct, and selective stability- indicating GC/MS procedure was developed for the determination of the anti-ischemic drug trimetazidine dihydrochloride (TMZ) in the presence of two of its related substances (potential impurities), namely, 2,3,4-trimethoxybenzyl alcohol (T1) and 2,3,4-trimethoxybenzaldehyde (T2). The method involved resolution of the undeilvatized compounds using a 100% dimethylpolysiloxane (Rtx-1) capillary column, and MS detection was carried out in the electron-impact mode. The peaks of the three compounds eluted at retention times 11.69, 11.92, and 15.47 min for T1, T2, and TMZ, respectively. Quantification of the parent drug TMZ was based on measuring its peak area. The reliability and analytical performance of the proposed method, including linearity, range, precision, accuracy, selectivity, detection, and quantification limits, were statistically validated. The calibration curve of TMZ was linear over the range 100-600 μg/mL. The proposed method was successfully applied to the assay of TMZ in several commercially available pharmaceutical formulations with recoveries not lessthan 96.2%. PMID:25632428

  17. Study on antitussive effects of eprazinone dihydrochloride on radiation induced inflammation of upper respiratory tract due to postoperative irradiation of breast cancer

    Discussion was made of antitussive effects of Eprazinone hydrochloride (Resplen) on radiation induced inflammation of the upper respiratory tract in 15 cases irradiated after an operation of breast cancer. Depth dose at 3 cm was 200 rads/day, and 5000 rad/25 times/5 weeks was irradiated with telecobalt. Mainly abnormal sensation and pain in the throat and cough appeared and patients complained of suffering from a common cold, because a part of irradiation field included the throat, trachea, and esophagus. At the same time as manifestation of symptoms, 120 mg/day of Eprazinone Dihydrochloride was administered. In 8 cases, other drugs were added to, or irradiation schedule was changed because the symptoms were not improved or were exaggerated. The symptoms disappeared up to 5000 rad irradiation in one case, up to 4000 - 5000 rad irradiation in 4 cases, and up to 4000 rad irradiation in 2 cases. It is impossible to decide effective rate of this drug because of shortage of clinical cases, but it is suggested that there is a significance to increase cases treated successively with this drug in future. (Tsunoda, M.)

  18. Alteration of gene expression in Pisum sativum tissue cultures caused by the free radical-generating agent 2,2`-azobis (2-amidinipropane) dihydrochloride

    Henkow, L. [Sveriges Lantbruksuniv., Inst. foer Vaextfoeraedling, Uppsala (Sweden); Strid, Aa.; Rydstroem, J. [Goeteborgs Univ. och Chalmers Tekniska Hoegskola, Inst. foer Biokemi och Biofysik, Goeteborg (Sweden); Berglund, T.; Ohlsson, A.B. [Kungliga Tekniska Hoegskolan, Inst. foer Biokemi och biokemisk Teknologi, Stockholm (Sweden)

    1996-04-01

    Root-differentiated tissue cultures (PS-R) from Pisum sativum (cv. Greenfeast) were exposed to a 5 mM solution of the free radical-generating compound 2,2`-azobis (2-amidinopropane) dihydrochloride (AAPH). The levels of mRNA transcripts for two genes were examined: chs2, encoding a chalcone synthase isozyme, and cab, encoding the chlorophyll a/b-binding protein of the light-harvesting antenna complex. In light-grown PS-R, cab mRNA transcript levels decreased to 14% of controls after 6 h of exposure, whereas chs2 mRNA levels increased 50-fold. In dark-grown PS-R, chs2 mRNA transcripts increased by 40-fold compared with the controls. Glutathione determination inlight-grown PS-R showed no substantial difference in total glutathione (GSH{sub tot}), whereas oxidized glutathione (GSSG) increased by 66% after 12 h of exposure. However, in dark-grown PS-R a decrease in both GSH{sub tot} and GSSG after 6 h was followed by an increase of about 70%, as compared with the controls, after 12 h of exposure. In conclusion AAPH generated oxidative stress, reflected in changed glutathione levels and induced expression of the chs2 gene of the flavonoid biosynthetic pathway and also caused a decreased level of mRNA for the photosynthetic cab gene. (au) 39 refs.

  19. Validated derivative and ratio derivative spectrophotometric methods for the simultaneous determination of levocetirizine dihydrochloride and ambroxol hydrochloride in pharmaceutical dosage form

    Ali, Omnia I. M.; Ismail, Nahla S.; Elgohary, Rasha M.

    2016-01-01

    Three simple, precise, accurate and validated derivative spectrophotometric methods have been developed for the simultaneous determination of levocetirizine dihydrochloride (LCD) and ambroxol hydrochloride (ABH) in bulk powder and in pharmaceutical formulations. The first method is a first derivative spectrophotometric method (1D) using a zero-crossing technique of measurement at 210.4 nm for LCD and at 220.0 nm for ABH. The second method employs a second derivative spectrophotometry (2D) where the measurements were carried out at 242.0 and 224.4 nm for LCD and ABH, respectively. In the third method, the first derivative of the ratio spectra was calculated and the first derivative of the ratio amplitudes at 222.8 and 247.2 nm was selected for the determination of LCD and ABH, respectively. Calibration graphs were established in the ranges of 1.0-20.0 μg mL- 1 for LCD and 4.0-20.0 μg mL- 1 for ABH using derivative and ratio first derivative spectrophotometric methods with good correlation coefficients. The developed methods have been successfully applied to the simultaneous determination of both drugs in commercial tablet dosage form.

  20. DEVELOPMENT AND VALIDATION OF FIRST ORDER DERIVATIVE UV SPECTROPHOTOMETRIC METHOD FOR SIMULTANEOUS ESTIMATION OF PROPRANOLOL HYDROCHLORIDE AND FLUNARIZINE DIHYDROCHLORIDE IN BULK AND COMBINED DOSAGE FORM

    Wagh Dipmala Dilip

    2013-06-01

    Full Text Available The first order derivative of UV spectrometry method for simultaneous determination of Propranolol hydrochloride (PRO and Flunarizine dihydrochloride (FLU in pure bulk drug and combined dosage form was found to be simple, accurate, fast, precise and reproducible. The first derivative values measured at 289nm for PRO and 253nm for FLU. The linearity for zero order derivative method was carried out by using the concentration range 4-28µg/ml for PRO and 3-7µg/ml for FLU. The coefficient correlation of PRO and FLU for zero order was found to be 0.9995 and 0.9991 respectively. At zero crossing point of PRO (289nm FLU showed a measurable derivative absorbance where as at the zero crossing point of FLU (253nm, PRO showed appreciable derivative absorbance value. The coefficient correlation of PRO and FLU for first order derivative was found to be 0.9991 and 0.9995 respectively. Precision study showed that % RSD was within the range of acceptable limits (<2. The % recovery for PRO and FLU was found to be in the range of 98-102% and 100-101% respectively. The percentage assay was found to be as 99.5 and 100.12% for PRO and FLU. The results of analysis have been validated as per ICH Q2 (R1 guidelines. This method has applied successfully for the determination of PRO and FLU in its combination with a high percentage of recovery good accuracy and precision.

  1. Anticancer Effect of Ursodeoxycholic Acid in Human Oral Squamous Carcinoma HSC-3 Cells through the Caspases

    Liang Pang

    2015-05-01

    Full Text Available Bear bile was used as a traditional medicine or tonic in East Asia, and ursodeoxycholic acid (UDCA is the most important compound in bear bile. Further, synthetic UDCA is also used in modern medicine and nutrition; therefore, its further functional effects warrant research, in vitro methods could be used for the fundamental research of its anticancer effects. In this study, the apoptotic effects of UDCA in human oral squamous carcinoma HSC-3 cells through the activation of caspases were observed by the experimental methods of MTT (3-(4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2-H-tetrazolium bromide assay, DAPI (4’,6-diamidino-2-phenylindole staining, flow cytometry analysis, RT-PCR (reverse transcription-polymerase chain reaction assay and Western blot assay after HSC-3 cells were treated by different concentrations of UDCA. With 0 to 400 μg/mL UDCA treatment, UDCA had strong growth inhibitory effects in HSC-3 cells, but had almost no effect in HOK normal oral cells. At concentrations of 100, 200 and 400 μg/mL, UDCA could induce apoptosis compared to untreated control HSC-3 cells. Treatment of 400 μg/mL UDCA could induce more apoptotic cancer cells than 100 and 200 μg/mL treatment; the sub-G1 DNA content of 400 μg/mL UDCA treated cancer cells was 41.3% versus 10.6% (100 μg/mL and 22.4% (200 μg/mL. After different concentrations of UDCA treatment, the mRNA and protein expressions of caspase-3, caspase-8, caspase-9, Bax, Fas/FasL (Fas ligand, TRAIL (TNF-related apoptosis-inducing ligand, DR4 (death receptor 4 and DR5 (death receptor 5 were increased in HSC-3 cells, and mRNA and protein expressions of Bcl-2 (B-cell lymphoma 2, Bcl-xL (B-cell lymphoma-extra large, XIAP (X-linked inhibitor of apoptosis protein, cIAP-1 (cellular inhibitor of apoptosis 1, cIAP-2 (cellular inhibitor of apoptosis 2 and survival were decreased. Meanwhile, at the highest concentration of 400 μg/mL, caspase-3, caspase-8, caspase-9, Bax, Fas/FasL, TRAIL, DR4, DR5, and

  2. Roselle improvement through conventional and mutation breeding

    Roselle (Hibiscus sabdariffa L.) from Malvaceae family is relatively a new crop in Malaysia. The origin is not fully known but believed to be from West Africa, although the plant is found native from India to Malaysia. The calyxes, stems and leaves are acid and closely resemble the cranberry (Vaccinium spp.) in flavour. Anthocyanins, which are now receiving a growing importance as natural food colorant, are responsible for the red to purple color of the calyx and other parts of the plant. The calyxes from the flowers are processed to produce juice for drink containing very high vitamin C (ascorbic acid), and also into jam, jelly and dried products. Interestingly, many other parts of the plant are also claimed to have various medicinal values. Presently, roselle is planted in Terengganu (175 ha in 2002) on bris soils, but its planting has spread to some parts of Kelantan, Pahang, Johor and also Sarawak. The number of roselle varieties available for planting is very limited; however, the effort carried out for roselle improvement thus far is equally very limited. There has been very little serious conventional breeding attempted, although varietal evaluation has had been carried out, particularly in form of agronomic trials. Since 1999, several studies on induced mutations have been attempted at UKM. A preliminary polyploidization study was conducted to determine the effects of colchicine concentrations of 0%, 0.04%, 0.08%, 0.12% and 0.16% and soaking times of 2 and 4 hours at room temperature (30 degree C) on 2-day old germinated seeds on morpho-agronomic traits (e.g. number of branches, internode length, leaf length, leaf width, number of flowers and days to flowering), ploidy level and pollen grain size in treated and also derived generations. Flow cytometric analyses of nuclear DNAAT content of leaf samples using LB01 lysis buffer and DNA specific fluorochrome DAPI (4',6-diamidino-2-phenylindole) staining were carried out using a flow cytometer at MINT, Bangi for

  3. Effect of two graded doses of whole-body X-irradiation and radioprotection by the use of S-phenethyl formamidino 4(N-ethyl isothioamide) morpholine dihydrochloride

    Hasan, S.S.; Chaturvedi, P.K.; Pandeya, S.N.

    1983-10-01

    The protection offered by a newly synthesized compound (S-phenethyl-formamidino-4(N-ethyl isothioamide) morpholine dihydrochloride) against radiation effects on DNA, RNA and protein biosynthetic processes in the brain, and on metabolites of 5-HT and nor-adrenalin, i.e., 5-HIAA and VMA, in the urine, including the radiobiological damage to thyroid and testes, was evaluated. The use of the compound prior to irradiation prevented radiation-induced changes in the thyroid and testes. The radiation-induced alterations in the pattern of DNA, RNA, protein in the brain, and in 5-HIAA and VMA in urine could be averted by treatment with this compound prior to each dose of X-irradiation.

