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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. 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...

  13. 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...

  14. 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...

  15. 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 (...

  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. 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...

  19. 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

  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. 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.

  10. 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...

  11. 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...

  12. 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...

  13. [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

  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 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.

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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

  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. 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.

  15. 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

  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. 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.

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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

  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. 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.

  19. 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

  20. 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.

  1. 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.

  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. 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...

  10. 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...

  11. 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...

  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. 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

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. 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

  19. 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

  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. 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

  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. 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.

  8. 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.

  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. 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

  12. 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

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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

  8. 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.

  9. 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.

  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. 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 %.

  16. 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.

  17. 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

  18. 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

  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. 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.

  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. 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

  8. 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

  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. 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

  11. 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

  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. 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

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. 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

  2. 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

  3. 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.

  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. 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

  8. 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.

  9. 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

  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. 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.)

  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. 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.

  18. 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.

  19. 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.

  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. 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

  2. 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

  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. 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)

  9. 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

  10. 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

  11. 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.

  12. 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.

  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. 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.

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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

  18. 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.

  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. 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

  3. 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

  4. 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.

  5. 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

  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

    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 ...

  8. 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.

  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. 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....

  11. 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.

  12. 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

  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. 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...

  2. 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 ...

  3. 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

  4. 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 ...

  5. 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 ...

  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)

  11. Stable carbocations from terpenoids. VII. Generation and transformations of carbodications with a cadalene skeleton

    For the case of certain sesquiterpenes isomeric stable carbodications were generated for the first time from dihydrochlorides and dienes having identical skeletons, with the same arrangement of the developing cationoid centers, and under identical conditions. Their spectral characteristics and the structures of the products from the quenching of the acidic solutions were studied. A mechanism is proposed for the formation of the dications from the dihydrochlorides. The effect of the conditions for the generation of the carbocations on the nature of the molecular rearrangements was studied

  12. Influence of Different Diets on Development of DMH-Induced Aberrant Crypt Foci and Colon Tumor Incidence in Wistar Rats

    Kristiansen, E.; Thorup, I.; Meyer, Otto A.

    1995-01-01

    The present study was undertaken to investigate certain dietary factors known to affect the development of colon cancer for their ability to modulate aberrant crypt foci (ACI;). Male Wistar rats were initiated with oral noses of dimethylhydrazine dihydrochloride (DMH-2HCl, 20 mg/kg body wt) once a...

  13. 21 CFR 520.2123a - Spectinomycin tablets.

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Spectinomycin tablets. 520.2123a Section 520.2123a... DRUGS, FEEDS, AND RELATED PRODUCTS ORAL DOSAGE FORM NEW ANIMAL DRUGS § 520.2123a Spectinomycin tablets. (a) Specifications. Each tablet contains spectinomycin dihydrochloride pentahydrate equivalent to...

  14. Drug: D09934 [KEGG MEDICUS

    Full Text Available D09934 Drug Naronapride dihydrochloride (USAN) C27H41ClN4O5. 2HCl 608.2299 610.0131...s Rhodopsin family Serotonin 5-HT4-receptor [HSA:3360] [KO:K04160] Naronapride D09934 Naronapride dihydrochl

  15. Silicon quantum dot sensors for an explosive taggant, 2,3-dimethyl-2,3-dinitrobutane (DMNB).

    Kim, Jin Soo; Cho, Bomin; Cho, Soo Gyeong; Sohn, Honglae

    2016-07-01

    Silicon quantum dots obtained by the reaction of magnesium silicide with ethylenediamine dihydrochloride were utilized to investigate the sensing mechanism and sensitivity for DMNB detection applications. Sensing DMNB provided us with evidence that Si QDs with a higher lying conduction band have better sensitivity compared to CdSe QDs. PMID:27188402

  16. Drug: D09887 [KEGG MEDICUS

    Full Text Available D09887 Drug Dexpramipexole dihydrochloride (USAN) C10H17N3S. 2HCl. H2O 301.0782 302.2642 D09887. ... gif Treatment of amyotrophic lateral s clerosis (ALS ) R(+) enantiomer of Pramipexole [DR: ...

  17. 21 CFR 529.400 - Chlorhexidine tablets and suspension.

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Chlorhexidine tablets and suspension. 529.400... Chlorhexidine tablets and suspension. (a) Specification. Each tablet and each 28-milliliter syringe of suspension contain 1 gram of chlorhexidine dihydrochloride.1 1 These conditions are NAS/NRC reviewed...

  18. Coloração de núcleos de esporos e hifas do cogumelo Agaricus blazei Nucleus stain of spores and hifas Ofagaricus blazei

    Cláudia Regina Gontijo Labory

    2003-04-01

    Full Text Available Com o objetivo de se estudar o comportamento nuclear durante o ciclo sexual do cogumelo Agaricus blazei , técnicas de coloração e a utilização de fluorocromos foram testadas, visando a uma padronização metodológica para estudos citogenéticos futuros. Das técnicas avaliadas neste trabalho (método de Feulgen, coloração com Giemsa e fluorescência-DAPI, a coloração com Giemsa e o fluorocromo DAPI apresentaram os melhores resultados para evidenciar núcleos. O corante Giemsa permitiu a visualização de compartimentos multinucleados. Com o DAPI, não foi possível a visualização dos septos, sendo necessária a utilização adicional de calcofluor, que tem a propriedade de corar a quitina que delimita os compartimentos das hifas.In order to study the nuclear behavior during the sexual cycle of Agaricus blazei , some coloration and fluorescent stain techniques were tested for a method standardization for fut ure cytogenetical works. Among the studied techniques (Schiff reactive, Giemsa, and fluorescent stain - DAPI, the Giemsa and fluorescent DAPI technique provided the best results. With Giemsa stain, it was possible to visualize multinucleate cell compartments. It was not possible to stain compartmental delimitation with DAPI stain, which was possible only with an additional fluorescent stain - calcofluor, which is specific for chitin.

  19. 1,5-diamino-2-pentyne is both a substrate and inactivator of plant copper amine oxidases.

    Lamplot, Zbynek; Sebela, Marek; Malon, Michal; Lenobel, René; Lemr, Karel; Havlis, Jan; Pec, Pavel; Qiao, Chunhua; Sayre, Lawrence M

    2004-12-01

    1,5-diamino-2-pentyne (DAPY) was found to be a weak substrate of grass pea (Lathyrus sativus, GPAO) and sainfoin (Onobrychis viciifolia, OVAO) amine oxidases. Prolonged incubations, however, resulted in irreversible inhibition of both enzymes. For GPAO and OVAO, rates of inactivation of 0.1-0.3 min(-1) were determined, the apparent KI values (half-maximal inactivation) were of the order of 10(-5) m. DAPY was found to be a mechanism-based inhibitor of the enzymes because the substrate cadaverine significantly prevented irreversible inhibition. The N1-methyl and N5-methyl analogs of DAPY were tested with GPAO and were weaker inactivators (especially the N5-methyl) than DAPY. Prolonged incubations of GPAO or OVAO with DAPY resulted in the appearance of a yellow-brown chromophore (lambda(max) = 310-325 nm depending on the working buffer). Excitation at 310 nm was associated with emitted fluorescence with a maximum at 445 nm, suggestive of extended conjugation. After dialysis, the color intensity was substantially decreased, indicating the formation of a low molecular mass secondary product of turnover. The compound provided positive reactions with ninhydrin, 2-aminobenzaldehyde and Kovacs' reagents, suggesting the presence of an amino group and a nitrogen-containing heterocyclic structure. The secondary product was separated chromatographically and was found not to irreversibly inhibit GPAO. MS indicated an exact molecular mass (177.14 Da) and molecular formula (C10H15N3). Electrospray ionization- and MALDI-MS/MS analyses yielded fragment mass patterns consistent with the structure of a dihydropyridine derivative of DAPY. Finally, N-(2,3-dihydropyridinyl)-1,5-diamino-2-pentyne was identified by means of 1H- and 13C-NMR experiments. This structure suggests a lysine modification chemistry that could be responsible for the observed inactivation. PMID:15606757

  20. Fast nuclear staining of head hair roots as a screening method for successful STR analysis in forensics

    Lepez, Trees; Vandewoestyne, Mado; Van Hoofstat, David; Deforce, Dieter

    2014-01-01

    The success rate of STR profiling of hairs found at a crime scene is quite low and negative results of hair analysis are frequently reported. To increase the success rate of DNA analysis of hairs in forensics, nuclei in hair roots can be counted after staining the hair root with DAPI. Two staining methods were tested: a longer method with two 1 h incubations in respectively a DAPI-and a wash-solution, and a fast, direct staining of the hair root on microscope slides. The two staining meth...

  1. Subsurface microbial ecology. Epi fluorescence direct counts; Ecologia microbica del sottosuolo: metodo di conta diretta in epifluorescenza

    Barra Caracciolo, A.; Silvestri, C.; Creo, C.; Izzo, G. [ENEA, Centro Ricerche Casaccia, Rome (Italy). Dipt. Ambiente

    1998-07-01

    To the aim of recognize the importance of microorganisms in affecting or even determining the fate of xenobiotics in the subsurface environment evaluating bacteria concentration in a subsurface ecosystem, the report discusses a soil sample treatment method which has been developed for epi fluorescence direct counting with DAPI. [Italian] Lo studio discute un metodo di trattamento del campione per la conta diretta in epifluorescenza con un marcatore selettivo per il DNA, il DAPI, al fine di quantificare la concentrazione batterica del sottosuolo e studiare il ruolo dei microrganismi nella biodegradazione delle molecole esogene, ancora poco indagato.

  2. Synthesis and Characterization of Novel Polyimide Gas Separation Membrane Material Systems

    Farr, Isaac Vincent

    1999-01-01

    Phenylindane monomers 5(6)-amino-1-(4-aminophenyl)-1,3,3-trimethylindane (DAPI), 5,6-diamino-1-(4-aminophenyl)-1,3,3-trimethylindane (TAPI) and 6-hydroxy- 1-(4-hydroxyphenyl)-1,3,3-trimethylindane (DHPI) were synthesized and characterized. DAPI, as well as other diamines, were then utilized in solution step polycondensation with a number of commercially available dianhydrides using either the two-step ester-acid solution imidization or the high temperature solution imidization routes. Hi...