  4. 光动力疗法联合瘤体输注树突状细胞对小鼠肝癌移植瘤的抑制作用及免疫效应的研究%Anti-tumor and Immunological Effects Induced by the Combination of Photodynamic Therapy and Dendritic Cells on Mouse Hepatoma

    白爽; 张南征; 杨宛莹; 刘军权; 周忠海; 孙蕾清; 陈复兴

    2012-01-01

    of splenocytes in the PDT and PIT groups was impressively greater than that of the DC and control groups(P <0.01 ,P<0. 01). Conclusions Photodynamic therapy (PDT) can restrain tumor growth and induce antitumor immune response, and can amplify the restraint on and host immune response against PDT-treated tumor when being used in conjunction with dendritic cell immunotherapy.%目的 探讨光动力疗法(photodynamic therapy,PDT)联合瘤体内输注树突状细胞(dendritic cell,DC)的光免疫疗法(photodynamic immuno-therapy,PIT)对小鼠Heps肝癌移植瘤的抑制作用及免疫效应.方法 体外培养昆明小鼠骨髓源性DC,4,6-二脒基-2-苯基吲哚(4,6-diamidino-2 -phenylindole,DAPI)荧光染色液标记DC备用.128只昆明小鼠皮下接种Heps肝癌细胞建立肿瘤模型,随机分为对照组、PDT组、DC组和PIT组.对照组小鼠瘤体内注射生理盐水,PDT组单纯PDT治疗,DC组小鼠瘤体内注射DAPI标记的DC,PIT组PDT联合瘤体内注射DAPI标记的DC细胞.治疗后定期测量各组肿瘤体积,记录各组小鼠生存时间,荧光显微镜下计数DC组及PIT组小鼠淋巴结中荧光细胞数目,流式细胞仪测定各组小鼠外周血T细胞亚群,LDH释放法测定各组小鼠脾细胞杀伤活性.结果 (1)与对照组相比,PDT组与PIT组治疗后肿瘤生长明显受抑;(2)PDT组与PIT组小鼠生存时间延长;(3)高倍镜视野下DC组较PIT组荧光细胞数增多(P<0.05);(4)治疗后72 h,PDT及PIT组小鼠外周血CD8+T细胞百分率均明显高于对照组和DC组(P<0.01、P<0.01),其中PIT组较PDT组增高明显(P<0.01),(5)PDT组与PIT组小鼠脾脏细胞杀伤活性较对照组和DC组明显增强(P<0.01,P<0.01).结论 PDT疗法能够抑制肿瘤生长并激发宿主免疫应答,联合输注DC可增强PDT对小鼠Heps肝癌移植瘤的抑制作用及免疫效应.

  5. Ammonia-treated N-(1-naphthyl) ethylenediamine dihydrochloride as a novel matrix for rapid quantitative and qualitative determination of serum free fatty acids by matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry

    Zhang, Yaping [Department of Biophysics and Structural Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, 5 Dongdan San Tiao, Beijing 100005 (China); Wang, Yanmin [Department of Clinical Laboratory, Heze Municipal Hospital, Shandong (China); Guo, Shuai; Guo, Yumei; Liu, Hui [Department of Biophysics and Structural Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, 5 Dongdan San Tiao, Beijing 100005 (China); Li, Zhili, E-mail: lizhili@ibms.pumc.edu.cn [Department of Biophysics and Structural Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, 5 Dongdan San Tiao, Beijing 100005 (China)

    2013-09-10

    Graphical abstract: -- Highlights: •A novel MALDI matrix for the detection of serum free fatty acids is ammonia-treated N-(1-naphthyl) ethylenediamine dihydrochloride. •Multiple point internal standard calibration curves were constructed for nine FFAs, respectively, with excellent correlation coefficients between 0.991 and 0.999. •The MALDI-MS approach was used to rapidly differentiate the patients with and without hyperglycemia and healthy controls. -- Abstract: The blood free fatty acids (FFAs), which provide energy to the cell and act as substrates in the synthesis of fats, lipoproteins, liposaccharides, and eicosanoids, involve in a number of important physiological processes. In the present study, matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR MS) with ammonia-treated N-(1-naphthyl) ethylenediamine dihydrochloride (ATNEDC) as a novel MALDI matrix in a negative ion mode was employed to directly quantify serum FFAs. Multiple point internal standard calibration curves between the concentration ratios of individual fatty acids to internal standard (IS, C{sub 17:0}) versus their corresponding intensity ratios were constructed for C{sub 14:0}, C{sub 16:1}, C{sub 16:0}, C{sub 18:0}, C{sub 18:1}, C{sub 18:2}, C{sub 18:3}, C{sub 20:4}, and C{sub 22:6}, respectively, in their mixture, with correlation coefficients between 0.991 and 0.999 and limits of detection (LODs) between 0.2 and 5.4 μM, along with the linear dynamic range of more than two orders of magnitude. The results indicate that the multiple point internal standard calibration could reduce the impact of ion suppression and improve quantification accuracy in the MALDI mode. The quantitative results of nine FFAs from 339 serum samples, including 161 healthy controls, 118 patients with hyperglycemia and 60 patients without hyperglycemia show that FFAs levels in hyperglycemic patient sera are significantly higher than those in healthy

  6. 盐酸曲美他嗪缓释片体外释放度研究%Preparation and in vitro release of trimetazidine dihydrochloride sustained-release tablets

    沈炳香; 聂松柳

    2011-01-01

    目的 以羟丙甲基纤维素为骨架材料制得盐酸曲美他嗪缓释片,并对释药机制进行探讨.方法 进行体外释放度试验,研究体外释放度测定方法,辅料和加速稳定性试验对释放度的影响.结果 制得的盐酸曲美他嗪缓释片具有明显的缓释作用,体外释放符合一级释药动力学规律.结论 制备盐酸曲美他嗪缓释片的方法简单,适于工业化生产.%Aim Trimetazidine dihydrochloride sustained-release tablets( TDST )were formulated with hydroxypropyl-methylcellulose as the matrixmateria and the release mechanism of the tablets were determined. Methods The effects of determination methods,main excipients and accelerated stability test on the sustained-release character were studied. Results The results indicated that the TDST had an expected sustained-release quality,and drug release pattern was m accord with the first order kinetics. Conclusion The method is simple for the preparation of TDST.and useful for the large-scale production.

  7. 5-氮杂胞嘧啶核苷诱导小鼠骨髓间充质干细胞凋亡%5-azacytidine induces apoptosis of bone marrow mesenchymal stem cells in mouse

    武俊芳; 吴艳芳; 李晓鹏; 张芬熙; 林俊堂; 高利洁; 崔柳苏

    2014-01-01

    Objective To investigate the effect of 5-azacytidine(5-AZA) on apoptosis of bone marrow mesenchymal stem cells(BMSCs).Methods BMSCs were isolated from bone marrow of mouse tibia and femur; the expression of MSC specific markers CD44 and CD90 in BMSCs was measured by immunofluorescence staining;BMSCs were cultured in vitro in the medium supplemented with 0,10 and 20 μmol/L 5-AZA for 48 hours.Cell apoptosis was measured with fluorescent labeled inhibitor of caspases (FLICA) apoptosis kit and 4',6-diamidino-2-phenylindole (DAPI) staining ;the expression of apoptosis-related proteins Annexin V and Caspase-3 in the treated BMSCs was detected by Western blot.Results In this study,BMSCs positively expressed MSC specific markers CD44 and CD90.DAPI staining and Caspase-3 staining both showed that 10 and 20 μmol/L 5-AZA markedly increased apoptotic rate of BMSCs;the apoptosis-positive rate in DAPI staining was (21.086 ± 2.601) %,(34.467 ± 3.724) % and (46.512 ± 3.864) %,the apoptosis-positive rate in Caspase-3 staining was (5.354 ± 0.735)%,(15.462 ± 2.385)% and (28.190 ± 4.190)% in the controls,10 and 20 μmol/L 5-AZA groups,and there were significant differences among the control group and 5-AZA treated groups(all P <0.01).Western blot assay showed that Annexin V and Caspase-3 were both markedly upregulated in 5-AZA treated cells;the relative level of Annexin V expression was(26.612 ±2.184)%,(42.873 ±4.313)% and (50.056 ± 4.457) %,the relative level of Caspase-3 expression was (19.231 ± 2.683) %,(38.618 ± 5.385) % and(91.235 ± 7.116)% in the controls,10 and 20 μmol/L 5-AZA groups,and there were significant differences among the control group and 5-AZA treated groups (all P < 0.01).Conclusion The commonly used doses of 5-AZA can induce apoptosis of BMSCs.%目的 研究5-氮杂胞嘧啶核苷(5-AZA)对小鼠骨髓间充质干细胞(BMSCs)凋亡的影响.方法 从小鼠股骨和胫骨骨髓中分离BMSCs;利用免疫荧光染色检

  8. Single-strand breaks in DNA of various organs of mice induced by methyl methanesulfonate and dimethylsulfoxide determined by the alkaline unwinding technique

    The method for determination of single-strand breaks (SSB) in DNA by the technique of alkaline unwinding and hydroxylapatite chromatography has been applied for cell nuclei from organs of mice. Male mice were given methyl methane-sulfonate (MMS) and dimethylsulfoxide (DMSO) by i.p. administration. Cell nuclei were prepared from various organs and then lysed in alkali. The amount of DNA was determined by fluorometry using 4',6-diamidino-2-phenylindole.2HCl. The relative level of SSB in DNA was determined in various organs (liver, kidney, lung, spleen, testis and brain) 1-24 h after administration of the agent. After MMS-treatment the number of SSB in DNA increased to about the same extent in all organs 1 h post-treatment but then decreased by time. The SSB persisted for the longest time in brain- and lung-DNA. DMSO induced SSB only in DNA of kidney

  9. Interference of ATP with the fluorescent probes YOYO-1 and YOYO-3 modifies the mechanical properties of intercalator-stained DNA confined in nanochannels

    Intercalating fluorescent probes are widely used to visualize DNA in studies on DNA-protein interactions. Some require the presence of adenosine triphosphate (ATP). We have investigated the mechanical properties of DNA stained with the fluorescent intercalating dyes YOYO-1 and YOYO-3 as a function of ATP concentrations (up to 2 mM) by stretching single molecules in nanofluidic channels with a channel cross-section as small as roughly 100 × 100 nm2. The presence of ATP reduces the length of the DNA by up to 11 %. On the other hand, negligible effects are found if DNA is visualized with the minor groove-binding probe 4′,6-diamidino-2-phenylindole. The apparent drop in extension under nanoconfinement is attributed to an interaction of the dye and ATP, and the resulting expulsion of YOYO-1 from the double helix. (author)

  10. 5-氮杂胞苷与盐酸洛拉曲克体外联合用药的抗癌增效作用观察%Synergistic anticancer wffect of 5-azacytidine combined with nolatrexed dihydrochloride in vitro

    涂洪谊; 陈卫民

    2006-01-01

    目的通过在体外分别对DNA甲基化转移酶(DNA methyltransferase,DNMT)抑制剂5-氮杂胞苷(5-azacytidine,5-aza-C)和新型脂溶性胸苷酸合成酶抑制剂盐酸洛拉曲克(nolatrexed dihydrochloride,Nolatrexed)联合用药于人大肠癌细胞LoVo和人肝癌细胞Hep3B的相互作用的性质观察,探讨DNMT抑制剂和胸苷酸合成酶抑制剂联合用药的可能性.方法使用MTT法测定5-aza-C和Nolatrexed单独用药或联合用药的抗癌活性,用抑制浓度的分数之和(a sum of fractional inhibitory concentration,SFIC)值及等效剂量分析方法(Isobologram)评价5-aza-C和Nolatrexed联合用药的作用性质.结果在5-aza-C和Nolatrexed联合用药时其SFIC值均小于或等于1,由此得到的等效剂量曲线图形表现为凹形.结论5-aza-C和Nolatrexed体外联合用药抗癌相互作用性质为明显的增效作用,DNMT抑制剂和胸苷酸合成酶抑制剂联合用药达到抗癌增效作用是可能的.

  11. Food restriction alters N'-propyl-4,5,6,7-tetrahydrobenzothiazole-2,6-diamine dihydrochloride (pramipexole)-induced yawning, hypothermia, and locomotor activity in rats: evidence for sensitization of dopamine D2 receptor-mediated effects.

    Collins, Gregory T; Calinski, Diane M; Newman, Amy Hauck; Grundt, Peter; Woods, James H

    2008-05-01

    Food restriction enhances sensitivity to the reinforcing effects of a variety of drugs of abuse including opiates, nicotine, and psychostimulants. Food restriction has also been shown to alter a variety of behavioral and pharmacological responses to dopaminergic agonists, including an increased sensitivity to the locomotor stimulatory effects of direct- and indirect-dopamine agonists, elevated extracellular dopamine levels in responses to psychostimulants, as well as suppression of agonist-induced yawning. Behavioral and molecular studies suggest that augmented dopaminergic responses observed in food-restricted animals result from a sensitization of the dopamine D2 receptor; however, little is known about how food restriction affects dopamine D3 receptor function. The current studies were aimed at better defining the effects of food restriction on D2 and D3 receptor function by assessing the capacity of N'-propyl-4,5,6,7-tetrahydrobenzothiazole-2,6-diamine dihydrochloride (pramipexole) to induce yawning, penile erection (PE), hypothermia, and locomotor activity in free-fed and food-restricted rats. Food restriction resulted in a suppression of pramipexole-induced yawning, a sensitized hypothermic response, and an enhanced locomotor response to pramipexole, effects that are suggestive of an enhanced D2 receptor activity; no effect on pramipexole-induced PE was observed. Antagonist studies further supported a food restriction-induced enhancement of the D2 receptor activity because the D2 antagonist 3-[4-(4-chlorophenyl)-4-hydroxypiperidin-l-yl]methyl-1H-indole (L741,626) recovered pramipexole-induced yawning to free-fed levels, whereas yawning and PE were suppressed following pretreatment with the D3 antagonist N-{4-[4-(2,3-dichlorophenyl)-piperazin-1-yl]-trans-but-2-enyl}-4-pyridine-2-yl-benzamide hydrochloride (PG01037). The results of the current studies suggest that food restriction sensitized rats to the D2-mediated effects of pramipexole while having no effect

  12. Protective effects of methanolic extract form fruits of Lycium ruthenicum Murr on 2,2′-azobis (2-amidinopropane dihydrochloride-induced oxidative stress in LLC-PK1 cells

    Jia-Le Song

    2014-01-01

    Full Text Available Background: Fruits of Lycium ruthenicum Murr is a health food and also used as a folk to treat heart disease, abnormal menstruation and menopause in Tibetan, China. However; whether L. ruthenicum Murr fruits methanolic extracts (LFME protect LLC-PK1 porcine renal tubules cells from AAPH-induced oxidative damage has not been investigated. Objective: To investigate the protective effects of L. ruthenicum Murr fruits methanolic extracts (LFME against 2, 2′- azobis (2-amidinopropane dihydrochloride (AAPH-induced oxidative damage in renal proximal tubule LLC-PK1 cells. Materials and Methods: LLC-PK1 cells were co-incubated with AAPH (1mM and different concentrations of LFMW together for 24 h. Cell viability was determined by MTT assay. Total intercellular reactive oxygen species (ROS levels and lipid peroxidation were measured using a fluorescent probe 2′, 7′-dichlorfluorescein-diacetate (DCFH-DA and the TBA reactive substance (TBARS assay, respectively. The endogenous antioxidant enzymes including catalase (CAT, superoxide dismutase (SOD, glutathione peroxidase (GSH-px and intercellular glutathione (GSH levels were determined using commercial assay kits according to the manufacturer′s instructions. Results: LFME did not show a significant cytotoxic effect and increased the viability of LLC-PK1 cells in a concentration-dependent manner. LFME also decreased the total intercellular levels of ROS, reduced lipid peroxidation and increased the GSH levels as well as the activities of endogenous antioxidant enzymes to protect LLC-PK1 cells against AAPH-induced oxidative damage. Conclusion: The results from the present study indicated that LFME is an effective ROS scavenger to protect LLC-PK1 cells against AAPH-induced oxidative damage through decreasing ROS generation, reducing lipid peroxidation and up-regulation of endogenous GSH levels and antioxidant enzymes.

  13. Effects of Photodynamic Therapy on the Localization of Transferred Natrual Killer Cells in Transplanted Heps Hepatoma%光动力疗法对输入的NK细胞在荷Heps肝癌鼠瘤内分布的影响

    杨宛莹; 张南征; 刘军权; 白爽; 周忠海; 陈复兴; 孙蕾清

    2012-01-01

    Objective To investigate the distributional regularity of natural killer cells ( NK) on Hepatoma-Heps-bearing mice in vivo, and whether photodynamic therapy (PDT) can promote the infiltration of NK cells in tumors. Methods The hepatoma model was established by subcutaneously inoculating Heps cells to 96 KM mice, who were then divided into the control, cell (NK) and photo immunotherapy (PIT) groups, each having 32 mice. The mice of the control group were only administered with NS. For the NK group, NK cells were labeled with 4, 6-diamidino-2-phenylindole (DAPI) and then injected into the mice tail veins. As to the PIT group, labeled NK cells were injected into the mice tail veins immediately after PDT light exposure. The tumors, lungs, spleens and livers were removed on day 1,2,4,6 and 8 after the treatment and observed for the distributional regularity under fluorescence microscope. The histopathological changes of tumors were observed via HE staining on day 2. Blood samples from the mice eyes were taken on day 1,2,4,6 and 12 after the treatment and the percentage of NK cells in peripheral blood was tested with flow cytometry. The cytotoxicity of the mice spleen cells were determined through lactate dehydrogenase (1,1)11) release assay on day 6. Results (1) On day 1, 2, 4, 6 and 8, labeled cells could be observed in all the said viscera organs of the treated mice. Labeled cells accumulated mostly in the tumors. On day 2 after the injection, much more labeled cells were seen in the tumors than at other time points (P < 0.01). The infiltration density of labeled cells in the PIT group was higher than that of the NK group (P < 0. 01). (2) On day 1, 2 and 4 after the treatment, the percentage of NK cells in mice peripheral blood of the NK group was larger than that of the PIT group (P<0. 01). (3) When T ratio being 10 = 1, 20 = 1 and 50:1, the cytotoxicity of spleen cells in the PIT group and NK group were far higher than that in the control group (P<0. 01), and that

  14. Variations in cell morphology in the canine cruciate ligament complex.

    Smith, K D; Vaughan-Thomas, A; Spiller, D G; Clegg, P D; Innes, J F; Comerford, E J

    2012-08-01

    Cell morphology may reflect the mechanical environment of tissues and influence tissue physiology and response to injury. Normal cruciate ligaments (CLs) from disease-free stifle joints were harvested from dog breeds with a high (Labrador retriever) and low (Greyhound) risk of cranial cruciate ligament (CCL) rupture. Antibodies against the cytoskeletal components vimentin and alpha tubulin were used to analyse cell morphology; nuclei were stained with 4',6-diamidino-2-phenylindole, and images were collected using conventional and confocal microscopy. Both cranial and caudal CLs contained cells of heterogenous morphologies. Cells were arranged between collagen bundles and frequently had cytoplasmic processes. Some of these processes were long (type A cells), others were shorter, thicker and more branched (type B cells), and some had no processes (type C cells). Processes were frequently shown to contact other cells, extending longitudinally and transversely through the CLs. Cells with longer processes had fusiform nuclei, and those with no processes had rounded nuclei and were more frequent in the mid-substance of both CLs. Cells with long processes were more commonly noted in the CLs of the Greyhound. As contact between cells may facilitate direct communication, variances in cell morphology between breeds at a differing risk of CCL rupture may reflect differences in CL physiology. PMID:22465617

  15. Capsaicin Inhibits Preferentially the NADH Oxidase and Growth of Transformed Cells in Culture

    Morre, D. James; Chueh, Pin-Ju; Morre, Dorothy M.

    1995-03-01

    A hormone- and growth factor-stimulated NADH oxidase of the mammalian plasma membrane, constitutively activated in transformed cells, was inhibited preferentially in HeLa, ovarian carcinoma, mammary adenocarcinoma, and HL-60 cells, all of human origin, by the naturally occurring quinone analog capsaicin (8-methyl-N-vanillyl-6-noneamide), compared with plasma membranes from human mammary epithelial, rat liver, normal rat kidney cells, or HL-60 cells induced to differentiate with dimethyl sulfoxide. With cells in culture, capsaicin preferentially inhibited growth of HeLa, ovarian carcinoma, mammary adenocarcinoma, and HL-60 cells but was largely without effect on the mammary epithelial cells, rat kidney cells, or HL-60 cells induced to differentiate with dimethyl sulfoxide. Inhibited cells became smaller and cell death was accompanied by a condensed and fragmented appearance of the nuclear DNA, as revealed by fluorescence microscopy with 4',6-diamidino-2-phenylindole, suggestive of apoptosis. The findings correlate capsaicin inhibition of cell surface NADH oxidase activity and inhibition of growth that correlate with capsaicin-induced apoptosis.

  16. In vitro study on the interaction of 4,4-dimethylcurcumin with calf thymus DNA

    The interaction of 4,4-dimethylcurcumin (DMCU), a synthesized analog of curcumin, with calf-thymus DNA (ct-DNA) was investigated using fluorescence, absorption, and circular dichroism (CD) spectroscopy, coupled with viscosity measurements and molecular docking techniques. DMCU was found to bind to ct-DNA with moderate binding affinity through groove binding as evidenced by a decrease in the absorption intensity in combination with no obvious change in the relative specific viscosity of ct-DNA and the CD spectrum. Thermodynamic analysis of the fluorescence data obtained at different temperatures suggested that the binding process was spontaneous and was primarily driven by hydrogen bonding and van der Waals forces. Furthermore, competitive binding experiments with ethidium bromide and 4′,6-diamidino-2-phenylindole as probes showed that DMCU could preferentially bind in the minor groove of double-stranded DNA. The results obtained from the molecular docking studies were consistent with these experimental results. This study explored the potential applicability of the spectroscopic properties of DMCU for studying its interactions with relevant biological or biomimicking targets. - Highlights: • 4,4-dimethylcurcumin (DMCU) has strong fluorescence characteristics. • DMCU could bind to DNA through groove binding. • Docking studies revealed that DMCU bound to the A–T region in the minor groove

  17. In vitro study on the interaction of 4,4-dimethylcurcumin with calf thymus DNA

    Liu, Bing-Mi, E-mail: liubingmi@163.com [Department of Pharmacy, Liaoning University, Shenyang 110036 (China); Bai, Chong-Liang [Centre for Molecular Science and Engineering, Northeastern University, Shenyang 110819 (China); Zhang, Jun; Liu, Yang; Dong, Bo-Yang; Zhang, Yi-Tong [Department of Pharmacy, Liaoning University, Shenyang 110036 (China); Liu, Bin, E-mail: liubinzehao@163.com [Department of Pharmacy, Liaoning University, Shenyang 110036 (China)

    2015-10-15

    The interaction of 4,4-dimethylcurcumin (DMCU), a synthesized analog of curcumin, with calf-thymus DNA (ct-DNA) was investigated using fluorescence, absorption, and circular dichroism (CD) spectroscopy, coupled with viscosity measurements and molecular docking techniques. DMCU was found to bind to ct-DNA with moderate binding affinity through groove binding as evidenced by a decrease in the absorption intensity in combination with no obvious change in the relative specific viscosity of ct-DNA and the CD spectrum. Thermodynamic analysis of the fluorescence data obtained at different temperatures suggested that the binding process was spontaneous and was primarily driven by hydrogen bonding and van der Waals forces. Furthermore, competitive binding experiments with ethidium bromide and 4′,6-diamidino-2-phenylindole as probes showed that DMCU could preferentially bind in the minor groove of double-stranded DNA. The results obtained from the molecular docking studies were consistent with these experimental results. This study explored the potential applicability of the spectroscopic properties of DMCU for studying its interactions with relevant biological or biomimicking targets. - Highlights: • 4,4-dimethylcurcumin (DMCU) has strong fluorescence characteristics. • DMCU could bind to DNA through groove binding. • Docking studies revealed that DMCU bound to the A–T region in the minor groove.

  18. Cell Biological Characterization of Male Meiosis and Pollen Development in Rice

    Chang-Bin CHEN; Yun-Yuan XU; Hong MA; Kang CHONG

    2005-01-01

    Little systematic analysis has been undertaken in rice (Oryza sativa L.) on the stages of male meiosis from leptotene to telophase Ⅱ or of pollen development from microspores to mature pollen grains.The present study describes multiple stages in detail from analysis of rice chromosome spreading with staining of 4',6-diamidino-2-phenylindole. The description of normal wild-type male meiosis provides an important morphological reference for analyses of meiotic mutants. Meiosis in rice is largely similar to those of the well characterizing model plants Arabidopsis thaliana L. and Zea mays L. However, rice meiosis differs from that in Arabidopsis in that rice meiosis I is followed by the formation of a cell plate, instead of an organelle band that forms between the two nuclei and persist through meiosis Ⅱ. This suggests a difference in the control of organelle biogenesis and distribution and cytokinesis. Our results should facilitate studies of rice meiosis and pollen development using molecular genetic and cell biological approaches.

  19. Bioactive chemicals from carrot (Daucus carota) juice extracts for the treatment of leukemia.

    Zaini, Rana; Clench, Malcolm R; Le Maitre, Christine L

    2011-11-01

    Overwhelming evidence indicates that consumption of fruits and vegetables with antioxidant properties correlates with reduced risk for cancers, including leukemia. Carrots contain beneficial agents, such as β-carotene and polyacetylenes, which could be effective in the treatment of leukemia. This study investigated the effect of carrot juice extracts on myeloid and lymphoid leukemia cell lines together with normal hematopoietic stem cells. Leukemia cell lines and nontumor control cells were treated with carrot juice extracts for up to 72 hours in vitro. Induction of apoptosis was investigated by using annexin V/propidium iodide staining followed by flow cytometric analysis, and results were confirmed by using 4'-6-diamidino-2-phenylindole morphology. Effects on cellular proliferation were investigated via cell cycle analysis and cell counts. Treatment of leukemia cell lines with carrot juice extract induced apoptosis and inhibited progression through the cell cycle. Lymphoid cell lines were affected to a greater extent than were myeloid cell lines, and normal hematopoietic stem cells were less sensitive than most cell lines. This study has shown that extracts from carrots can induce apoptosis and cause cell cycle arrest in leukemia cell lines. The findings suggest that carrots may be an excellent source of bioactive chemicals for the treatment of leukemia. PMID:21864090

  20. Specific antibodies induce apoptosis in Trypanosoma cruzi epimastigotes.

    Fernández-Presas, Ana María; Tato, Patricia; Becker, Ingeborg; Solano, Sandra; Copitin, Natalia; Kopitin, Natalia; Berzunza, Miriam; Willms, Kaethe; Hernández, Joselin; Molinari, José Luis

    2010-05-01

    The susceptibility of Trypanosoma cruzi epimastigotes to lysis by normal or immune sera in a complement-dependent reaction has been reported. Mouse immune sera depleted complement-induced damage in epimastigotes characterized by morphological changes and death. The purpose of this work was to study the mechanism of death in epimastigotes exposed to decomplemented mouse immune serum. Epimastigotes were maintained in RPMI medium. Immune sera were prepared in mice by immunization with whole crude epimastigote extracts. Viable epimastigotes were incubated with decomplemented normal or immune sera at 37 degrees C. By electron microscopy, agglutinated parasites showed characteristic patterns of membrane fusion between two or more parasites; this fusion also produced interdigitation of the subpellicular microtubules. Apoptosis was determined by flow cytometry using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and annexin V assays. Nuclear features were examined by 4'-,6-diamidino-2'-phenylindole diHCI cytochemistry that demonstrated apoptotic nuclear condensation. Caspase activity was also measured. TUNEL results showed that parasites incubated with decomplemented immune sera took up 26% of specific fluorescence as compared to 1.3% in parasites incubated with decomplemented normal sera. The Annexin-V-Fluos staining kit revealed that epimastigotes incubated with decomplemented immune sera exposed phosphatidylserine on the external leaflet of the plasma membrane. The incubation of parasites with immune sera showed caspase 3 activity. We conclude that specific antibodies are able to induce agglutination and apoptosis in epimastigotes, although the pathway is not elucidated. PMID:20237802