  3. Antiviral treatment is more effective than smallpox vaccination upon lethal monkeypox virus infection

    Stittelaar, K. J.; Neyts, J.; Naesens, L.; Amerongen van, G.; Lavieren van, R. F.; Holý, Antonín; De Clercq, E.; Niesters, H. G. M.; Fries, E.; Maas, Ch.; Mulder, P. G. H.; Zeijst van der, B. A. M.; Osterhaus, D. M. E.

    2006-01-01

    Roč. 439, č. 7077 (2006), s. 745-748. ISSN 0028-0836 Institutional research plan: CEZ:AV0Z40550506 Keywords : monkeypox virus * vaccination * HPMPC * HPMPO-DAPy Subject RIV: CC - Organic Chemistry Impact factor: 26.681, year: 2006

  4. High resolution DNA flow cytometry of boar sperm cells in identification of boars carrying cytogenetic aberrations

    Larsen, Jacob; Christensen, Knud; Larsen, Jørgen K;

    2004-01-01

    The cytogenetic quality of boars used for breeding determines the litter outcome and thus has large economical consequences. Traditionally, quality controls based on the examination of simple karyograms are time consuming and sometimes give uncertain results. As an alternative, the use of high......-resolution DNA flow cytometry on DAPI-stained sperm cell nuclei (CV...

  5. Issue Cover (June 2016).

    2016-06-01

    Cover legend: Yeast cells were labeled with the fluorescent viability dyes propidium iodide (Red) and DiBAC4(3) (green) and the nucleus was stained with DAPI (blue). Cells were visualized using wide-field fluorescent microscopy. See Chadwick et al. Traffic 2016; 17(6):689-703. Read the full article on doi:10.1111/tra.12391. PMID:27174376

  6. Histologic analysis of rabbit liver cancer treated by bulk ultrasound ablation

    Karunakaran, Chandra Priya; Rudich, Steven M.; Alqadah, Amel; Burgess, Mark T.; Narmoneva, Daria A.; Mast, T. Douglas

    2012-10-01

    VX2 rabbit liver cancer, treated in vivo using bulk ultrasound ablation by miniaturized image-ablate arrays, was histologically analyzed using TTC vital stain and DAPI nucleic acid stain. VX2 cells were implanted into rabbit liver lobes and allowed to grow for 11-21 days. Liver lobes containing solid VX2 tumors were then treated with 4.8 MHz, 22.5-38.5 W/cm2 in situ intensity, unfocused ultrasound for exposure times of 20-120 s. After animal sacrifice, thermal lesions were bisected along the imaging/treatment plane, one face stained with TTC, and the other with DAPI. Levels of TTC uptake (no uptake, partial uptake, and complete uptake) in liver parenchyma corresponded to three discrete regions of tan, pink and red color. By processing images of DAPI-stained parenchymal tissue from these three regions, cellular damage was quantified. A viability index parameter incorporating the size and shape of DAPI-stained nuclei correlated significantly with levels of TTC uptake, and thus with local tissue viability. For ablation of normal liver, viability indices for parenchymal regions of no TTC uptake and partial TTC uptake were significantly different from those for viable tissue. For ablation of VX2 tumor, differences in viability index between regions of no TTC uptake and complete TTC uptake were smaller, but significant overall.

  7. Taxonomic revision of Sorbus subgenus Aria occurring in the Czech Republic

    Lepší, M.; Lepší, P.; Koutecký, P.; Bílá, J.; Vít, Petr

    2015-01-01

    Roč. 87, č. 2 (2015), s. 109-162. ISSN 0032-7786 R&D Projects: GA ČR GB14-36079G Institutional support: RVO:67985939 Keywords : apomixis * DAPI flow cytometry * hybridization Subject RIV: EF - Botanics Impact factor: 4.104, year: 2014

  8. Heterochromatin differentiation in holocentric chromosomes of Rhynchospora (Cyperaceae

    André L.L. Vanzela

    2000-06-01

    Full Text Available Holocentric chromosomes of six species of Rhynchospora, R. ciliata, R. pubera, R. riparia and R. barbata (2n = 10, R. nervosa (2n = 30 and R. globosa (2n = 36, were stained with CMA3/DAPI fluorochromes or treated with C-banding and sequentially stained with Giemsa or CMA3/DAPI. Variability in banding pattern was found among the species studied. Heterochromatin was observed on terminal and interstitial chromosome regions, indicating that the holocentric chromosomes of Rhynchospora show a heterochromatin distribution pattern similar to those plant monocentric chromosomes.Cromossomos holocêntricos de seis espécies de Rhynchospora (R. ciliata, R. pubera, R. riparia e R. barbata (2n = 10, R. nervosa (2n = 30 and R. globosa (2n = 36 foram corados com os fluorocromos CMA3/DAPI ou tratados para bandeamento C e seqüencialmente corados com Giemsa ou CMA3/DAPI. Variabilidade no padrão de bandas foi encontrada entre as espécies estudadas. A heterocromatina foi observada em regiões terminais e intersticiais dos cromossomos, indicando que os cromossomos holocêntricos de Rhynchospora mostram um padrão de distribuição de heterocromatina similar àqueles dos cromossomos monocêntricos de plantas.

  9. Quantization of dextromethorphan and levocetirizine in combined dosage form using a novel validated RP-HPLC method

    Shalini Joshi

    2012-01-01

    Full Text Available The present study reveals a simple isocratic RP-HPLC method for the simultaneous determination of dextromethorphan hydrobromide and levocetirizine dihydrochloride in a cough syrup. The separation of these compounds was achieved within 10 min on a Phenomenex (USA C 18 analytical column, 250Χ4.0 mm i.d., using an isocratic mobile phase consisting of potassium dihydrogen phosphate buffer (pH 2.5 - acetonitrile- tetrahydrofuran (70:25:5, v/v/v. The analysis was performed at a flow rate of 1.2 ml/min and at a detection wavelength of 232 nm. Percentage recovery and RSD were 100.36% and 0.05% for levocetirizine dihydrochloride, 100.35% and 0.27% for dextromethorphan hydrobromide respectively. Quantification of the components in syrup formulation was calculated against the peak areas of freshly prepared standard solutions. The method was validated as per ICH guidelines.

  10. Quantization of Dextromethorphan and Levocetirizine in Combined Dosage form Using a Novel Validated RP-HPLC Method.

    Joshi, Shalini; Bhatia, C; Bal, C S; Rawat, M S M

    2012-01-01

    The present study reveals a simple isocratic RP-HPLC method for the simultaneous determination of dextromethorphan hydrobromide and levocetirizine dihydrochloride in a cough syrup. The separation of these compounds was achieved within 10 min on a Phenomenex (USA) C(18) analytical column, 250×4.0 mm i.d., using an isocratic mobile phase consisting of potassium dihydrogen phosphate buffer (pH 2.5) - acetonitrile- tetrahydrofuran (70:25:5, v/v/v). The analysis was performed at a flow rate of 1.2 ml/min and at a detection wavelength of 232 nm. Percentage recovery and RSD were 100.36% and 0.05% for levocetirizine dihydrochloride, 100.35% and 0.27% for dextromethorphan hydrobromide respectively. Quantification of the components in syrup formulation was calculated against the peak areas of freshly prepared standard solutions. The method was validated as per ICH guidelines. PMID:23204629

  11. Motoneuron organisation of the muscles of the spinal accessory complex of the sheep investigated with the fluorescent retrograde tracer technique.

    P. Clavenzani; Scapolo, P A; Callegari, E; Barazzoni, A M; Petrosino, G.; Lucchi, M L; Bortolami, R

    1994-01-01

    Retrograde transport of the fluorescent tracers Diamidino Yellow dihydrochloride and Fast Blue was used to determine the location of the spinal nucleus of the accessory nerve in the sheep. We also considered whether in this species the sternocephalic, brachiocephalic, omotransversarius and trapezius muscles, i.e. the muscles of the spinal accessory complex, are supplied by more than one population of motoneurons. The spinal accessory nucleus extends as a single column of neurons from C1 to C7...

  12. The European standard series. European Environmental and Contact Dermatitis Research Group (EECDRG)

    Bruynzeel, D P; Andersen, Klaus Ejner; Camarasa, J G;

    1995-01-01

    Changes to the European standard series which have taken place since the last officially recommended alterations in 1988, are explained. New to the series is the sesquiterpene lactone mix. The PPD black rubber mix and the quinoline mix have been replaced by single components; one of the p-hydroxy......-hydroxybenzoates has been left out of the paraben mix. Ethylenediamine dihydrochloride has been dropped from the series....

  13. Altered membrane lipid dynamics and chemoprevention by non-steroidal anti inflammatory drugs during colon carcinogenesis Alteración de la dinámica de los lípidos de membrana y quimioprevención mediante los fármacos antiinflamatorios no esteroideos en la carcinogénesis de colon

    S. Singh Kanwar; V. Vaish; S. Nath Sanya

    2011-01-01

    The present work focuses on the anti-neoplastic role of non steroidal anti-inflammatory drugs (NSAIDs) in modulating the biophysical parameters of the colonic membranes in 1,2-dimethylhydrazine dihydrochloride (DMH) induced carcinogenesis. The steady-state fluorescence polarization technique was applied to assess membrane fluidity, membrane polarity and lipid phase states. The decline in cholesterol content, biosynthesis and cholesterol: phospholipids ratio with DMH treatment indicates more f...

  14. SELECTION OF GROWTH STIMULATORS AMONG THE PYRIDINE SUBSTITUTES OF MERCAPTO ACIDS

    Petrusha Yu.Yu.; Omelyanchik L.A.