  1. Formation of volutin granules in Corynebacterium glutamicum.

    Pallerla, Srinivas Reddy; Knebel, Sandra; Polen, Tino; Klauth, Peter; Hollender, Juliane; Wendisch, Volker F; Schoberth, Siegfried M

    2005-02-01

    Volutin granules are intracellular storages of complexed inorganic polyphosphate (poly P). Histochemical staining procedures differentiate between pathogenic corynebacteria such as Corynebacterum diphtheriae (containing volutin) and non-pathogenic species, such as C. glutamicum. Here we report that strains ATCC13032 and MH20-22B of the non-pathogenic C. glutamicum also formed subcellular entities (18-37% of the total cell volume) that had the typical characteristics of volutin granules: (i) volutin staining, (ii) green UV fluorescence when stained with 4',6-diamidino-2-phenylindole, (iii) electron-dense and rich in phosphorus when determined with transmission electron microscopy and X-ray microanalysis, and (iv) 31P NMR poly P resonances of isolated granules dissolved in EDTA. MgCl2 addition to the growth medium stimulated granule formation but did not effect expression of genes involved in poly P metabolism. Granular volutin fractions from lysed cells contained polyphosphate glucokinase as detected by SDS-PAGE/MALDI-TOF, indicating that this poly P metabolizing enzyme is present also in intact poly P granules. The results suggest that formation of volutin is a more widespread phenomenon than generally accepted. PMID:15668011

  2. Flow sorting of the Y sex chromosome in the dioecious plant Melandrium album

    Veuskens, J.; Jacobs, M.; Negrutiu, I. [Free Univ. of Brussels (Belgium)] [and others

    1995-12-01

    The preparation of stable chromosome suspensions and flow cytometric sorting of both the Y sex chromosome of the white campion, Melandrium album, and the deleted Y chromosome of an asexual mutant, 5K63, is described. The principle has been to maintain transformed roots in vitro, synchronize and block mitosis, reduce cells to protoplasts, and lyse these to release chromosomes. Such in vitro material, unlike many cell suspensions, showed a stable karyotype. Factors critical to producing high-quality chromosome suspensions from protoplasts include osmolality of isolation solutions and choice of spindle toxin and of lysis buffer. Agrobacterium rhizogenes transformed young growing root cultures were synchronized at G1/S with 50 {mu}M aphidicolin for 24 h and released to a mitotic block with 30 {mu}M oryzalin for 11 h. Protoplast preparations from such tissue routinely had metaphase indices reaching 15%. Suspensions of intact metaphase chromosomes, with few chromatids, were obtained by lysing swollen mitotic protoplasts in a citric acid/disodium phosphate buffer. Except for the presence of clumps of autosomal chromosomes near the X and Y chromosome zones, monoparametric histograms of fluorescence intensities of suspensions stained with 4{prime},6-diamidino-2-phenylindole showed profiles similar to theoretical flow karyotypes. Two types of Y chromosomes, one full-length and one partially deleted (from the asexual mutant), could be sorted at 90% purity (21-fold enrichment of Y). These results are discussed in the context of sex determination and differentiation in higher plants. 45 refs., 6 figs., 2 tabs.

  3. Single Walled Carbon Nanotubes Exhibit Dual-Phase Regulation to Exposed Arabidopsis Mesophyll Cells

    Yuan, Hengguang; Hu, Shanglian; Huang, Peng; Song, Hua; Wang, Kan; Ruan, Jing; He, Rong; Cui, Daxiang

    2011-12-01

    Herein we are the first to report that single-walled carbon nanotubes (SWCNTs) exhibit dual-phase regulation to Arabidopsis mesophyll cells exposed to different concentration of SWCNTs. The mesophyll protoplasts were prepared by enzyme digestion, and incubated with 15, 25, 50, 100 μg/ml SWCNTs for 48 h, and then were observed by optical microscopy and transmission electron microscopy, the reactive oxygen species (ROS) generation was measured. Partial protoplasts were stained with propidium iodide and 4'-6- diamidino-2-phenylindole, partial protoplasts were incubated with fluorescein isothiocyanate-labeled SWCNTs, and observed by fluorescence microscopy. Results showed that SWCNTs could traverse both the plant cell wall and cell membrane, with less than or equal to 50 μg/ml in the culture medium, SWCNTs stimulated plant cells to grow out trichome clusters on their surface, with more than 50 μg/ml SWCNTs in the culture medium, SWCNTs exhibited obvious toxic effects to the protoplasts such as increasing generation of ROS, inducing changes of protoplast morphology, changing green leaves into yellow, and inducing protoplast cells' necrosis and apoptosis. In conclusion, single walled carbon nanotubes can get through Arabidopsis mesophyll cell wall and membrane, and exhibit dose-dependent dual-phase regulation to Arabidopsis mesophyll protoplasts such as low dose stimulating cell growth, and high dose inducing cells' ROS generation, necrosis or apoptosis.

  4. Single Walled Carbon Nanotubes Exhibit Dual-Phase Regulation to Exposed Arabidopsis Mesophyll Cells

    Huang Peng

    2011-01-01

    Full Text Available Abstract Herein we are the first to report that single-walled carbon nanotubes (SWCNTs exhibit dual-phase regulation to Arabidopsis mesophyll cells exposed to different concentration of SWCNTs. The mesophyll protoplasts were prepared by enzyme digestion, and incubated with 15, 25, 50, 100 μg/ml SWCNTs for 48 h, and then were observed by optical microscopy and transmission electron microscopy, the reactive oxygen species (ROS generation was measured. Partial protoplasts were stained with propidium iodide and 4'-6- diamidino-2-phenylindole, partial protoplasts were incubated with fluorescein isothiocyanate-labeled SWCNTs, and observed by fluorescence microscopy. Results showed that SWCNTs could traverse both the plant cell wall and cell membrane, with less than or equal to 50 μg/ml in the culture medium, SWCNTs stimulated plant cells to grow out trichome clusters on their surface, with more than 50 μg/ml SWCNTs in the culture medium, SWCNTs exhibited obvious toxic effects to the protoplasts such as increasing generation of ROS, inducing changes of protoplast morphology, changing green leaves into yellow, and inducing protoplast cells' necrosis and apoptosis. In conclusion, single walled carbon nanotubes can get through Arabidopsis mesophyll cell wall and membrane, and exhibit dose-dependent dual-phase regulation to Arabidopsis mesophyll protoplasts such as low dose stimulating cell growth, and high dose inducing cells' ROS generation, necrosis or apoptosis.

  5. Weightlessness induced apoptosis in normal thyroid cells and papillary thyroid carcinoma cells via extrinsic and intrinsic pathways.

    Kossmehl, Peter; Shakibaei, Mehdi; Cogoli, Augusto; Infanger, Manfred; Curcio, Francesco; Schönberger, Johann; Eilles, Christoph; Bauer, Johann; Pickenhahn, Holger; Schulze-Tanzil, Gundula; Paul, Martin; Grimm, Daniela

    2003-09-01

    Apoptosis plays a pivotal role in development, tissue homeostasis, cancer, immune defense, and response to weightlessness. It can be initiated by external signals via death receptors, but may also emerge from mitochondria. We exposed mitochondria-rich thyroid carcinoma cells (ONCO-DG1 cell line) and normal thyroid cells (HTU-5) to conditions of simulated microgravity. After 24 h, 10% of the cancer cells had entered a Fas-dependent apoptotic pathway, but destruction and redistribution of mitochondria, microtubuli disruption, and caspase-3 activation were also detected, demonstrating the activation of extrinsic as well as intrinsic pathways. Furthermore, ONCO-DG1 cells grown on the clinostat showed elevated amounts of Bax, but reduced quantities of bcl-2. In addition, signs of apoptosis became detectable, as assessed by terminal deoxynucleotidyl transferase-mediated dUTP digoxigenin nick end labeling, 4',6-diamidino-2-phenylindole staining, and 85-kDa apoptosis-related cleavage fragments. These fragments resulted from enhanced 116-kDa poly(ADP-ribose)polymerase activity and apoptosis. Apoptosis was also detected in normal HTU-5 cells, as demonstrated by electron microscopy, activation of caspase-3, increases in Fas and Bax, and elevation of 85-kDa apoptosis-related cleavage fragments resulting from enhanced poly(ADP-ribose) polymerase activity. Gravitational unloading affects the mitochondria and thereby may trigger apoptosis in thyroid cells subjected to weightlessness by clinorotation. PMID:12933692

  6. Dynamic expression profiles from static cytometry data: component fitting and conversion to relative, "same scale" values.

    Jayant Avva

    Full Text Available BACKGROUND: Cytometry of asynchronous proliferating cell populations produces data with an extractable time-based feature embedded in the frequency of clustered, correlated events. Here, we present a specific case of general methodology for calculating dynamic expression profiles of epitopes that oscillate during the cell cycle and conversion of these values to the same scale. METHODS: Samples of K562 cells from one population were labeled by direct and indirect antibody methods for cyclins A2 and B1 and phospho-S10-histone H3. The same indirect antibody was used for both cyclins. Directly stained samples were counter-stained with 4'6-diamidino-2-phenylindole and indirectly stained samples with propidium to label DNA. The S phase cyclin expressions from indirect assays were used to scale the expression of the cyclins of the multi-variate direct assay. Boolean gating and two dimensional, sequential regions set on bivariate displays of the directly conjugated sample data were used to untangle and isolate unique, unambiguous expression values of the cyclins along the four-dimensional data path through the cell cycle. The median values of cyclins A2 and B1 from each region were correlated with the frequency of events within each region. RESULTS: The sequential runs of data were plotted as continuous multi-line linear equations of the form y = [(y(i+1-y(i/(x(i+1-x(i]x + y(i-[(y(i+1-y(i/(x(i+1-x(i]x(i (line between points (x(i,y(i and (x(i+1, y(i+1 to capture the dynamic expression profile of the two cyclins. CONCLUSIONS: This specific approach demonstrates the general methodology and provides a rule set from which the cell cycle expression of any other epitopes could be measured and calculated. These expression profiles are the "state variable" outputs, useful for calibrating mathematical cell cycle models.

  7. Dynamic Expression Profiles from Static Cytometry Data: Component Fitting and Conversion to Relative, “Same Scale” Values

    Avva, Jayant; Weis, Michael C.; Sramkoski, R. Michael; Sreenath, Sree N.; Jacobberger, James W.

    2012-01-01

    Background Cytometry of asynchronous proliferating cell populations produces data with an extractable time-based feature embedded in the frequency of clustered, correlated events. Here, we present a specific case of general methodology for calculating dynamic expression profiles of epitopes that oscillate during the cell cycle and conversion of these values to the same scale. Methods Samples of K562 cells from one population were labeled by direct and indirect antibody methods for cyclins A2 and B1 and phospho-S10-histone H3. The same indirect antibody was used for both cyclins. Directly stained samples were counter-stained with 4′6-diamidino-2-phenylindole and indirectly stained samples with propidium to label DNA. The S phase cyclin expressions from indirect assays were used to scale the expression of the cyclins of the multi-variate direct assay. Boolean gating and two dimensional, sequential regions set on bivariate displays of the directly conjugated sample data were used to untangle and isolate unique, unambiguous expression values of the cyclins along the four-dimensional data path through the cell cycle. The median values of cyclins A2 and B1 from each region were correlated with the frequency of events within each region. Results The sequential runs of data were plotted as continuous multi-line linear equations of the form y  =  [(yi+1−yi)/(xi+1−xi)]x + yi−[(yi+1−yi)/(xi+1−xi)]xi (line between points (xi,yi) and (xi+1, yi+1)) to capture the dynamic expression profile of the two cyclins. Conclusions This specific approach demonstrates the general methodology and provides a rule set from which the cell cycle expression of any other epitopes could be measured and calculated. These expression profiles are the “state variable” outputs, useful for calibrating mathematical cell cycle models. PMID:22808005

  8. Neuroprotective role of thymoquinone against 1-methyl-4-phenylpyridinium-induced dopaminergic cell death in primary mesencephalic cell culture

    Radad, Khaled S.; Al-Shraim, Mubarak M.; Moustafa, Mahmoud F.; Rausch, Wolf-Dieter

    2015-01-01

    Objectives: To investigate potential mechanisms mediating the neuroprotective effect of thymoquinone (TQ) on dopaminergic neurons. Methods: This study was conducted in the Chemistry and Biochemistry Institute, University of Veterinary Medicine, Vienna, Austria between June and August 2013. Primary cultures were prepared from embryonic mouse mesencephala (OFI/SPF) at gestation day 14. Four sets of cultures were kept untreated, treated with TQ on the eighth day in vitro (DIV) for 4 days, treated with 1-methyl-4-phenylpyridinium (MPP+) on the tenth DIV for 48 hours and co-treated with thymoquinone and MPP+. On the twelfth DIV, cultures were subjected to immunohistochemistry against tyrosine hydroxylase and fluorescent staining using LysoTracker® Deep Red, 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethyl benzimidazolylcarbocyanine (JC-1) and 4’,6-diamidino-2-phenylindole stains. Results: The MPP+ decreased the number of dopaminergic neurons by 40%, and increased the release of lactate dehydrogenase (LDH) into the culture medium. The TQ significantly rescued dopaminergic neurons and decreased the release of LDH at the concentrations of 0.1 and 1 µM. The TQ significantly shifted the red fluorescent intensity of the LysoTracker® Deep Red, increased the mitochondrial membrane potential as it increased the red:green florescent ratio of JC-1, and decreased MPP+-induced apoptotic cell death. Conclusion: The TQ protects dopaminergic neurons in primary mesencephalic culture by enhancing lysosomal degradation that clears damaged mitochondria and inhibits mitochondria-mediated apoptotic cell death. PMID:25630775

  9. Molecular signalling mediating the protective effect of A1 adenosine and mGlu3 metabotropic glutamate receptor activation against apoptosis by oxygen/glucose deprivation in cultured astrocytes.

    Ciccarelli, Renata; D'Alimonte, Iolanda; Ballerini, Patrizia; D'Auro, Mariagrazia; Nargi, Eleonora; Buccella, Silvana; Di Iorio, Patrizia; Bruno, Valeria; Nicoletti, Ferdinando; Caciagli, Francesco

    2007-05-01

    Astrocyte death may occur in neurodegenerative disorders and complicates the outcome of brain ischemia, a condition associated with high extracellular levels of adenosine and glutamate. We show that pharmacological activation of A(1) adenosine and mGlu3 metabotropic glutamate receptors with N(6)-chlorocyclopentyladenosine (CCPA) and (-)2-oxa-4-aminocyclo-[3.1.0]hexane-4,6-dicarboxylic acid (LY379268), respectively, protects cultured astrocytes against apoptosis induced by a 3-h exposure to oxygen/glucose deprivation (OGD). Protection by CCPA and LY379268 was less than additive and was abrogated by receptor blockade with selective competitive antagonists or pertussis toxin. Both in control astrocytes and in astrocytes exposed to OGD, CCPA and LY379268 induced a rapid activation of the phosphatidylinositol-3-kinase (PI3K) and extracellular signal-regulated kinases 1 and 2 (ERK1/2)/mitogen-activated protein kinase (MAPK) pathways, which are known to support cell survival. In cultures exposed to OGD, CCPA and LY379268 reduced the activation of c-Jun N-terminal kinase and p38/MAPK, reduced the levels of the proapoptotic protein Bad, increased the levels of the antiapoptotic protein Bcl-X(L), and were highly protective against apoptotic death, as shown by nuclear 4'-6-diamidino-2-phenylindole staining and measurements of caspase-3 activity. All of these effects were attenuated by treatment with 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126) and 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002), which inhibit the MAPK and the PI3K pathways, respectively. These data suggest that pharmacological activation of A(1) and mGlu3 receptors protects astrocytes against hypoxic/ischemic damage by stimulating the PI3K and ERK1/2 MAPK pathways. PMID:17293559

  10. Efficacy of two peroxygen-based disinfectants for inactivation of Cryptosporidium parvum oocysts.

    Quilez, Joaquin; Sanchez-Acedo, Caridad; Avendaño, Catalina; del Cacho, Emilio; Lopez-Bernad, Fernando

    2005-05-01

    Two commercial peroxygen-based disinfectants containing hydrogen peroxide plus either peracetic acid (Ox-Virin) or silver nitrate (Ox-Agua) were tested for their ability to inactivate Cryptosporidium parvum oocysts. Oocysts were obtained from naturally infected goat kids and exposed to concentrations of 2, 5, and 10% Ox-Virin or 1, 3, and 5% Ox-Agua for 30, 60, and 120 min. In vitro excystation, vital dyes (4',6'-diamidino-2-phenylindole and propidium iodide), and infectivity in neonatal BALB/c mice were used to assess the viability and infectivity of control and disinfectant-treated oocysts. Both disinfectants had a deleterious effect on the survival of C. parvum oocysts, since disinfection significantly reduced and in some cases eliminated their viability and infectivity. When in vitro assays were compared with an infectivity assay as indicators of oocyst inactivation, the excystation assay showed 98.6% inactivation after treatment with 10% Ox-Virin for 60 min, while the vital-dye assay showed 95.2% inactivation and the infectivity assay revealed 100% inactivation. Treatment with 3% Ox-Agua for 30 min completely eliminated oocyst infectivity for mice, although we were able to observe only 74.7% inactivation as measured by excystation assays and 24.3% with vital dyes (which proved to be the least reliable method for predicting C. parvum oocyst viability). These findings indicate the potential efficacy of both disinfectants for C. parvum oocysts in agricultural settings where soil, housing, or tools might be contaminated and support the argument that in comparison to the animal infectivity assay, vital-dye and excystation methods overestimate the viability of oocysts following chemical disinfection. PMID:15870337

  11. Characterization of Interspecific Hybrids Between Oryza sativa L. and Three Wild Rice Species of China by Genomic In Situ Hybridization

    Guang-Xuan Tan; Zhi-Yong Xiong; Hua-Jun Jin; Gang Li; Li-Li Zhu; Li-Hui Shu; Guang-Cun He

    2006-01-01

    In the genus Oryza, interspecific hybrids are useful bridges for transferring the desired genes from wild species to cultivated rice (Oryza sativa L.). In the present study, hybrids between O. sativa (AA genome)and three Chinese wild rices, namely O. rufipogon (AA genome), O. officinalis (CC genome), and O. meyeriana (GG genome), were produced. Agricultural traits of the F1 hybrids surveyed were intermediate between their parents and appreciably resembled wild rice parents. Except for the O. sativa × O. rufipogon hybrid,the other F1 hybrids were completely sterile. Genomic in situ hybridization (GISH) was used for hybrid verification. Wild rice genomic DNAs were used as probes and cultivated rice DNA was used as a block. With the exception of O. rufipogon chromosomes, this method distinguished the other two wild rice and cultivated rice chromosomes at the stage of mitotic metaphase with different blocking ratios. The results suggest that a more distant phylogenetic relationship exists between O. meyeriana and O. sativa and that O. rufipogon and O. sativa share a high degree of sequence homology. The average mitotic chromosome length of O. officinalis and O. meyeriana was 1.25- and 1.51-fold that of O. sativa, respectively. 4',6'-Diamidino2-phenylindole staining showed that the chromosomes of O. officinalis and O. meyeriana harbored more heterochromatin, suggesting that the C and G genomes were amplified with repetitive sequences compared with the A genome. Although chromocenters formed by chromatln compaction were detected with wild rice-specific signals corresponding to the C and G genomes in discrete domains of the F1 hybrid interphase nuclei, the size and number of O. meyeriana chromocenters were bigger and greater than those of O. officinalis. The present results provide an important understanding of the genomic relationships and a tool for the transfer of useful genes from three native wild rice species in China to cultivars.

  12. Determination of ACC-induced cell-programmed death in roots of Vicia faba ssp. minor seedlings by acridine orange and ethidium bromide staining.

    Byczkowska, Anna; Kunikowska, Anita; Kaźmierczak, Andrzej

    2013-02-01

    Fluorescence staining with acridine orange (AO) and ethidium bromide (EB) showed that nuclei of cortex root cells of 1-aminocyclopropane-1-carboxylic acid (ACC)-treated Vicia faba ssp. minor seedlings differed in color. Measurement of resultant fluorescence intensity (RFI) showed that it increased when the color of nuclear chromatin was changed from green to red, indicating that EB moved to the nuclei via the cell membrane which lost its integrity and stained nuclei red. AO/EB staining showed that changes in color of the nuclear chromatin were accompanied by DNA condensation, nuclei fragmentation, and chromatin degradation which were also shown after 4,6-diamidino-2-phenylindol staining. These results indicate that ACC induced programmed cell death. The increasing values of RFI together with the corresponding morphological changes of nuclear chromatin were the basis to prepare the standard curve; cells with green unchanged nuclear chromatin were alive while those with dark orange and bright red nuclei were dead. The cells with nuclei with green-yellow, yellow-orange, and bright orange chromatin with or without their condensation and fragmentation chromatin were dying. The prepared curve has became the basis to draw up the digital method for detection and determination of the number of living, dying, and dead cells in an in planta system and revealed that ACC induced death in about 20% of root cortex cells. This process was accompanied by increase in ion leakage, shortening of cells and whole roots, as well as by increase in weight and width of the apical part of roots and appearance of few aerenchymatic spaces while not by internucleosomal DNA degradation. PMID:22350735

  13. Dioscin-induced autophagy mitigates cell apoptosis through modulation of PI3K/Akt and ERK and JNK signaling pathways in human lung cancer cell lines.

    Hsieh, Ming-Ju; Tsai, Te-Lung; Hsieh, Yih-Shou; Wang, Chau-Jong; Chiou, Hui-Ling

    2013-11-01

    Our previous study has revealed that dioscin, a compound with anti-inflammatory, lipid-lowering, anticancer and hepatoprotective effects, may induce autophagy in hepatoma cells. Autophagy is a lysosomal degradation pathway that is essential for cell survival and tissue homeostasis. In this study, the role of autophagy and related signaling pathways during dioscin-induced apoptosis in human lung cancer cells was investigated. Results from 4'-6-diamidino-2-phenylindole and annexin-V/PI double-staining assay showed that caspase-3- and caspase-8-dependent, and dose-dependent apoptoses were detected after a 24-h dioscin treatment. Meanwhile, autophagy was detected as early as 12 h after an exposure to low-dose dioscin, as indicated by an up-regulated expression of LC3-II and beclin-1 proteins. Blockade of autophagy with bafilomycin A1 or 3-methyladenine sensitized the A549 and H1299 cells to apoptosis. Treatment of A549 and H1299 cells with dioscin caused a dose-dependent increase in ERK1/2 and JNK1/2 activity, accompanied with a decreased PI3K expression and decreased phosphorylation of Akt and mTOR. Taken together, this study demonstrated for the first time that autophagy occurred earlier than apoptosis during dioscin-induced human lung cancer cell line apoptosis. Dioscin-induced autophagy via ERK1/2 and JNK1/2 pathways may provide a protective mechanism for cell survival against dioscin-induced apoptosis to act as a cytoprotective reaction. PMID:23552851

  14. Green synthesis of silver and gold nanoparticles from Gymnema sylvestre leaf extract: study of antioxidant and anticancer activities

    The present study reports the biological synthesis of silver and gold nanoparticles from Gymnema sylvestre leaf extract and their in vitro free radical scavenging efficacy as well as antiproliferative effect in Hep2 cells. The formation of silver (GYAgNPs) and gold nanoparticles (GYAuNPs) was confirmed by UV–visible spectroscopy. The average size of synthesized GYAgNPs and GYAuNPs was found to be 33 and 26 nm, respectively, by DLS particle size analyzer. TEM analysis indicated spherical shape of GYAgNPs and GYAuNPs and in EDX analysis they produced strong signal for silver and gold, respectively. Both GYAgNPs and GYAuNPs exhibited strong in vitro free radical quenching ability and their activity was comparable to that of GYLE. The cytotoxic effect of GYAgNPs and GYAuNPs in Hep2 cells was examined by MTT assay in which GYAgNPs displayed an IC50 value of 121 µg ml−1, while GYAuNPs produced up to 38 % of inhibition at the maximum concentration of 250 µg ml−1 used in this study. Distinct morphological changes were observed in Hep2 cells following treatment with GYAgNPs and GYAuNPs at 24 h, and orange-colored apoptotic bodies were located by acridine orange and ethidium bromide double-staining technique. Also, there was increase in the levels of reactive oxygen species in treated cells as indicated by 2′,7′-dichlorofluorescin diacetate staining. Further, nuclear changes like chromatin condensation/fragmentation were also observed by propidium iodide and 4′,6-diamidino-2-phenylindole dilactate staining methods. These findings support that the antiproliferative effects of GYAgNPs and GYAuNPs in Hep2 cells are mediated through induction of apoptosis

  15. Green synthesis of silver and gold nanoparticles from Gymnema sylvestre leaf extract: study of antioxidant and anticancer activities

    Nakkala, Jayachandra Reddy; Mata, Rani; Bhagat, Ekta; Sadras, Sudha Rani, E-mail: dr.ssrlab@gmail.com, E-mail: sadrassudha@gmail.com [Pondicherry University, Department of Biochemistry and Molecular Biology, School of Life Sciences (India)

    2015-03-15

    The present study reports the biological synthesis of silver and gold nanoparticles from Gymnema sylvestre leaf extract and their in vitro free radical scavenging efficacy as well as antiproliferative effect in Hep2 cells. The formation of silver (GYAgNPs) and gold nanoparticles (GYAuNPs) was confirmed by UV–visible spectroscopy. The average size of synthesized GYAgNPs and GYAuNPs was found to be 33 and 26 nm, respectively, by DLS particle size analyzer. TEM analysis indicated spherical shape of GYAgNPs and GYAuNPs and in EDX analysis they produced strong signal for silver and gold, respectively. Both GYAgNPs and GYAuNPs exhibited strong in vitro free radical quenching ability and their activity was comparable to that of GYLE. The cytotoxic effect of GYAgNPs and GYAuNPs in Hep2 cells was examined by MTT assay in which GYAgNPs displayed an IC{sub 50} value of 121 µg ml{sup −1}, while GYAuNPs produced up to 38 % of inhibition at the maximum concentration of 250 µg ml{sup −1} used in this study. Distinct morphological changes were observed in Hep2 cells following treatment with GYAgNPs and GYAuNPs at 24 h, and orange-colored apoptotic bodies were located by acridine orange and ethidium bromide double-staining technique. Also, there was increase in the levels of reactive oxygen species in treated cells as indicated by 2′,7′-dichlorofluorescin diacetate staining. Further, nuclear changes like chromatin condensation/fragmentation were also observed by propidium iodide and 4′,6-diamidino-2-phenylindole dilactate staining methods. These findings support that the antiproliferative effects of GYAgNPs and GYAuNPs in Hep2 cells are mediated through induction of apoptosis.