    2013-01-01

    We tracked the grow-stimulating activity of new synthesized pyridinesubstitutes of L- cysteine and others mercaptoacids. We also studied the influence of the synthesized conections on cels growth and fision of Cucumis sp. sprouts (cultivar "Competitor"). The obtained results confirm the perspective selection of new efective and ecologicaly safe grow-stimulators of agricultural culture from these chemical substances. We sugested that dihydrochloride S-(pyridine-4-il)-L-cysteine cou...

  15. Development, validation and comparison of NIR and Raman methods for the identification and assay of poor-quality oral quinine drops.

    Mbinze Kidenge, Jérémie(*); Sacre, Pierre-Yves; Yemoa, Achille; Mavar Tayey Mbay, Jean; Habyalimana, Védaste; Kalenda, Nick; HUBERT, Philippe; Marini Djang'Eing'A, Roland; Ziemons, Eric

    2015-01-01

    Poor quality antimalarial drugs are one of the public’s major health problems in Africa. The depth of this problem may be explained in part by the lack of effective enforcement and the lack of efficient local drug analysis laboratories. To tackle part of this issue, two spectroscopic methods with the ability to detect and to quantify quinine dihydrochloride in children’s oral drops formulations were developed and validated. Raman and Near Infrared (NIR) spectroscopy were selected for the drug...

  16. Co-occurrence of the Cyanotoxins BMAA, DABA and Anatoxin-a in Nebraska Reservoirs, Fish, and Aquatic Plants

    Al-Sammak, Maitham; Hoagland, Kyle; Cassada, David; Snow, Daniel

    2014-01-01

    Several groups of microorganisms are capable of producing toxins in aquatic environments. Cyanobacteria are prevalent blue green algae in freshwater systems, and many species produce cyanotoxins which include a variety of chemical irritants, hepatotoxins and neurotoxins. Production and occurrence of potent neurotoxic cyanotoxins β-N-methylamino-l-alanine (BMAA), 2,4-diaminobutyric acid dihydrochloride (DABA), and anatoxin-a are especially critical with environmental implications to public and...

  17. Drug: D10105 [KEGG MEDICUS

    Full Text Available D10105 Drug Daclatasvir hydrochloride (JAN); Daclatasvir dihydrochloride (USAN) C40...H50N8O6. 2HCl 810.3387 811.7969 D10105.gif Treatment of hepatitis C [DS:H00413] HCV NS5A inhibitor [CPD:C182...92] ko05160 Hepatitis C CAS: 1009119-65-6 PubChem: 135626823 LigandBox: D10105 ATOM 56 1 C8x C 21.4200 -21.4

  18. Induction of pigment formation in phenolic compounds by gamma-irradiated sodium chloride

    A spectrophotometric study is reported on the pigment formation of free radicals produced from o-dianisidine dihydrochloride and pyrogallol by the action of γ-irradiated sodium chloride. The species liberated during dissolution of the γ-irradiated salt also greatly enhance the rate of catalytic formation of the pigment due to peroxidase enzyme in the presence of hydrogen peroxide. The G values for these systems are compared. (author) 7 refs.; 4 figs

  19. Synthesis and Applications of (-)-(S)-3-Aminoquinuclidine- Derived Thiourea

    Rolava, E; Turks, M

    2015-01-01

    A synthesis of enantiopure thiourea organocatalyst based on (-)-(S)-3-aminoquinuclidine dihydrochloride was developed with quantitative product yield. The catalyst was tested in different reactions: asymmetric Michael addition of ketones and malonates to nitroalkenes, nitromethane 1,4-addition to trans-chalcone, and Friedel-Crafts alkylation of indoles with trans-β-nitrostyrene. The novel thiourea proved to catalyze the aforementioned reactions and expected products were ...

  20. Effect of Chinese Medical Herbs-Huiru Yizeng Yihao on Hyperprolactinemia and Hyperplasia of Mammary Gland in Mice

    Wang, Xiong; Chen, Yong-Gang; Ma, Li; Li, Zhi-hui; Li, Ju-yi; Liu, Xin-guo; Zou, Ji-li; Wu, Jin-hu

    2013-01-01

    The study investigated the pharmacodynamism and mechanism of Chinese medicinal formula-Huiru Yizeng Yihao (NO.1 HRYZ) on the model rats of hyperpro-lactinemia and the model rats of hyperplasia of mammary gland (HMG), and studied the internal connection between hyperprolactinemia and HMG.. The hyperprolactinemia rat models were established by injecting metoclopramide dihydrochloride in the back of rats. The model rat of HMG was prepared by injecting estradiol in the thigh muscle of the rats an...

  1. Effect of trimetazidine on membrane damage induced by oxygen free radicals in human red cells.

    Maridonneau-Parini, I; Harpey, C.

    1985-01-01

    The effect of trimetazidine, 1-(2, 3, 4 trimethoxybenzyl)piperazine di-hydrochloride, on membrane damage induced by oxygen free radicals in red cells was studied in seven healthy volunteers after oral administration. Red cells collected prior to and after a 7 day treatment period with trimetazidine were incubated in the presence of phenazine methosulphate (an intracellular oxygen free radical generator) and diethyldithiocarbamate (a Cu-Zn superoxide dismutase inhibitor). The loss of intracell...

  2. Taste Masked Orally Disintegrating Pellets of Antihistaminic and Mucolytic Drug: Formulation, Characterization, and In Vivo Studies in Human

    Taj, Yasmeen; Pai, Roopa S; V Kusum Devi; Singh, Gurinder

    2014-01-01

    The main aim of the present study was to evaluate the potential of orally disintegrating pellets (ODPs) as an approach for taste masking of bitter drugs, namely, Ambroxol hydrochloride (A-HCl) and Cetirizine dihydrochloride (C-DHCl). Pellets were prepared by extrusion/spheronization with Eudragit EPO, kyron T-134, Kyron T-314, mannitol, sorbitol, MCC (Avicel PH-101), sucralose, chocolate flavor, and 5% xanthum gum. The prepared pellets were characterized for percentage yield, drug content, pa...

  3. The antidepressant- and anxiolytic-like activities of new xanthone derivative with piperazine moiety in behavioral tests in mice

    Pytka, Karolina; Żmudzka, Elżbieta; Lustyk, Klaudia; Rapacz, Anna; Olczyk, Adrian; Gałuszka, Adam; Waszkielewicz, Anna; Marona, Henryk; Sapa, Jacek; Barbara, Filipek

    2016-01-01

    Objectives: Xanthones are flavonoids with numerous activities, including antioxidant, antidepressant., or anxiolytic-like. Therefore, the aim of our study was to determine antidepressant- and anxiolytic-like properties of four xanthone derivatives (3-chloro-5-[(4-methylpiperazin-1-yl)methyl]-9H-xanthen-9-one dihydrochloride [HBK-5], 6-methoxy-2-[(4-methylpiperazin-1-yl) methyl]-9H-xanthen-9-one dihydrochloride, 2-[(4-benzylpiperazin-1-yl) methyl]-6-methoxy-9H-xanthen-9-one dihydrochloride, 2-{[4-(2-methoxyphenyl) piperazin-1-yl] methyl}-9H-xanthen-9-one hydrochloride), as well as the influence on cognitive and motor function of active compounds, using animal models. Materials and Methods: To determine the antidepressant-like activity, we used forced swim test (FST) and tail suspension test (TST) in mice. We evaluated anxiolytic-like properties in the four-plate test in mice. We studied the influence on cognitive and motor function in passive avoidance step-through and chimney tests, respectively. Results: The antidepressant-like activity (in both FST and TST) showed only HBK-5. Moreover, the compound was also active in the four-plate test, which suggests that it possessed anxiolytic-like properties. HBK-5 did not cause any cognitive and motor deficits in mice at antidepressant- and anxiolytic-like doses. Conclusions: HBK-5 may have potential in the treatment of depression or anxiety disorders, but this issue needs further studies. PMID:27298499

  4. In situ identification of polyphosphate- and polyhydroxyalkanoate-accumulating traits for microbial populations in a biological phosphorus removal process

    Liu, W.-T.; Nielsen, Alex Toftgaard; Wu, JH;

    2001-01-01

    Polyphosphate- and polyhydroxyalkanoate (PHA)- accumulating traits of predominant microorganisms in an efficient enhanced biological phosphorus removal (EBPR) process were investigated systematically using a suite of non-culture-dependent methods. Results of 16S rDNA clone library and fluorescence......, electron microscopy with energy-dispersive X-ray analysis was used to validate the staining specificity of 4,6-diamino-2-phenylindole (DAPI) for intracellular polyphosphate and revealed the composition of polyphosphate granules accumulated in predominant bacteria as mostly P, Ca and Na. As a result, DAPI...... and PHA staining procedures could be combined with FISH to identify directly the polyphosphate- and PHA-accumulating traits of different phylogenetic groups. Members of Accumulibacter phosphatis and the novel gamma-proteobacterial group were observed to accumulate both polyphosphate and PHA. In addition...

  5. Nerve growth factor promotes in vitro proliferation of neural stem cells from tree shrews

    Liu-lin Xiong; Zhi-wei Chen; Ting-hua Wang

    2016-01-01

    Neural stem cells promote neuronal regeneration and repair of brain tissue after injury, but have limited resources and proliferative ability in vivo. We hypothesized that nerve growth factor would promotein vitro proliferation of neural stem cells derived from the tree shrews, a primate-like mammal that has been proposed as an alternative to primates in biomedical translational research. We cultured neural stem cells from the hippocampus of tree shrews at embryonic day 38, and added nerve growth factor (100 μg/L) to the culture medium. Neural stem cells from the hippocampus of tree shrews cultured without nerve growth factor were used as controls. After 3 days, lfuorescence mi-croscopy after DAPI and nestin staining revealed that the number of neurospheres and DAPI/nestin-positive cells was markedly greater in the nerve growth factor-treated cells than in control cells. These ifndings demonstrate that nerve growth factor promotes the proliferation of neural stem cells derived from tree shrews.