  16. Effect of magnetic nanoparticles on apoptosis and cell cycle induced by wogonin in Raji cells

    Wang XM

    2012-02-01

    Full Text Available Lei Wang1,2,*, Haijun Zhang1,2,*, Baoan Chen1,2, Guohua Xia1,2, Shuai Wang1,2, Jian Cheng1,2, Zeye Shao1,2, Chong Gao1,2, Wen Bao1,2, Liang Tian1,2, Yanyan Ren1,2, Peipei Xu1,2, Xiaohui Cai1,2, Ran Liu1,2, Xuemei Wang3 1Department of Hematology and Oncology, Zhongda Hospital, Medical School, 2Faculty of Oncology, Medical School, 3State Key Laboratory of Bioelectronics (Chien-Shiung Wu Laboratory, Southeast University, Nanjing, China*These authors contributed equally to this workAbstract: Traditional Chinese medicine is gradually becoming a new source of anticancer drugs. One such example is wogonin, which is cytotoxic to various cancer cell lines in vitro. However, due to its low water solubility, wogonin is restricted to clinical administration. Recently, the application of drug-coated magnetic nanoparticles (MNPs to increase water solubility of the drug and to enhance its chemotherapeutic efficiency has attracted much attention. In this study, wogonin was conjugated with the drug delivery system of MNPs by mechanical absorption polymerization to fabricate wogonin-loaded MNPs. It was demonstrated that MNPs could strengthen wogonin-induced cell inhibition, apoptosis, and cell cycle arrest in Raji cells by methylthiazol tetrazolium assay, flow cytometer assay, and nuclear 4',6-diamidino-2-phenylindole staining. Furthermore, the molecular mechanisms of these phenomena were explored by western blot, in which the protein levels of caspase 8 and caspase 3 were increased significantly while those of survivin and cyclin E were decreased significantly in wogonin-MNPs group. These findings suggest that the combination of wogonin and MNPs provides a promising strategy for lymphoma therapy.Keywords: wogonin, magnetic nanoparticles, Raji cell, apoptosis, cell cycle, caspase 8, caspase 3, survivin, cyclin E

  17. Quantification of Campylobacter spp. in chicken rinse samples by using flotation prior to real-time PCR.

    Wolffs, Petra; Norling, Börje; Hoorfar, Jeffrey; Griffiths, Mansel; Rådström, Peter

    2005-10-01

    Real-time PCR is fast, sensitive, specific, and can deliver quantitative data; however, two disadvantages are that this technology is sensitive to inhibition by food and that it does not distinguish between DNA originating from viable, viable nonculturable (VNC), and dead cells. For this reason, real-time PCR has been combined with a novel discontinuous buoyant density gradient method, called flotation, in order to allow detection of only viable and VNC cells of thermotolerant campylobacters in chicken rinse samples. Studying the buoyant densities of different Campylobacter spp. showed that densities changed at different time points during growth; however, all varied between 1.065 and 1.109 g/ml. These data were then used to develop a flotation assay. Results showed that after flotation and real-time PCR, cell concentrations as low as 8.6 x 10(2) CFU/ml could be detected without culture enrichment and amounts as low as 2.6 x 10(3) CFU/ml could be quantified. Furthermore, subjecting viable cells and dead cells to flotation showed that viable cells were recovered after flotation treatment but that dead cells and/or their DNA was not detected. Also, when samples containing VNC cells mixed with dead cells were treated with flotation after storage at 4 or 20 degrees C for 21 days, a similar percentage resembling the VNC cell fraction was detected using real-time PCR and 5-cyano-2,3-ditolyl tetrazolium chloride-4',6'-diamidino-2-phenylindole staining (20% +/- 9% and 23% +/- 4%, respectively, at 4 degrees C; 11% +/- 4% and 10% +/- 2%, respectively, at 20 degrees C). This indicated that viable and VNC Campylobacter cells could be positively selected and quantified using the flotation method. PMID:16204485

  18. Radiosensitization Effect of STI-571 on Pancreatic Cancer Cells In Vitro

    Purpose: To examine STI-571-induced radiosensitivity in human pancreatic cancer cells in vitro. Methods and Materials: Three human pancreatic cancer cell lines (Bxpc-3, Capan-1, and MiaPaCa-2) exhibiting different expression levels of c-Kit and platelet-derived growth factor receptor β (PDGFRβ) and showing different K-ras mutation types were used. For evaluation of the antitumor activity of STI-571 in combination with radiation, clonogenic survival assays, Western blot analysis, and the annexin V/propidium iodide assay with microscopic evaluation by 4',6-diamidino-2-phenylindole were conducted. Results: Dramatic phosphorylated (p)-c-Kit and p-PDGFRβ attenuation, a modest dose- and time-dependent growth inhibition, and significant radiosensitization were observed after STI-571 treatment in view of apoptosis, although the levels of growth inhibition and increased radiosensitization were different according to cell lines. The grades of radiosensitivity corresponded to the attenuation levels of p-c-Kit and p-PDGFRβ by STI-571, particularly to those of p-c-Kit, and the radiosensitivity was partially affected by K-ras mutation in pancreatic cancer cells. Among downstream pathways associated with c-Kit or PDGFRβ, p-PLCγ was more closely related to radiosensitivity compared with p-Akt1 or p-extracellular signal-regulated kinase 1. Conclusion: STI-571 enhances radiation response in pancreatic cancer cells. This effect is affected by the attenuation levels of p-c-Kit or p-PDGFRβ, and K-ras mutation status. Among them, p-c-Kit plays more important roles in the radiosensitivity in pancreatic cancer compared with p-PDGFRβ or K-ras mutation status.

  19. FORMULATION AND EVALUATION OF EXTENDED RELEASE MATRIX TABLETS OF TRIMETAZIDINE DIHYDROCHLORIDE

    Mogili Dinesh; Manivannan, R.; Radhakrishna; Abhishek.P; Swetha Reddy

    2013-01-01

    Oral ingestion has long been the most convenient and commonly employed route of drug delivery. Indeed, for Extended release systems, the oral route of administration has by far received the most attention with respect to research on physiological and drug constraints as well as design and testing of products. The primary objective of the extended release (Matrix) drug delivery system is to ensure safety and to improve efficacy of drug as well as patient compliance. The present invention provi...

  20. Formulation and Evaluation of Taste Masked Cetrizine Dihydrochloride Orally Disintegrating Tablets: A Research

    Smita Kolhe

    2013-03-01

    Full Text Available Oral routes of drug administration have wide acceptance up to 50-60% of total dosage forms. ODTs dissolve or disintegrate instantly on the patients tongue or buccal mucosa and leaves easy-to-swallow residue. It is suited for tablets undergoing high first pass metabolism and is used for reducing dosing frequency. New ODT technologies address many pharmaceutical and patient needs such as, it denotes its importance in case of pediatric, geriatric and patients suffering from nausea or repeated emesis conditions, bed-ridden patients having dysphagia. So taste masking is essential criteria following the advanced method of use of ion exchange resins. Research in developing orally disintegrating systems has been aimed to investigate different excipients as well as techniques to meet number of challenges. Orally disintegrating tablets are the safest, convenient and highly economical formulations. They show satisfactory absorption from oral mucosa; ultimately immediate pharmacological action. This research deals with formulation of orally disintegrating tablets by direct compression inorder to achieve a better dissolution rate and further improving the bioavailability of the drug. The result showed rapid dissolution of drug with the use of superdisintegrants, Ac-di-sol (Disintegration Time [DT] 5-6 sec. as compared to sodium starch glycolate (DT 8-9 sec. and polyplasdone (DT 8-10 sec..

  1. DESIGN DEVELOPMENT AND EVALUATION OF TRIMETAZIDINE DIHYDROCHLORIDE FLOATING BILAYER M.R TABLETS

    Biswajit Biswal

    2011-07-01

    Full Text Available Modified release tablet are coated or uncoated tablet containing auxiliary substance or prepared by the procedure that, separately or together are design to modify the rate or place at which the active ingredient are released. Modified release (MR DDS is an attempt to sustain drug blood concentration at relatively constant and effective level in the body by spatial placement or temporal delivery. Thus controlled release drug delivery system (CRDDS offer various advantages viz. reduce blood level fluctuations, minimize drug accumulation, employ less total drug, improve patient compliance, and minimize local and systemic side effects.

  2. Chromosomal evidence for a putative cryptic species in the Gymnotus carapo species-complex (Gymnotiformes, Gymnotidae

    De Souza Augusto CP

    2008-11-01

    Full Text Available Abstract Background In this study we examined the karyotypes of morphologically indistinguishable populations of the electric knifefish Gymnotus carapo sensu stricto from the Eastern Amazon of Brazil. These were identified unambiguously on the basis of external morphology, meristics, and pigmentation. Results Specimens from one of five localities exhibited a karyotype previously not documented for Gymnotus species in the Amazon basin: 2n = 40 (34M/SM+6ST/A. Samples from the other four localities exhibited a different karyotype: 2n = 42 (30M/SM+12ST/A, which we had previously described. Specimens from all five localities presented constitutive heterochromatin in the centromeric region of almost all chromosomes, including in the distal and interstitial regions. Staining with 4'6-Diamidino-2-phenylindole revealed C-positive banding. In both karyotypes the Nucleolar Organizer Region (NOR was located on the short arm of pair 20, and Chromomycin A3 stained the NORs. Fluorescent in situ hybridization with telomeric probes showed an Interstitial Telomeric Sequence (ITS in the proximal short arm of a metacentric pair in the 2n = 40 karyotype. Conclusion The difference between the two karyotypes on the diploid number and chromosome morphology can be explained by rearrangements of the fusion-fission type and also by pericentric inversions. The presence of ITS in a metacentric pair of the 2n = 40 karyotype suggests that the difference in the diploid number of the karyotypes results from a fusion. The consistent 2n = 42 karyotype at four localities suggests an interbreeding population. However, because fusion-fission and pericentric inversions of this nature typically result in reproductive isolation, we speculate that the form with the 2n = 40 karyotype is a different species to that of the 2n = 42 form. Nonetheless, we did not observe evident differences in external morphology, meristics and pigmentation between the two forms, which suggest that they

  3. Synergistic antitumoral activity and induction of apoptosis by novel pan Bcl-2 proteins inhibitor apogossypolone with adriamycin in human hepatocellular carcinoma

    Jin-xia MI; Guang-feng WANG; Heng-bang WANG; Xiao-qing SUN; Xin-yan NI; Xiong-wen ZHANG; Jia-ming TANG; Da-jun YANG

    2008-01-01

    Aim: To investigate the in vitro and in vivo activities and related mechanism of apogossypoione (ApoG2) alone or in combination with adriamycin (ADM) against human hepatocellular carcinoma (HCC). Methods: The IC50 of ApoG2 in vitro was tested by WST assay, and the synergistic effect was analyzed using the CalcuSyn method. Cell apoptosis was determined using 4',6-diamidino-2-phenylindole staining and flow cytometric analysis. Western blotting was used to determine the expression of apoptosis-related proteins. In vivo activity was evaluated in the xenograft model in nude mice, and apoptosis in tumor tissues was determined by terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling (TUNEL) assay. Results: The IC50 of ApoG2 in HCC cells was 17.28-30.63 μmol/L. When ApoG2 was combined with ADM, in-creased cytotoxicity and apoptosis were observed in SMMC-7721 cells compared to treatment with ApoG2 alone. The Western blotting results indicated that the ApoG2 induced apoptosis in SMMC-7721 cells by downregulating anti-apoptotic proteins Bcl-2, Mcl-1, and Bcl-XL, up-regulating pro-apoptotic protein Noxa, and promoting the activities of caspases-9 and -3. The tumor growth of xenograft SMMC-7721 was inhibited in nude mice when ApoG2 was administered orally without causing damage to the normal tissues. The in vivo study also indicated an increasing anti-tumoral effect when ApoG2 at 100 or 200 mg/kg dosages were used together with ADM at 5.5 mg/kg, with relative tumor proliferation rate (T/C) values of 0.456 and 0.323, respectively. Apoptosis induced in vivo by ApoG2 alone or combined with ADM was confirmed by TUNEL assay in tumor tissues. Conclusion: ApoG2 is a potential non-toxic target agent that induces apoptosis by upregulating Noxa, while inhibiting anti-apoptotic proteins and pro-moting the effect of chemotherapy agent ADM in HCC.

  4. Effect of modeled reduced gravity conditions on bacterial morphology and physiology

    Vukanti Raja

    2012-01-01

    Full Text Available Abstract Background Bacterial phenotypes result from responses to environmental conditions under which these organisms grow; reduced gravity has been demonstrated in many studies as an environmental condition that profoundly influences microorganisms. In this study, we focused on low-shear stress, modeled reduced gravity (MRG conditions and examined, for Escherichia coli and Staphlyococcus aureus, a suite of bacterial responses (including total protein concentrations, biovolume, membrane potential and membrane integrity in rich and dilute media and at exponential and stationary phases for growth. The parameters selected have not been studied in E. coli and S. aureus under MRG conditions and provide critical information about bacterial viability and potential for population growth. Results With the exception of S. aureus in dilute Luria Bertani (LB broth, specific growth rates (based on optical density of the bacteria were not significantly different between normal gravity (NG and MRG conditions. However, significantly higher bacterial yields were observed for both bacteria under MRG than NG, irrespective of the medium with the exception of E. coli grown in LB. Also, enumeration of cells after staining with 4',6-diamidino-2-phenylindole showed that significantly higher numbers were achieved under MRG conditions during stationary phase for E. coli and S. aureus grown in M9 and dilute LB, respectively. In addition, with the exception of smaller S. aureus volume under MRG conditions at exponential phase in dilute LB, biovolume and protein concentrations per cell did not significantly differ between MRG and NG treatments. Both E. coli and S. aureus had higher average membrane potential and integrity under MRG than NG conditions; however, these responses varied with growth medium and growth phase. Conclusions Overall, our data provides novel information about E. coli and S. aureus membrane potential and integrity and suggest that bacteria are

  5. Reprogramming of cassava (Manihot esculenta) microspores towards sporophytic development.

    Perera, P I P; Ordoñez, C A; Dedicova, B; Ortega, P E M

    2014-01-01

    Gametes have the unique potential to enter the sporophytic pathway, called androgenesis. The plants produced are usually haploid and recombinant due to the preceding meiosis and they can double their chromosome number to form doubled haploids, which are completely homozygous. Availability of the doubled haploids facilitates mapping the genes of agronomically important traits, shortening the time of the breeding process required to produce new hybrids and homozygous varieties, and saving the time and cost for inbreeding. This study aimed to test the feasibility of using isolated and in vitro cultured immature cassava (Manihot esculenta) microspores to reprogramme and initiate sporophytic development. Different culture media and different concentrations of two ion components (Cu(2+) and Fe(2+)) were tested in two genotypes of cassava. External structural changes, nuclear divisions and cellular changes during reprogramming were analysed by scanning electron microscopy, by staining with 4',6-diamidino-2-phenylindole, and through classical histology and transmission electron microscopy. In two cassava genotypes, different developmental stages of microspores were found to initiate sporophytic cell divisions, that is, with tetrads of TMS 60444 and with mid or late uni-nucleate microspores of SM 1219-9. In the modified NLN medium (NLNS), microspore enlargements were observed. The medium supplemented with either sodium ferrous ethylene-diamine-tetraacetic acid (NaFeEDTA) or CuSO4·5H2O induced sporophytic cell division in both genotypes. A low frequency of the reprogramming and the presence of non-responsive microspores among the responsive ones in tetrads were found to be related to the viability and exine formation of the microspores. The present study clearly demonstrated that reprogramming occurs much faster in isolated microspore culture than in anther culture. This paves the way for the development of an efficient technique for the production of homozygous lines in

  6. Morphologic characterization of isolated bovine early preantral follicles during short-term individual in vitro culture.

    Jorssen, E P A; Langbeen, A; Marei, W F A; Fransen, E; De porte, H F M; Leroy, J L M R; Bols, P E J

    2015-07-15

    To provide new insights in the molecular mechanism controlling preantral follicular development and to unravel the needs to support in vitro follicular development of early-stage preantral follicles (PAFs), there is a need for alternative in vitro bovine follicle culture methods. In this study, we aimed to characterize follicular dynamics using an IVC system of isolated and individually cultured bovine early PAFs during 10 days to generate individual follicle follow-up data. Preantral follicles (ovaries and cultured individually for 10 days. Individual follicle morphology, growth, survival, quality, and cell proliferation were evaluated in time by combining noninvasive and invasive assessment methods. The PAFs were light microscopically evaluated during culture to assess follicular dynamics, stained with neutral red to determine follicle viability, stained with 4',6-diamidino-2-phenylindole and terminal deoxynucleotidyl transferase dUTP nick end labeling to evaluate cell proliferation and follicle quality, and processed for histologic evaluation to assess follicle morphology. On the basis of their morphology, follicles were subdivided in three categories, with category 1 follicles showing the best morphologic features. On Day 0, only category 1 follicles were selected, but follicle categories were reassigned on evaluation Days 1, 2, 4, 7, or 10. Although 67% of the follicles survived 10 days of IVC, the number of follicles exhibiting a normal morphology decreased significantly from Day 7 onward and the apoptotic index increased significantly from Day 10. Both category 1 and 2 follicles showed a significant increase in follicular diameter (Day 10: 21.80 ± 0.86 and 11.82 ± 0.80, respectively). This increase in follicular diameter showed to be correlated with an increase in the total cell number. In conclusion, this culture system showed to support follicular development until Day 10, although the proportion of follicles showing normal morphologic features and the

  7. Cytogenetic data of Partamona peckolti (Hymenoptera, Apidae, Meliponini by C banding and fluorochrome staining with DA/CMA3 and DA/DAPI

    Brito Rute Magalhães

    2003-01-01

    Full Text Available The stingless bees of the Partamona genus have been studied taxonomically, ecologically and behaviourally, but cytogenetic studies are still rare. The objective of this study was to obtain cytogenetic data to contribute to Partamona peckolti species characterization. Heterochromatin was localized in all chromosome pericentromeric regions but some blocks could be visualized on some large chromosomes arms. A large heterozygous DA-CMA3-positive band was observed on one large chromosome arm, but was completely absent when C banding was applied before fluorochrome staining, with only one small positive band being visualized. Sequential DA-CMA3-NOR staining of interphase nuclei provided coincident positive responses. This suggests that DA-CMA3-positive bands of P. peckolti correspond to nucleolar organizer regions, as previously confirmed for another Partamona species by FISH.

  8. Cytogenetic data of Partamona peckolti (Hymenoptera, Apidae, Meliponini) by C banding and fluorochrome staining with DA/CMA3 and DA/DAPI

    2003-01-01

    The stingless bees of the Partamona genus have been studied taxonomically, ecologically and behaviourally, but cytogenetic studies are still rare. The objective of this study was to obtain cytogenetic data to contribute to Partamona peckolti species characterization. Heterochromatin was localized in all chromosome pericentromeric regions but some blocks could be visualized on some large chromosomes arms. A large heterozygous DA-CMA3-positive band was observed on one large chromosome arm, but ...

  9. Formation of primary pit connection during conchocelis phase of Porphyra yezoensis (Bangiophyceae, Rhodophyta)

    SHUAI Li; JIANG Ming; DUAN Delin

    2006-01-01

    The formation of pit connection during conchocelis phase of Porphyra yezoensis Ueda was observed and examined with transmission electron microscope (TEM) and epifluorence microscope. It is indicated that the pit connection was formed in late stage of conchocelis phase and the early stages of conchosporangial cell development, and disappeared in bispore stage. The pit connection contained a thin membrane layer at outer pit plug. Stained with 4′, 6′-diamidino-2-phenylidole dihydrochloride hydrate (DAPI), transferring of DNA or RNA between adjacent cells were observed in late stage of conchocelis development, it was deduced that pit connection might serve as a channel for signal transduction and genetic substance transportation in conchocelis phase.

  10. Sapropterin dihydrochloride use in pregnant women with phenylketonuria: an interim report of the PKU MOMS sub-registry.

    Grange, Dorothy K; Hillman, Richard E; Burton, Barbara K; Yano, Shoji; Vockley, Jerry; Fong, Chin-To; Hunt, Joellen; Mahoney, John J; Cohen-Pfeffer, Jessica L

    2014-05-01

    For pregnant women with phenylketonuria (PKU), maintaining blood phenylalanine (Phe)360μmol/L. Severe adverse events identified by the investigators as possibly related to sapropterin use were premature labor (N=1) and spontaneous abortion (N=1) for the women and hypophagia for the offspring [premature birth (35w4d), N=1]. One congenital malformation (cleft palate) of unknown etiology was reported as unrelated to sapropterin. Although there is limited information regarding the use of sapropterin during pregnancy, these sub-registry data show that sapropterin was generally well-tolerated and its use during pregnancy was associated with lower mean blood Phe. Because the teratogenicity of elevated maternal blood Phe is without question, sapropterin should be considered as a treatment option in pregnant women with PKU who cannot achieve recommended ranges of blood Phe with dietary therapy alone. PMID:24667082

  11. Two randomised phase II trials of subcutaneous interleukin-2 and histamine dihydrochloride in patients with metastatic renal cell carcinoma

    Donskov, F; Middleton, M; Fode, K;

    2005-01-01

    Histamine inhibits formation and release of phagocyte-derived reactive oxygen species, and thereby protects natural killer and T cells against oxidative damage. Thus, the addition of histamine may potentially improve the efficacy of interleukin-2 (IL-2). Two randomised phase II trials of IL-2 with...... randomised phase III trial is warranted to clarify the potential role of adding histamine to IL-2 in mRCC....

  12. Synthesis of o-Chlorophenols via an Unexpected Nucleophilic Chlorination of Quinone Monoketals Mediated by N, N′-Dimethylhydrazine Dihydrochloride

    Yin, Zhiwei; Zhang, Jinzhu; Wu, Jing; Green, Riana; Li, Sihan; Zheng, Shengping

    2014-01-01

    An unexpected nucleophilic chlorination of a quinone monoketal while carrying out a pyrazolidine synthesis has led to a general preparation of multisubstituted phenols. The products are obtained in good to high yields under mild conditions. The bridged pyrazolidines that were the original targets are obtained in the presence of a protic solvent.

  13. Distribution and differentiation of mesenchymal stem cells in tumor tissue

    ZHAO Hai-feng; CHEN Jun; XU Zhi-shun; ZHANG Ke-qin

    2009-01-01

    Background Tumor has an ability to become enriched in mesenchymal stem cells (MSCs) and of guiding MSCs to migrate to tumor tissue. But there are lack of relevant reports on the distribution and differentiation of MSCs in tumor tissue and the effect on tumor growth after MSCs engrafted in tumor tissue. In this study, we observed the distribution of bone marrow MSCs in tumor tissue and the possibility of MSCs differentiating into myofibroblast under the induction of local tumor microenvironment.Methods Twenty-four New Zealand rabbits were randomly classified into the control group and the test group. MSCs were isolated and cultured for each animal, vx-2 tumor tissue was transplanted under the bladder mucosa of each animal. One week after the transplantation, the self F2 passage MSCs marked by 4',6-diamidino-2-phenylindole were transplanted into tumor tissue in the test group while only Dulbecco's modified Eagle's medium-low glucose was infused into the control group. Ultrasonography was performed for each animal 1,2, 3 and 4 week(s) after the vx-2 tumor mass was transplanted. The maximum bladder tumor diameter of each animal was recorded and the mean value of each group was calculated. One animal from each group was sacrificed in the third week and the remaining animals in the fourth week to observe the tumor development. Another animal treated the same as the test group was sacrificed to observe the distribution of MSCs in tumor tissue one week after self MSCs transplantation. Immunofluorescence was used to trace MSCs in tumor tissue. The double labeling immunofluorescence for α-smooth muscle actin (α-SMA) and vimentin was performed to identify whether the MSCs can differentiate into myofibroblast.Results The ultrasonography showed no tumor mass one week after the vx-2 tumor mass transplantation. The mean maximum tumor diameter of the control group and test group was (0.70±0.14) cm and (0.78±0.14) cm, respectively, and there was no significant difference (t=1

  14. DNA staining with the fluorochromes EtBr, DAPI and YOYO-1 in the comet assay with tobacco plants after treatment with ethyl methanesulphonate, hyperthermia and DNase-I

    Gichner, Tomáš; Mukherjee, A.; Velemínský, Jiří

    2006-01-01

    Roč. 605, 1-2 (2006), s. 17-21. ISSN 1383-5718 R&D Projects: GA ČR GA521/05/0500 Institutional research plan: CEZ:AV0Z50380511 Keywords : DNA migration * Nicotiana tabacum * Single-cell gel electrophoresis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.122, year: 2006

  15. Research on Dapi of Song Dynasty:From the Perspective of Execution Rate%宋代大辟研究--从宋代死刑的执行率角度考察

    杨高凡

    2014-01-01

    The proportion of the death penalty cases, which was carried out accounted for only 1/10 of all the death sentences in Song Dynasty. Because the rulers believed in“Wusong” and Renzheng of Confucianism, paid attention to the review of death penalty cases and used to frequent pardon crime in Song Dynasty. The low rate of implement was the symbol of the stable rule of a country. But it di-rectly resulted in the lack of the legal credibility.%宋代每年判决的大辟案件较多,但最后实施真刑的比例大约只占十分之一,其原因在于宋朝君臣尊崇儒家仁政思想、受“无讼”思想的长期影响、死刑复核制度健全、频繁赦宥天下。死刑案件执行率低是宋代推行仁政、法制健全的表现,但死刑执行率低,容易导致缺乏权威性和威慑力。