  6. Occurrence of B chromosomes in Tetragonisca Latreille, 1811 (Hymenoptera, Apidae, Meliponini): A new contribution to the cytotaxonomy of the genus

    Adriane Barth; Anderson Fernandes; Silvia das Graças Pompolo; Marco Antônio Costa

    2011-01-01

    Tetragonisca angustula and Tetragonisca fiebrigi have recently been listed as valid species. This study aimed to cytogenetically investigate both species, emphasizing the new registry of B chromosomes in the tribe Meliponini. We analyzed colonies of T. angustula and T. fiebrigi collected at Tangará da Serra, Mato Grosso, Brazil, through conventional Giemsa staining, C-banding, and base-specific fluorochrome staining (CMA3/DAPI). T. angustula showed 2n = 34 chromosomes in females and n = 17 in...

  7. New occurrence of B chromosomes in Partamona helleri (Friese, 1900) (Hymenoptera, Meliponini)

    Cinthia Caroline Cardoso Martins; Olivia Maria Pereira Duarte; Ana Maria Waldschmidt; Rogério Marco de Oliveira Alves; Marco Antônio Costa

    2009-01-01

    Cytogenetic analyses of the stingless bee Partamona helleri collected in the state of Bahia, Northeast Brazil revealed the chromosome numbers n = 18 in the haploid males and 2n = 35 in the diploid females. All karyotypes displayed one large acrocentric B chromosome, which differs from the minute B chromosomes previously described in the populations from southeastern Brazil. Giemsa staining, C-banding and DAPI/CMA3 fluorochrome staining also revealed a remarkable interpopulational divergence r...

  8. Cytogenetic studies in four cultivated Amaranthus (Amaranthaceae) species

    Bonasora, Marisa Graciela; Poggio, Lidia; Greizerstein, Eduardo José

    2013-01-01

    Abstract In the present study, the chromosomes numbers were confirmed, 2n = 34 for Amaranthus cruentus Linnaeus, 1759, and 2n = 32 for Amaranthus hypochondriacus Linnaeus, 1753, Amaranthus mantegazzianus Passer, 1864, and Amaranthus caudatus Linnaeus, 1753. The distribution and variability of constitutive heterochromatin were detailed using DAPI-CMA3 banding technique. The position of the nucleolus organizer region (NOR) was observed using Ag-NOR banding (active loci) and fluorescent in situ ...

  9. Cytogenetic studies in four cultivated Amaranthus (Amaranthaceae) species

    Marisa Bonasora; Lidia Poggio; Eduardo Greizerstein

    2013-01-01

    In the present study, the chromosomes numbers were confirmed, 2n = 34 for Amaranthus cruentus Linnaeus, 1759, and 2n = 32 for A. hypochondriacus Linnaeus, 1753, A. mantegazzianus Passer, 1864, and A. caudatus Linnaeus, 1753. The distribution and variability of constitutive heterochromatin were detailed using DAPI-CMA3 banding technique. The position of the nucleolus organizer region (NOR) was observed using Ag-NOR banding (active loci) and fluorescent in situ hybridization (rDNA-FISH) in the ...

  10. Retinal incorporation and differentiation of mesenchymal stem cells intravitreally injected in the injured retina of rats Incorporação e diferenciação retiniana de células tronco mesenquimais intravítreas em ratos

    Paula Castanheira; Leonardo Torquetti; Marcio Bittar Nehemy; Alfredo Miranda Goes

    2008-01-01

    PURPOSE: To evaluate the pattern of retinal integration and differentiation of mesenchymal stem cells (MSCs) injected into the vitreous cavity of rat eyes with retinal injury. METHODS: Adult rat retinas were submitted to laser damage followed by transplantation of DAPI-labeled BM-MSCs grafts. To assess the integration and differentiation of BM-MSCs in laser-injured retina, host retinas were evaluated 2.4 and 8 weeks after injury/transplantation. RESULTS: Our results demonstrated that the graf...

  11. Aggregation and biofilm formation of bacteria isolated from domestic drinking water

    Ramalingam, B.; R. Sekar; Boxall, J. B.; Biggs, C.

    2013-01-01

    The objective of this study was to investigate the autoaggregation, coaggregation and biofilm formation of four bacteria namely Sphingobium, Xenophilus, Methylobacterium and Rhodococcus isolated from drinking water. Auto and coaggregation studies were performed by both qualitative (DAPI staining) and semi-quantitative (visual coaggregation) methods and biofilms produced by either pure or dual-cultures were quantified by crystal violet method. Results from the semi-quantitative ...

  12. Metagenomic analysis of soil microbial communities

    Đokić Lidija; Savić M.; Narančić Tanja; Vasiljević Branka

    2010-01-01

    Ramonda serbica and Ramonda nathaliae, rare resurrection plants growing in the Balkan Peninsula, produce a high amount of phenolic compounds as a response to stress. The composition and size of bacterial communities in two rhizosphere soil samples of these plants were analyzed using a metagenomic approach. Fluorescent in situ hybridization (FISH) experiments together with DAPI staining showed that the metabolically active bacteria represent only a small fraction, approximately 5%, of total so...

  13. The Distribution of Cytoplasm and Nuclei within the Extra-radical Mycelia in Glomus intraradices, a Species of Arbuscular Mycorrhizal Fungi

    Lee, Jaikoo

    2011-01-01

    Nuclear distribution within the extra-radical fungal structures and during spore production in the arbuscular mycorrhizae fungus Glomus intraradices was examined using an in vitro monoxenic culture system. A di-compartmental monoxenic culture system was modified using a nitrocellulose membrane and a coverglass slip for detailed observations. Nuclear distribution was observed using the fluorescent DNA binding probes SYBR Green I and DAPI. Both septate and non-septate mycelial regions were obse...

  14. Description of the karyotype of Rhagomys rufescens Thomas, 1886 (Rodentia, Sigmodontinae) from Southern Brazil Atlantic forest

    André Filipe Testoni; Sérgio Luiz Althoff; André Paulo Nascimento; Francisco Steiner-Souza; Ives José Sbalqueiro

    2010-01-01

    Rhagomys rufescens (Rodentia: Sigmodontinae) is an endemic species of the Atlantic forest from Southern and Southeastern Brazil. Some authors consider Rhagomys as part of the tribe Thomasomyini; but its phylogenetic relationships remain unclear. Chromosomal studies on eight specimens of Rhagomys rufescens revealed a diploid number of 2n = 36 and a number of autosome arms FN = 50. GTG, CBG and Ag-NOR banding and CMA3 /DAPI staining were performed on metaphase chromosomes. Eight biarmed and nin...

  15. シロイヌナズナに対する倍数化処理の減数分裂期染色体に及ぼす影響に関する研究

    岩元, 明敏; IWAMOTO, Akitoshi(Department of Biology, Tokyo Gakugei University); 杉山, 宗隆; Sugiyama, Munetaka; 安積, 良隆; Azumi, Yoshitaka

    2008-01-01

    We observed chromosome behavior during meiosis of Arabidopsis thaliana justafter polyploidization. Two strains of A. thaliana, Columbia (Col) and Landsberg erecta(Ler), were treated with colchicine to induce polyploid lines, and the treated generationswere analyzed. Flow cytometric analysis revealed that a tetraploid line was obtained fromthe treated Col and an octoploid line from the treated Ler. Meiotic chromosomes of pollenmother cells of these polyploid lines were visualized by DAPI stain...

  16. Breaking the pumping speed barrier in mass spectrometry: discontinuous atmospheric pressure interface.

    Gao, Liang; Cooks, R Graham; Ouyang, Zheng

    2008-06-01

    The performance of mass spectrometers with limited pumping capacity is shown to be improved through use of a discontinuous atmospheric pressure interface (DAPI). A proof-of-concept DAPI interface was designed and characterized using a miniature rectilinear ion trap mass spectrometer. The interface consists of a simple capillary directly connecting the atmospheric pressure ion source to the vacuum mass analyzer region; it has no ion optical elements and no differential pumping stages. Gases carrying ionized analytes were pulsed into the mass analyzer for short periods at high flow rates rather than being continuously introduced at lower flow rates; this procedure maximized ion transfer. The use of DAPI provides a simple solution to the problem of coupling an atmospheric pressure ionization source to a miniature instrument with limited pumping capacity. Data were recorded using various atmospheric pressure ionization sources, including electrospray ionization (ESI), nano-ESI, atmospheric pressure chemical ionization (APCI), and desorption electrospray ionization (DESI) sources. The interface was opened briefly for ion introduction during each scan. With the use of the 18 W pumping system of the Mini 10, limits of detection in the low part-per-billion levels were achieved and unit resolution mass spectra were recorded. PMID:18461971

  17. Karyotype relationships among Anastrepha bistrigata, A. striata and A. serpentina (Diptera, Tephritidae

    Denise Selivon

    2007-01-01

    Full Text Available The species of Anastrepha are arranged into 17 intrageneric groups. Recently, it was proposed that two species of the striata group, Anastrepha striata and A. bistrigata, might be realocated to serpentina group. Anastrepha bistrigata and A. serpentina have an X1X2Y/X1X1 X2X2 sex chromosome system while A. striata has a XY/XX system. It was previously proposed that the karyotype of A. bistrigata could be derived from that of A. striata by an Y:A fusion, and that the karyotype of A. serpentina would be derived from another, hypothetical karyotype. In the present report sequential staining with DAPI and chromomycin A3 (CMA3, followed by C-banding, revealed that the C-banded heterochromatic blocks of the sex chromosomes of A. bistrigata have different affinities to fluorochromes in comparison to the chromosomes of A. striata, from which they have hypothetically derived. The chromosomes of A. serpentina show substantial differences in their cytochemical properties compared to their A. bistrigata and A. striata counterparts. The FISH technique showed that the ribosomal gene sequences are located in DAPI- or DAPI/CMA3-positive heterochromatic blocks of the sex chromosomes, one site on the Y chromosome and one site on the X chromosome (X1 in A. bistrigata and A. serpentina. The data suggest that the karyotype of A. striata and A. bistrigata could be derived from a common ancestral karyotype, while the A. serpentina karyotype probably has a distinct origin.

  18. Molecular cytogenetic characterization of the dioecious Cannabis sativa with an XY chromosome sex determination system.

    Mikhail G Divashuk

    Full Text Available Hemp (Cannabis sativa L. was karyotyped using by DAPI/C-banding staining to provide chromosome measurements, and by fluorescence in situ hybridization with probes for 45 rDNA (pTa71, 5S rDNA (pCT4.2, a subtelomeric repeat (CS-1 and the Arabidopsis telomere probes. The karyotype has 18 autosomes plus a sex chromosome pair (XX in female and XY in male plants. The autosomes are difficult to distinguish morphologically, but three pairs could be distinguished using the probes. The Y chromosome is larger than the autosomes, and carries a fully heterochromatic DAPI positive arm and CS-1 repeats only on the less intensely DAPI-stained, euchromatic arm. The X is the largest chromosome of all, and carries CS-1 subtelomeric repeats on both arms. The meiotic configuration of the sex bivalent locates a pseudoautosomal region of the Y chromosome at the end of the euchromatic CS-1-carrying arm. Our molecular cytogenetic study of the C. sativa sex chromosomes is a starting point for helping to make C. sativa a promising model to study sex chromosome evolution.

  19. Comparative cytogenetic study on two species of the genus Entedon Dalman, 1820 (Hymenoptera, Eulophidae using DNA-binding fluorochromes and molecular and immunofluorescent markers

    Nadezhda Bolsheva

    2012-02-01

    Full Text Available Karyotypes of Entedon cionobius Thomson, 1878 and E. cioni Thomson, 1878 (Hymenoptera: Eulophidae were studied using DNA-binding ligands with different base specificity (propidium iodide, chromomycin A3, methyl green and DAPI; all these ligands, except for the last one, were used for the first time in parasitic wasps, C-banding, fluorescence in situ hybridization (FISH with a 45S rDNA probe and 5-methylcytosine immunodetection. Female karyotypes of both species contain five pairs of relatively large metacentric chromosomes and a pair of smaller acrocentric chromosomes (2n = 12. As in many other Hymenoptera, males of both Entedon Dalman, 1820 species have haploid chromosome sets (n = 6. Fluorochrome staining revealed chromosome-specific banding patterns that were similar between the different fluorochromes, except for the CMA3- and PI-positive and DAPI-negative band in the pericentromeric regions of the long arms of both acrocentric chromosomes. The obtained banding patterns were virtually identical in both species and allowed for the identification of each individual chromosome. C-banding revealed a pattern similar to DAPI staining, although centromeric and telomeric regions were stained more intensively using the former technique. FISH detected a single rDNA site in the same position on the acrocentric chromosomes as the bright CMA3-positive band. Immunodetection of 5-methylcytosine that was performed for the first time in the order Hymenoptera revealed 5-methylcytosine-rich sites in the telomeric, centromeric and certain interstitial regions of most of the chromosomes.

  20. Efficacy of turkey herpesvirus vaccine when administered simultaneously with fowl pox vaccine.

    Eidson, C S; Villegas, P; Kleven, S H

    1975-11-01

    The efficacy of the turkey herpesvirus (HVT) vaccine in protecting chickens challenged with virulent Marek's disease (MD) virus was unaffected by the presence of either the chick embryo fowl pox vaccine or fowl pox vaccine derived from cell culture. Conversely the HVT vaccine did not affect the efficacy of the fowl pox vaccine in chickens challenged with pathogenic fowl pox virus. A combination of spectinomycin dihydrochloride pentahydrate and lincomycin hydrochloride monohydrate as well as spectinomycin sulfate tetrahydrate were found to be compatible with the HVT and fowl pox vaccines as demonstrated by resistance after challenge with virulent MD virus or fowl pox virus. PMID:180502

  1. Intramuscular loading dose of quinine for falciparum malaria: pharmacokinetics and toxicity

    1986-01-01

    In a study of intramuscular injection of quinine eight adults with moderately severe falciparum malaria resistant to chloroquine were treated with quinine dihydrochloride, being given a loading dose of 20 mg salt (16.7 mg base)/kg followed by three or four eight hourly maintenance doses of 10 mg salt (8.3 mg base)/kg injected into the anterior thigh. All patients responded to treatment. Fever and parasite clearance times (mean (SD) 60 (23) h and 53 (22) h respectively) were comparable with th...

  2. A green process for the preparation of 11-{4-[2-(2-hydroxyethoxyethyl]-1-piperazinyl}dibenzo[b,f][1,4]thiazepine

    GANESH D. MAHALE

    2008-04-01

    Full Text Available A green process for the synthesis of 11-{4-[2-(2-hydroxyethoxyethyl]-1-piperazinyl}dibenzo[b,f][1,4]thiazepine by the reaction of 11-(1-piperazinyldibenzo[b,f][1,4]thiazepine or its dihydrochloride salt with 2-(2-chloroethoxyethanol in the presence of an inorganic base and water is reported (conversion 99.9 % in a short time and without any impurities. The metal halides and phase transfer catalyst increase the rate of reaction, especially in water as the solvent.

  3. In vitro efficacy of octenidine and polihexanide against biofilms composed of Pseudomonas aeruginosa

    Siebert, Jörg

    2007-12-01

    Full Text Available The removal and inactivation of biofilms through wound cleansing solutions with and without antimicrobial supplements (octenidine dihydrochloride, polihexanide have been investigated in a laboratory model with Pseudomonas aeruginosa. Plastic slides of polycarbonate grown over with P. aeruginosa for 1 week were incubated with the cleansing solutions for 60 min. Removal and inactivation of the biofilm were determined by staining with crystal violet and by plating, respectively. No inhibition occurred by supplementing the cleansing solutions with octenidine or polyhexanide. By using octenidine and polihexanide a pronounced decrease in colony numbers of the biofilm was achieved compared to pure salt solutions (NaCl, Ringer.

  4. Unilateral sudden hearing loss: a rare symptom of Moyamoya disease.

    Gül, Fatih; Berçin, Sami; Müderris, Togay; Yalçıner, Gökhan; Ünal, Özkan; Kırış, Muzaffer

    2016-01-01

    A 38-year-old female patient experienced a sudden onset of unilateral sensorineural hearing loss due to Moyamoya disease. A detailed summary of audiological and neurological findings indicated that the sudden hearing loss might be due to Moyamoya disease resulting in occlusion of posterior and middle cerebral arteries. Intravenous prednisolone and trimetazidine dihydrochloride may improve hearing thresholds and speech understanding. To our knowledge, this is the first article in the literature reporting a case of sudden hearing loss as the first manifestation of Moyamoya disease in a young adult. PMID:26890714

  5. Drug: D09326 [KEGG MEDICUS

    Full Text Available D09326 Drug Carmegliptin dihydrochloride (USAN) C20H28FN3O3. 2HCl 449.1648 450.3749 D09326.gif Treatme... dipeptidyl-peptidase 4 (DPP4) [HSA:1803] [KO:K01278] Carmegliptin D09326 Carme...K01278] hsa04974(1803) Protein digestion and absorption Target-based classification of drugs [BR:br08310] Enzymes Hydrolases...nt of type 2 diabetes [DS:H00409] dipeptidyl-peptidase 4 (DPP4) inhibitor [HSA:1803] [KO:

  6. Subchronic oral toxicity and metabolite profiling of the p53 stabilizing agent, CP-31398, in rats and dogs

    Johnson, William D.; Muzzio, Miguel; Detrisac, Carol J.; Kapetanovic, Izet M.; Kopelovich, Levy; McCormick, David L.

    2011-01-01

    CP-31398 (N′-[2-[2-(4-methoxyphenyl)ethenyl]-4-quinazolinyl]-N,N-dimethyl-1,3-propanediamine dihydrochloride) is a styrylquinazoline that stabilizes the DNA binding conformation of p53, thereby maintaining the activity of p53 as a transcription factor and tumor suppressor. In consideration of the potential use of p53 stabilizers for cancer prevention and therapy, 28-day studies (with recovery) were performed to characterize the toxicity of CP-31398 in rats and dogs. In the rat study, groups o...

  7. Different effects of short- and long-chained fructans on large intestinal physiology and carcinogen-induced aberrant crypt foci in rats

    Poulsen, Morten; Molck, Anne-Marie; Jacobsen, Bodil Lund

    2002-01-01

    the development of ACF: microflora, short-chain fatty acids, pH, and cell proliferation. A 3-wk pretreatment period with both fructans was included. Feeding the long-chained fructan (5% or 15%) significantly inhibited the numbers of small and total ACF after 5 and 10 wk. The short-chained fructan (15......-type fructan on 1,2-dimethylhydrazine dihydrochloride-induced aberrant crypt foci (ACF) in the rat colon. In addition, the present study investigated the influence of chain length, dietary level (5% or 15%), and duration of feeding (5 or 10 wk) on the following intestinal parameters supposed to be involved in...

  8. The sigma-1 receptor enhances brain plasticity and functional recovery after experimental stroke

    Ruscher, Karsten; Shamloo, Mehrdad; Rickhag, Karl Mattias;

    2011-01-01

    )piperazine dihydrochloride, an agonist of the sigma-1 receptor, starting two days after injury, enhanced the recovery of lost sensorimotor function without decreasing infarct size. The sigma-1 receptor was found in the galactocerebroside enriched membrane microdomains of reactive astrocytes and in neurons. Sigma-1 receptor...... induced sigma-1 receptor expression and actin dependent membrane raft formation, the latter blocked by sigma-1 receptor small interfering RNA silencing and pharmacological inhibition. We conclude that sigma-1 receptor activation stimulates recovery after stroke by enhancing cellular transport...