  16. Comparison of various staining methods for the detection of Cryptosporidium in cell-free culture.

    Boxell, Annika; Hijjawi, Nawal; Monis, Paul; Ryan, Una

    2008-09-01

    The complete development of Cryptosporidium in host cell-free medium first described in 2004, represented a significant advance that can facilitate many aspects of Cryptosporidium research. A current limitation of host cell-free cultivation is the difficulty involved in visualising the life-cycle stages as they are very small in size, morphologically difficult to identify and dispersed throughout the media. This is in contrast to conventional cell culture methods for Cryptosporidium, where it is possible to focus on the host cells and view the foci of infection on the host cells. In the present study, we compared three specific and three non-specific techniques for visualising Cryptosporidium parvum life-cycle stages in cell-free culture; antibody staining using anti-sporozoite and anti-oocyst wall antibodies (Sporo-Glo and Crypto Cel), fluorescent in-situ hybridization (FISH) using a Cryptosporidium specific rRNA oligonucleotide probe and the non-specific dyes; Texas Red, carboxyfluorescein diacetate succinimidyl ester (CFSE) and 4,6' diamino-2-phenylindole dihydrochloride (DAPI). Results revealed that a combination of Sporo-Glo and Crypto Cel staining resulted in easy and reliable identification of all life-cycle stages. PMID:18547565

  17. Distribution of marine bacteria and their environmental factors in Xiangshan Bay%象山港海域细菌的分布特征及其环境影响因素

    杨季芳; 王海丽; 陈福生; 屠霄霞; 陈吉刚; 冯辉强

    2011-01-01

    Marine bacteria in water or sediment are often closely linked to environmental conditions in their surrounding, thus influencing their distribution and abundance. In order to estimate the distribution of marine bacteria in Xiangshan Bay , undisturbed overlying seawater, sediment and surface seawater bacteria were analysed by staining with 4. 6-Diamidino-2Phenylindole and observed with Epifluorescence Microscope ( EFM ). Environmental parameters such as seawater temperature ( ST) , salinity ( SS) , pH, dissolved oxygen ( DO) , chemical oxygen demand ( COD) as well as NO:-N,N02--N , DIN , NH+4-N , DIP, TN , PO3-4 -P and chlorophyll a ( Chla) values were measured at the same time. Samples were collected with a cylindrical high fidelity gravity sampler instead of the conventional grab sampler. Bacteria abundance in July 2007 , October 2007 , January 2008 and April 2008 , representing the situation for summer,autumn, winter and spring, ranged from 1. 50 ×lO5to 9. 78 ×l010 cells/mL ( cells/g) , with a mean of 2. 76× 109 cells/mL (cells/g). Significantly high values could be observed for bacteria abundance in July ( summer) compared to other sampling time. A red tide outbreak during the sampling event in April 2008 could have contributed to the lowest bacteria abundance value observed for the whole study period. In vertical direction , the number of bacteria in the overlying water or in the sediment was higher than that in the surface seawater. The mean number of bacteria abundance in overlying water, in the sediment and in surface seawater was 9. 70×107cells/mL, 7. 40×109cells/mL and 4. 61 ×109cells/mL respectively. In horizontal direction, we draw the contour maps of the number of bacteria with the software ‘ surfer 8.0 ’ . According to the contour map we found that the number of bacteria decreased from the top to the mouth of the bay with higher abundance near the breeding areas. Very high values of bacteria abundance was also observed near power plant

  18. c-Jun N-terminal kinase is required for vitamin E succinate-induced apoptosis in human gastric cancer cells

    Kun Wu; Yan Zhao; Gui-Chang Li; Wei-Ping Yu

    2004-01-01

    AIM: To investigate the roles of c-Jun N-terminal kinase (JNK)signaling pathway in vitamin E succinate-induced apoptosis in human gastric cancer SGC-7901 cells.METHODS: Human gastric cancer cell lines (SGC-7901)were treated with vitamin E succinate (VES) at 5, 10, 20 mg/L.Succinic acid and vitamin E were used as vehicle controls and condition medium only as an untreated (UT) control.Apoptosis was observed by 4′, 6-diamidine-2′-phenylindole dihydrochloride (DAPI) staining for morphological changes and by DNA fragmentation for biochemical alterations.Western blot analysis was applied to measure the expression ofJNK and phosphorylated JNK. After the cells were transiently transfected with dominant negative mutant of JNK (DNJNK) followed by treatment of VES, the expression of JNK and c-Jun protein was determined.RESULTS: The apoptotic changes were observed after VES treatment by DNA fragmentation. DNA ladder in the 20 mg/L VES group was more clearly seen than that in 10 mg/L VES group and was not detected following treatment of UT control, succinate and vitamin E. VES at 5, 10 and 20 mg/L increased the expression of p-JNK by 2.5-, 2.8- and 4.2-fold, respectively. VES induced the phosphorylation of JNK beginning at 1.5 h and produced a sustained increase for 24 h with the peak level at 12 h. Transient transfection of DN-JNK blocked VES-triggered apoptosis by 52%. DN-JNK significantly increased the level of JNK, while decreasing the expression of VES-induced c-Jun protein.CONCLUSION: VES-induced apoptosis in human gastric cancer SGC-7901 cells involves JNK signaling pathway via c-Jun and its downstream transcription factor.

  19. Drug: D08806 [KEGG MEDICUS

    Full Text Available D08806 Mixture, Drug Histamine dihydrochloride - human normal immunoglobulin mixt; ...Histaglobin (TN) Histamine dihydrochloride [DR:D04444], Human normal immunoglobulin [DR:D06458] Therapeutic ...category: 6399 Therapeutic category of drugs in Japan [BR:br08301] 6 Agents against pathologic organisms and... parasites 63 Biological preparations 639 Miscellaneous 6399 Others D08806 Histamine dihydrochloride - human normal immunoglobulin mixt PubChem: 96025489 ...

  20. Drug: D08461 [KEGG MEDICUS

    Full Text Available D08461 Drug Quinine dihydrochloride; Quinine (TN) C20H24N2O2. 2HCl 396.1371 397.3386 D08461.gif Antiprotozoa... USP drug classification [BR:br08302] Antiparasitics Antiprotozoals Quinine D08461 Quinine dihydrochloride A

  1. Drug: D02548 [KEGG MEDICUS

    Full Text Available PROTOZOAL DISEASES P01AX Other agents against amoebiasis and other protozoal diseases P01AX05 Mepacrine D02...548 Mepacrine dihydrochloride Antiinfectives [BR:br08307] Antiparasitics Agents against Amebiasis and other antiprotozoa

  2. Phenylketonuria (PKU)

    ... may benefit from a medication called sapropterin dihydrochloride (brand name Kuvan®) that treats the disorder. 2 NICHD. ( ... Advance Birth Defects Research September Is Newborn Screening Awareness Month All related news Home Contact Accessibility Web ...

  3. Drug: D01798 [KEGG MEDICUS

    Full Text Available D01798 Drug Sapropterin hydrochloride (JAN); Sapropterin dihydrochloride (USAN); Biopten (TN); K ... gif Treatment of mild-to-moderate phenylketonuria (PKU ) Therapeutic category: 3999 ATC code: A16AX07 Ther ...

  4. 77 FR 63290 - Foreign-Trade Zone 121-Albany, NY; Notification of Proposed Production Activity; Albany Molecular...

    2012-10-16

    ... Rensselaer, New York, (Board Order 698, 7/20/1994, 59 FR 18318, 7/28/1994). AMRI is now requesting to produce an active pharmaceutical ingredient, dexpramipexole dihydrochloride monohydrate, under...

  5. Drug: D02536 [KEGG MEDICUS

    Full Text Available D02536 Drug Pyrithioxine hydrochloride (JAN); Pyrithioxine dihydrochloride hydrate; Pyritinol di ... OANALEPTICS N06B PSYCHOSTIMULANTS, AGENTS USED FOR ADHD ... AND NOOTROPICS N06BX Other psychostimulants and no ...

  6. [Comparative cytogenetic study of the tetraploid Matricaria chamomilla L. and Matricaria inodora L].

    Samatadze, T E; Amosova, A V; Mel'nikova, N V; Suslina, S N; Zagumennikova, T N; Zelenin, A V; Bykov, V A; Muravenko, O N

    2014-01-01

    A comparative cytogenetic study of the autotetraploid breed of Matricaria chamomilla L. (M. recutita L.) and Matricaria inodora L. was carried out by DAPI-banding, fluorescent hybridization in situ (FISH) with 26S and 5S rDNA probes, and analysis of meiosis. All chromosomes were identified in both karyotypeson the basis of DAPI-banding images and 26S and 5S rDNA distribution, and species-specific idiograms were composed for both M. chamomilla and M. indora taking into account the polymorphous variants of DAPI-banding images, showing the location of the 26S and 5S rDNA sites. PMID:25735163

  7. Intra- and interspecific chromosome polymorphisms in cultivated Cichorium L. species (Asteraceae)

    Bernardes, Ebenézer C.S.; Benko-Iseppon, Ana M.; Santelmo Vasconcelos; Reginaldo de Carvalho; Brasileiro-Vidal, Ana C.

    2013-01-01

    Endive (Cichorium endivia L.) and chicory (C. intybus L.) both have 2n = 18, but until now, there has been no detailed karyomorphological characterization. The present work evaluated five accessions of each species using FISH with rDNA probes and fluorochrome staining with CMA and DAPI. Both species presented distinct banding patterns after fluorochrome staining: while endive had proximal CMA++/DAPI- bands in the short arms of pairs 1, 2 and 3, chicory had proximal CMA-positive bands in chrom...

  8. The synthesis of bisguanidinoalkanes and guanidinoalkanes, N- or N'-substitutes with pyrimidines, as analogues of chlorhexidine

    A series of N,N''' -alkanediylbis[N'-(5-halopyrimidin-2-yl)guanidine] salts has been synthesized along with N,N'''-(trans-cyclohexane-1,4-diyl)bis[N'-(5-chloropyrimidin-2-yl)guanidine], N,N'''-(cis-cyclohexane-1,4-diyl)bis[N'-(5-chloropyrimidin-2-yl)guanidine] dihydrochloride and N-(cis-4-aminocyclohexan-1-yl)-N'-(5-chloropyrimidin-2-yl)guanidine dihydrochloride. Furthermore, a series of N-(alkan-1-yl)-N'-(5-chloropyrimidin-2yl)guanidine hydrochlorides and N-(6-aminohexan-1-yl)-N'-(5-chloropyrimidin-2-yl)guanidine dihydrochloride were synthesized. This series of compounds was prepared by displacement reactions of 2-methylsulfonylpyrimidines with bisguanidinoalkanes or by condensation of 5-chloro-2-cyanoaminopyrimidine (5-chloropyrimidin-2-ylcyanamide) with alkylamines. 19 refs

  9. The synthesis of bisguanidinoalkanes and guanidinoalkanes, N- or N`-substitutes with pyrimidines, as analogues of chlorhexidine

    Elmes, B.C.; Holan, G.; Wernert, G.T.; Winkler, D.A. [Commonwealth Scientific and Industrial Research Organisation (CSIRO), Melbourne, VIC (Australia). Div. of Materials Science

    1996-12-31

    A series of N,N``` -alkanediylbis[N`-(5-halopyrimidin-2-yl)guanidine] salts has been synthesized along with N,N```-(trans-cyclohexane-1,4-diyl)bis[N`-(5-chloropyrimidin-2-yl)guanidine], N,N```-(cis-cyclohexane-1,4-diyl)bis[N`-(5-chloropyrimidin-2-yl)guanidine] dihydrochloride and N-(cis-4-aminocyclohexan-1-yl)-N`-(5-chloropyrimidin-2-yl)guanidine dihydrochloride. Furthermore, a series of N-(alkan-1-yl)-N`-(5-chloropyrimidin-2yl)guanidine hydrochlorides and N-(6-aminohexan-1-yl)-N`-(5-chloropyrimidin-2-yl)guanidine dihydrochloride were synthesized. This series of compounds was prepared by displacement reactions of 2-methylsulfonylpyrimidines with bisguanidinoalkanes or by condensation of 5-chloro-2-cyanoaminopyrimidine (5-chloropyrimidin-2-ylcyanamide) with alkylamines. 19 refs.

  10. The synthesis of 3H-putrescine and subsequent biosynthesis of 3H-jacobine, a pyrrolizidine alkaloid from Senecio jacobaea

    A new method was developed for the preparation of tritiated putrescine dihydrochloride ([2,3-3H]-1,4-diaminobutane dihydro-chloride) from succinonitrile (1,4-butanedinitrile) and 3H2O, with a radiochemical yield of 16%. Tritiated jacobine and other pyrrolizidine alkaloids were then biosynthesized in Senecio jacobaea using 3H-putrescine-2HCl as the precursor with a radiochemical yield of 0.9% into total pyrrolizidine alkaloids. Jacobine accounted for 36% of the total. This synthetic method provides a relatively inexpensive source for the preparation of these labelled compounds. (author)