  9. Inhibitors of cellular kinases with broad-spectrum antiviral activity for hemorrhagic fever viruses.

    Mohr, Emma L; McMullan, Laura K; Lo, Michael K; Spengler, Jessica R; Bergeron, Éric; Albariño, César G; Shrivastava-Ranjan, Punya; Chiang, Cheng-Feng; Nichol, Stuart T; Spiropoulou, Christina F; Flint, Mike

    2015-08-01

    Host cell kinases are important for the replication of a number of hemorrhagic fever viruses. We tested a panel of kinase inhibitors for their ability to block the replication of multiple hemorrhagic fever viruses. OSU-03012 inhibited the replication of Lassa, Ebola, Marburg and Nipah viruses, whereas BIBX 1382 dihydrochloride inhibited Lassa, Ebola and Marburg viruses. BIBX 1382 blocked both Lassa and Ebola virus glycoprotein-dependent cell entry. These compounds may be used as tools to understand conserved virus-host interactions, and implicate host cell kinases that may be targets for broad spectrum therapeutic intervention. PMID:25986249

  10. Prevention of myocardial enzyme release by ranolazine in a primate model of ischaemia with reperfusion.

    Allely, M. C.; Alps, B J

    1990-01-01

    In control anaesthetized baboons subjected to 30 min occlusion of the left anterior descending coronary artery, followed by 5.5 h reperfusion, total plasma levels for creatine kinase (CK) and lactate dehydrogenase (LDH) were markedly elevated in a time-related manner. In a second group of baboons pretreated 10 min prior to ischaemia with ranolazine [(+/-)-N-(2,6-dimethyl-phenyl)-4[2-hydroxy-3-(2-methoxyphenoxy)propyl]-1 - piperazine acetamide dihydrochloride; RS-43285-193] at 500 micrograms k...

  11. Polyurethane-based scaffolds for myocardial tissue engineering

    Chiono, Valeria; Mozetic, Pamela; Boffito, Monica; Sartori, Susanna; Gioffredi, Emilia; Silvestri, Antonella; Rainer, Alberto; Giannitelli, Sara Maria; Trombetta, Marcella; Nurzynska, Daria; Di Meglio, Franca; Castaldo, Clotilde; Miraglia, Rita; Montagnani, Stefania; Ciardelli, Gianluca

    2014-01-01

    Bi-layered scaffolds with a 0°/90° lay-down pattern were prepared by melt-extrusion additive manufacturing (AM) using a poly(ester urethane) (PU) synthesized from poly(ε-caprolactone) diol, 1,4-butandiisocyanate and l-lysine ethyl ester dihydrochloride chain extender. Rheological analysis and differential scanning calorimetry of the starting material showed that compression moulded PU films were in the molten state at a higher temperature than 155°C. The AM processing temperature was set at 1...

  12. Visualization of vasodynamics using THz imaging with applications to allergy testing (Conference Presentation)

    Sung, Shijun; Bajwa, Neha; Grundfest, Warren; Grundfest, Zachary

    2016-03-01

    This paper explores vasodynamics in response to histamine injection using reflective THz imaging. Histamine is a major contributor to allergic disease. Elevations in tissue histamine levels have been observed during anaphylaxis and experimental allergic responses of the skin, nose, and airways. In the skin specifically, vasodilation, vascular permeability, and pruritus is controlled by the release and resorption of histamine. These properties are leveraged in skin prick testing for allergies where histamine dihydrochloride is injected as a positive control to confirm allergen susceptibility prior to the administration of candidate allergens. Subjective parameters such as skin coloration, irritation, and bulging as a consequence of histamine injection and histamine release are well characterized. However limited quantitative metrics on the body's edematous response are available due to the lack of imaging diagnostics that can map surface tissue water content (TWC). THz imaging was used to explore the utility of reflective THz imaging to quantify edematous responses to histamine. Rat models were injected with varying concentrations of histamine dihydrochloride and the resultant edematous response arising from perturbed vasodymanics was mapped. Significant build up and dissipation of surface tissue water content was observed and THz frequency contrast was seen to correlate with visual appearance in some cases and in others reveal tissue water content variations not discernable with the naked eye. The results suggest that THz imaging may be a valuable tool in quantifying the degree of allergic responses and assist in detecting hypersensitivity.

  13. In vitro dissolution and in vivo bioequivalence evaluation of two brands of trimetazidine tablets.

    Helmy, Sally A; Mansour, Noha O

    2014-03-01

    Trimetazidine is an effective anti-anginal agent and anti-ischaemic effect. The objective of this study was to assess the in vitro dissolution and to evaluate the bioavailability of two brands of trimetazidine dihydrochloride tablets. Prior to the in vivo PKs study, an in vitro comparative dissolution test was performed for 2 oral brands of trimetazidine dihydrochloride tablets (20 mg). In vivo PKs study was evaluated in 24 healthy male volunteers after a single dose oral administration in an open, randomized, two-way crossover study with a washout period of 1 week. After an overnight fast, human volunteers were randomly allocated to receive a single dose of either test or reference product. Blood samples were collected over a 24-hour period following drug administration and plasma was analyzed for trimetazidine concentrations using a validated high-performance liquid chromatography assay method. The PK parameters Cmax , AUC0-t , AUC0-∞ , tmax , and t1/2 were determined from plasma concentration-time profiles. The 90% confidence intervals for the ratio of log transformed values of Cmax , AUC0-t , and AUCt-∞ of the test product over those of reference were within the acceptable range (0.8-1.25) for bioequivalence. As a result, the 2 trimetazidine formulations are considered bioequivalent and thus could be prescribed interchangeably in the medical practice based on its PK effect and biopharmaceutical performance. PMID:27128458

  14. Angiogenesis is repressed by ethanol exposure during chick embryonic development.

    Wang, Guang; Zhong, Shan; Zhang, Shi-Yao; Ma, Zheng-Lai; Chen, Jian-Long; Lu, Wen-Hui; Cheng, Xin; Chuai, Manli; Lee, Kenneth Ka Ho; Lu, Da-Xiang; Yang, Xuesong

    2016-05-01

    It is now known that excess alcohol consumption during pregnancy can cause fetal alcohol syndrome to develop. However, it is not known whether excess ethanol exposure could directly affect angiogenesis in the embryo or angiogenesis being indirectly affected because of ethanol-induced fetal alcohol syndrome. Using the chick yolk sac membrane (YSM) model, we demonstrated that ethanol exposure dramatically inhibited angiogenesis in the YSM of 9-day-old chick embryos, in a dose-dependent manner. Likewise, the anti-angiogenesis effect of ethanol could be seen in the developing vessel plexus (at the same extra-embryonic regions) during earlier stages of embryo development. The anti-angiogenic effect of ethanol was found associated with excess reactive oxygen species (ROS) production; as glutathione peroxidase activity increased while superoxide dismutase 1 and 2 activities decreased in the YSMs. We further validated this observation by exposing chick embryos to 2,2'-azobis-amidinopropane dihydrochloride (a ROS inducer) and obtained a similar anti-angiogenesis effect as ethanol treatment. Semiquantitative reverse transcription-polymerase chain reaction analysis of the experimental YSMs revealed that expression of angiogenesis-related genes, vascular endothelial growth factor and its receptor, fibroblast growth factor 2 and hypoxia-inducible factor, were all repressed following ethanol and 2,2'-azobis-amidinopropane dihydrochloride treatment. In summary, our results suggest that excess ethanol exposure inhibits embryonic angiogenesis through promoting superfluous ROS production during embryo development. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26177723

  15. A sensitive fluorimetric method for determination of trace amounts of nitrite based on luminescence energy transfer

    An efficient luminescence energy transfer (LET) system based on terbium(III)-sodium hexametaphosphate (Tb/SHMP) chelates as donor and 4-((4-(2-aminoethylamino)naphthalen-1-yl)diazenyl)benzenesulfonic acid dihydrochloride (ANDBS) as acceptor was developed for sensitive determination of trace nitrite. Stable and strong fluorescence Tb/SHMP chelates were prepared in aqueous solution. Based on Griess Reaction, ANDBS was generated by the quantitative reaction of nitrite, sulfanilamide and N-(1-naphtyl)-ethylenediamine dihydrochloride (N1NED). The degree of the overlap was effective for LET between the emission spectrum of Tb/SHMP chelates and the absorption spectrum of ANDBS. Based on the luminescence intensity quenching of Tb/SHMP chelates in proportion to the trace amounts of nitrite, a new assay for the selective and sensitive determination of nitrite was developed. Under the optimum conditions, the linear calibration graph was obtained with a linear range of 0.00040-0.20 μg mL-1 (R=0.99657). The detection limit of NO2- was 0.00010 μg mL-1 (R=0.99657). The method was applied successfully to the determination of nitrite for synthetic samples.

  16. Indications and agent selection for the antiseptic therapy of secondarily healing wounds

    Assadian, Ojan

    Full Text Available For chronic wounds to become primarily healing wounds, not only the appropriate surgical procedure - especially wound debridement - but also the treatment of the causative basic disease must be taken into consideration. In both the prophylaxis of wound infections and the treatment of existing wound infections, wound antiseptics play a crucial role. As long as a clinically manifest infection is confined to the wound and has not spread hematogenically, the indication for application of wound antiseptics is given. Should systemic involvement exist, antimicrobial chemotherapy is additionally required. Antiseptics must only be used following careful determination of indication. Impaired wound healing can otherwise result. For chronically poorly healing wounds and burns, polihexanide is the agent of first choice. However, based on new in-vitro findings, octenidine dihydrochloride is superior to polihexanide in terms of therapeutic spectrum. These findings enable a broader application of octenidine dihydrochloride to include use on chronic wounds at lower concentrations than usual. The following agents must be viewed critically in terms of their use as wound antiseptics: chlorhexidine, 8-chinolinol, nitrofural, phenol derivates, quats, silver nitrate, silver sulphadiazine, tosylchloramide sodium, triclosan, and hydrogen peroxide. As wound antiseptics, dyes and organic mercury compounds are obsolete.

  17. A sensitive fluorimetric method for determination of trace amounts of nitrite based on luminescence energy transfer

    Wang Lun, E-mail: wanglun@mail.ahnu.edu.c [Anhui Key Laboratory of Chemo-Biosensing, College of Chemistry and Materials Science, Anhui Normal University, Wuhu 241000 (China); Chen Jingguo; Chen Hongqi; Zhou Cailing; Ling Bo; Fu Jie [Anhui Key Laboratory of Chemo-Biosensing, College of Chemistry and Materials Science, Anhui Normal University, Wuhu 241000 (China)

    2011-01-15

    An efficient luminescence energy transfer (LET) system based on terbium(III)-sodium hexametaphosphate (Tb/SHMP) chelates as donor and 4-((4-(2-aminoethylamino)naphthalen-1-yl)diazenyl)benzenesulfonic acid dihydrochloride (ANDBS) as acceptor was developed for sensitive determination of trace nitrite. Stable and strong fluorescence Tb/SHMP chelates were prepared in aqueous solution. Based on Griess Reaction, ANDBS was generated by the quantitative reaction of nitrite, sulfanilamide and N-(1-naphtyl)-ethylenediamine dihydrochloride (N1NED). The degree of the overlap was effective for LET between the emission spectrum of Tb/SHMP chelates and the absorption spectrum of ANDBS. Based on the luminescence intensity quenching of Tb/SHMP chelates in proportion to the trace amounts of nitrite, a new assay for the selective and sensitive determination of nitrite was developed. Under the optimum conditions, the linear calibration graph was obtained with a linear range of 0.00040-0.20 {mu}g mL{sup -1} (R=0.99657). The detection limit of NO{sub 2}{sup -} was 0.00010 {mu}g mL{sup -1} (R=0.99657). The method was applied successfully to the determination of nitrite for synthetic samples.

  18. The diversity of karyotypes and genomes within section Syllinum of the Genus Linum (Linaceae revealed by molecular cytogenetic markers and RAPD analysis.

    Nadezhda L Bolsheva

    Full Text Available The wide variation in chromosome number found in species of the genus Linum (2n = 16, 18, 20, 26, 28, 30, 32, 36, 42, 72, 84 indicates that chromosomal mutations have played an important role in the speciation of this taxon. To contribute to a better understanding of the genetic diversity and species relationships in this genus, comparative studies of karyotypes and genomes of species within section Syllinum Griseb. (2n = 26, 28 were carried out. Elongated with 9-aminoacridine chromosomes of 10 species of section Syllinum were investigated by C- and DAPI/С-banding, CMA and Ag-NOR-staining, FISH with probes of rDNA and of telomere repeats. RAPD analysis was also performed. All the chromosome pairs in karyotypes of the studied species were identified. Chromosome DAPI/C-banding patterns of 28-chromosomal species were highly similar. Two of the species differed from the others in chromosomal location of rDNA sites. B chromosomes were revealed in all the 28-chromosomal species. Chromosomes of Linum nodiflorum L. (2n = 26 and the 28-chromosomal species were similar in DAPI/C-banding pattern and localization of several rDNA sites, but they differed in chromosomal size and number. The karyotype of L. nodiflorum was characterized by an intercalary site of telomere repeat, one additional 26S rDNA site and also by the absence of B chromosomes. Structural similarities between different chromosome pairs in karyotypes of the studied species were found indicating their tetraploid origin. RAPD analysis did not distinguish the species except L. nodiflorum. The species of section Syllinum probably originated from a common tetraploid ancestor. The 28-chromosomal species were closely related, but L. nodiflorum diverged significantly from the rest of the species probably due to chromosomal rearrangements occurring during evolution.

  19. Comparison of genomes of eight species of sections Linum and Adenolinum from the genus Linum based on chromosome banding, molecular markers and RAPD analysis.

    Muravenko, Olga V; Yurkevich, Olga Yu; Bolsheva, Nadezhda L; Samatadze, Tatiana E; Nosova, Inna V; Zelenina, Daria A; Volkov, Alexander A; Popov, Konstantin V; Zelenin, Alexander V

    2009-03-01

    Karyotypes of species sects. Linum and Adenolinum have been studied using C/DAPI-banding, Ag-NOR staining, FISH with 5S and 26S rDNA and RAPD analysis. C/DAPI-banding patterns enabled identification of all homologous chromosome pairs in the studied karyotypes. The revealed high similarity between species L. grandiflorum (2n = 16) and L. decumbens by chromosome and molecular markers proved their close genome relationship and identified the chromosome number in L. decumbens as 2n = 16. The similarity found for C/DAPI-banding patterns between species with the same chromosome numbers corresponds with the results obtained by RAPD-analysis, showing clusterization of 16-, 18- and 30-chromosome species into three separate groups. 5S rDNA and 26S rDNA were co-localized in NOR-chromosome 1 in the genomes of all species investigated. In 30-chromosome species, there were three separate 5S rDNA sites in chromosomes 3, 8 and 13. In 16-chromosome species, a separate 5S rDNA site was also located in chromosome 3, whereas in 18-chromosome species it was found in the long arm of NOR-chromosome 1. Thus, the difference in localization of rDNA sites in species with 2n = 16, 2n = 30 and 2n = 18 confirms taxonomists opinion, who attributed these species to different sects. Linum and Adenolinum, respectively. The obtained results suggest that species with 2n = 16, 2n = 18 and 2n = 30 originated from a 16-chromosome ancestor. PMID:18500654

  20. The diversity of karyotypes and genomes within section Syllinum of the Genus Linum (Linaceae) revealed by molecular cytogenetic markers and RAPD analysis.

    Bolsheva, Nadezhda L; Zelenin, Alexander V; Nosova, Inna V; Amosova, Alexandra V; Samatadze, Tatiana E; Yurkevich, Olga Yu; Melnikova, Nataliya V; Zelenina, Daria A; Volkov, Alexander A; Muravenko, Olga V

    2015-01-01

    The wide variation in chromosome number found in species of the genus Linum (2n = 16, 18, 20, 26, 28, 30, 32, 36, 42, 72, 84) indicates that chromosomal mutations have played an important role in the speciation of this taxon. To contribute to a better understanding of the genetic diversity and species relationships in this genus, comparative studies of karyotypes and genomes of species within section Syllinum Griseb. (2n = 26, 28) were carried out. Elongated with 9-aminoacridine chromosomes of 10 species of section Syllinum were investigated by C- and DAPI/С-banding, CMA and Ag-NOR-staining, FISH with probes of rDNA and of telomere repeats. RAPD analysis was also performed. All the chromosome pairs in karyotypes of the studied species were identified. Chromosome DAPI/C-banding patterns of 28-chromosomal species were highly similar. Two of the species differed from the others in chromosomal location of rDNA sites. B chromosomes were revealed in all the 28-chromosomal species. Chromosomes of Linum nodiflorum L. (2n = 26) and the 28-chromosomal species were similar in DAPI/C-banding pattern and localization of several rDNA sites, but they differed in chromosomal size and number. The karyotype of L. nodiflorum was characterized by an intercalary site of telomere repeat, one additional 26S rDNA site and also by the absence of B chromosomes. Structural similarities between different chromosome pairs in karyotypes of the studied species were found indicating their tetraploid origin. RAPD analysis did not distinguish the species except L. nodiflorum. The species of section Syllinum probably originated from a common tetraploid ancestor. The 28-chromosomal species were closely related, but L. nodiflorum diverged significantly from the rest of the species probably due to chromosomal rearrangements occurring during evolution. PMID:25835524

  1. Focus on Chirality of HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitors

    Valeria Famiglini

    2016-02-01

    Full Text Available Chiral HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs are of great interest since one enantiomer is often more potent than the corresponding counterpart against the HIV-1 wild type (WT and the HIV-1 drug resistant mutant strains. This review exemplifies the various studies made to investigate the effect of chirality on the antiretroviral activity of top HIV-1 NNRTI compounds, such as nevirapine (NVP, efavirenz (EFV, alkynyl- and alkenylquinazolinone DuPont compounds (DPC, diarylpyrimidine (DAPY, dihydroalkyloxybenzyloxopyrimidine (DABO, phenethylthiazolylthiourea (PETT, indolylarylsulfone (IAS, arylphosphoindole (API and trifluoromethylated indole (TFMI The chiral separation, the enantiosynthesis, along with the biological properties of these HIV-1 NNRTIs, are discussed.

  2. The Molecular Cytogenetic Characterization of Pistachio (Pistacia vera L.) Suggests the Arrest of Recombination in the Largest Heteropycnotic Pair HC1

    Sola-Campoy, Pedro J.; Francisca Robles; Trude Schwarzacher; Carmelo Ruiz Rejón; Roberto de la Herrán; Rafael Navajas-Pérez

    2015-01-01

    This paper represents the first molecular cytogenetic characterization of the strictly dioecious pistachio tree (Pistacia vera L.). The karyotype was characterized by fluorescent in situ hybridization (FISH) with probes for 5S and 45S rDNAs, and the pistachio specific satellite DNAs PIVE-40, and PIVE-180, together with DAPI-staining. PIVE-180 has a monomeric unit of 176-178 bp and high sequence homology between family members; PIVE-40 has a 43 bp consensus monomeric unit, and is most likely a...

  3. Pronounced in vitro and in vivo antiretroviral activity of 5-substituted 2,4-diamino-6-[2-(phosphonomethoxy)ethoxy] pyrimidines

    Balzarini, J.; Schols, D.; Van Laethem, K.; De Clercq, E.; Hocková, Dana; Masojídková, Milena; Holý, Antonín

    2007-01-01

    Roč. 59, č. 1 (2007), s. 80-86. ISSN 0305-7453 R&D Projects: GA MŠk 1M0508; GA AV ČR IBS4055109; GA AV ČR 1QS400550501 Grant ostatní: ISEP/FORTIS(XE) GOA/2005/19 Institutional research plan: CEZ:AV0Z40550506 Keywords : acyclic nucleoside phosphonates * antiretroviral drugs * HIV * MSV * PMEO-DAPy Subject RIV: CC - Organic Chemistry Impact factor: 4.038, year: 2007

  4. Primo Vascular System Accompanying a Blood Vessel from Tumor Tissue and a Method to Distinguish It from the Blood or the Lymph System

    Jaekwan Lim; Sungwoo Lee; Zhendong Su; Hong Bae Kim; Jung Sun Yoo; Kwang-Sup Soh; Sungchul Kim; Yeon Hee Ryu

    2013-01-01

    A primo vessel was observed in the abdominal cavity in the lung cancer mouse model, and its function as an extra metastatic path was observed. In this work, we found a primo vessel accompanying a blood vessel emanating from a tumor in the skin. We also presented simple and efficient criteria to distinguish a primo vessel from a blood or a lymph vessel and from a nerve. The criteria for using DAPI and Phalloidin will be useful in clinical situations to find and identify the primo vessels among...

  5. Cardiac Fas-Dependent and Mitochondria-Dependent Apoptosis after Chronic Cocaine Abuse

    Cher-Ming Liou; Shiow-Chwen Tsai; Chia-Hua Kuo; Hua Ting; Shin-Da Lee

    2014-01-01

    To evaluate whether chronic cocaine abuse will increase cardiac Fas-dependent and mitochondria-dependent apoptotic pathways, thirty-two male Wistar rats at 3–4 months of age were randomly divided into a vehicle-treated group (phosphate-buffered saline, PBS, 0.5 mL, SQ per day) and a cocaine-treated group (Cocaine, 10 mg/kg, SQ per day). After 3 months of treatment, the excised left ventricles were measured by H&E staining, Western blotting, DAPI staining and TUNEL assays. More cardiac TU...

  6. [Genetic polymorphism of flax Linum usitatissimum based on use of molecular cytogenetic markers].

    Rachinskaia, O A; Lemesh, V A; Muravenko, O V; Iurkevich, O Iu; Guzenko, E V; Bol'sheva, N L; Bogdanova, M V; Samatadze, T E; Popov, K V; Malyshev, S V; Shostak, N G; Heller, K; Khotyleva, L V; Zelenin, A V

    2011-01-01

    Using a set of approaches based on the use of molecular cytogenetic markers (DAPI/C-banding, estimation of the total area of DAPI-positive regions in prophase nuclei, FISH with 26S and 5S rDNA probes) and the microsatellite (SSR-PCR) assay, we studied genomic polymorphism in 15 flax (Linum usitatissimum L.) varieties from different geographic regions belonging to three directions of selection (oil, fiber, and intermediate flaxes) and in the k-37 x Viking hybrid. All individual chromosomes have been identified in the karyotypes of these varieties on the basis of the patterns of differential DAPI/C-banding and the distribution of 26S and 5S rDNA, and idiograms of the chromosomes have been generated. Unlike the oil flax varieties, the chromosomes in the karyotypes of the fiber flax varieties have, as a rule, pericentromeric and telomeric DAPI-positive bands of smaller size, but contain larger intercalary regions. Two chromosomal rearrangements (chromosome 3 inversions) were discovered in the variety Luna and in the k-37 x Viking hybrid. In both these forms, no colocalization of 26S rDNA and 5S rDNA on the satellite chromosome was detected. The SSR assay with the use of 20 polymorphic pairs of primers revealed 22 polymorphic loci. Based on the SSR data, we analyzed genetic similarity of the flax forms studied and constructed a genetic similarity dendrogram. The genotypes studied here form three clusters. The oil varieties comprise an independent cluster. The genetically related fiber flax varieties Vita and Luna, as well as the landrace Lipinska XIII belonging to the intermediate type, proved to be closer to the oil varieties than the remaining fiber flax varieties. The results of the molecular chromosomal analysis in the fiber and oil flaxes confirm their very close genetic similarity. In spite of this, the combined use of the chromosomal and molecular markers has opened up unique possibilities for describing the genotypes of flax varieties and creating their genetic

  7. Characterization of the bacterial community associated with body wall lesions of Tripneustes gratilla (Echinoidea) using culture-independent methods.

    Becker, Pierre T; Gillan, David C; Eeckhaut, Igor

    2009-02-01

    The bacterial community associated with skin lesions of the sea urchin Tripneustes gratilla was investigated using 16S ribosomal RNA gene cloning and fluorescent in situ hybridization (FISH). All clones were classified in the Alphaproteobacteria, Gammaproteobacteria and Cytophaga-Flexibacter-Bacteroides (CFB) bacteria. Most of the Alphaproteobacteria were related to the Roseobacter lineage and to bacteria implicated in marine diseases. The majority of the Gammaproteobacteria were identified as Vibrio while CFB represented only 9% of the total clones. FISH analyses showed that Alphaproteobacteria, CFB bacteria and Gammaproteobacteria accounted respectively for 43%, 38% and 19% of the DAPI counts. The importance of the methods used is emphasized. PMID:19041326

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

    Pellicer, J.; Garcia, Sònia; Garnatje, Teresa; Hidalgo, O.; Siljak-Yakovlev, S.; Vallès, Joan

    2008-01-01

    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 y con 4',6-diamidino-2-fenilindol (DAPI), así como hibridación in situ fluorescente (FISH) del ADN ribosómico 5S y 18S-5.8S-26S. Se han calculado los datos morfomé...

  9. Primo Vascular System Accompanying a Blood Vessel from Tumor Tissue and a Method to Distinguish It from the Blood or the Lymph System

    Jaekwan Lim

    2013-01-01

    Full Text Available A primo vessel was observed in the abdominal cavity in the lung cancer mouse model, and its function as an extra metastatic path was observed. In this work, we found a primo vessel accompanying a blood vessel emanating from a tumor in the skin. We also presented simple and efficient criteria to distinguish a primo vessel from a blood or a lymph vessel and from a nerve. The criteria for using DAPI and Phalloidin will be useful in clinical situations to find and identify the primo vessels among the blood vessels, lymph vessels, or nerves in the tissue surrounding a tumor such as a melanoma or breast cancer.

  10. Characterizing the chromosomes of the platypus (Ornithorhynchus anatinus).

    McMillan, Daniel; Miethke, Pat; Alsop, Amber E; Rens, Willem; O'Brien, Patricia; Trifonov, Vladimir; Veyrunes, Frederic; Schatzkamer, Kyriena; Kremitzki, Colin L; Graves, Tina; Warren, Wesley; Grützner, Frank; Ferguson-Smith, Malcolm A; Graves, Jennifer A Marshall

    2007-01-01

    Like the unique platypus itself, the platypus genome is extraordinary because of its complex sex chromosome system, and is controversial because of difficulties in identification of small autosomes and sex chromosomes. A 6-fold shotgun sequence of the platypus genome is now available and is being assembled with the help of physical mapping. It is therefore essential to characterize the chromosomes and resolve the ambiguities and inconsistencies in identifying autosomes and sex chromosomes. We have used chromosome paints and DAPI banding to identify and classify pairs of autosomes and sex chromosomes. We have established an agreed nomenclature and identified anchor BAC clones for each chromosome that will ensure unambiguous gene localizations. PMID:18185982

  11. Melittin induces apoptotic features in Candida albicans

    Melittin is a well-known antimicrobial peptide with membrane-active mechanisms. In this study, it was found that Melittin exerted its antifungal effect via apoptosis. Candida albicans exposed to Melittin showed the increased reactive oxygen species (ROS) production, measured by DHR-123 staining. Fluorescence microscopy staining with FITC-annexin V, TUNEL and DAPI further confirmed diagnostic markers of yeast apoptosis including phosphatidylserine externalization, and DNA and nuclear fragmentation. The current study suggests that Melittin possesses an antifungal effect with another mechanism promoting apoptosis.

  12. Melittin induces apoptotic features in Candida albicans

    Park, Cana [School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, 1370 Sankyuk-dong, Puk-ku, Daegu 702-701 (Korea, Republic of); Lee, Dong Gun, E-mail: dglee222@knu.ac.kr [School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, 1370 Sankyuk-dong, Puk-ku, Daegu 702-701 (Korea, Republic of)

    2010-03-26

    Melittin is a well-known antimicrobial peptide with membrane-active mechanisms. In this study, it was found that Melittin exerted its antifungal effect via apoptosis. Candida albicans exposed to Melittin showed the increased reactive oxygen species (ROS) production, measured by DHR-123 staining. Fluorescence microscopy staining with FITC-annexin V, TUNEL and DAPI further confirmed diagnostic markers of yeast apoptosis including phosphatidylserine externalization, and DNA and nuclear fragmentation. The current study suggests that Melittin possesses an antifungal effect with another mechanism promoting apoptosis.

  13. Cytogenetic analysis and chromosomal characteristics of the polymorphic 18S rDNA in the fish Prochilodus lineatus (Characiformes, Prochilodontidae)

    Marcelo Ricardo Vicari; Mara Cristina de Almeida; Luiz Antonio Carlos Bertollo; Orlando Moreira-Filho; Roberto Ferreira Artoni

    2006-01-01

    We used differential staining techniques (BSG, GTG, AgNO3, DAPI and CMA3 banding) and fluorescent in situ hybridization (FISH) with 5S and 18S probes to investigated the karyotypic and cytogenetic chracteristics of Prochilodus lineatus specimens from a population in Vila Velha state park (Parque Estadual de Vila Velha, Ponta Grossa, Paraná state, southern Brazil). The specimens studied showed the same karyotype as that found in other P. lineatus populations, i.e. 2n = 54 biarmed chromosomes (...

  14. Chromosome landmarks and autosome-sex chromosome translocations in Rumex hastatulus, a plant with XX/XY1Y2 sex chromosome system.

    Grabowska-Joachimiak, Aleksandra; Kula, Adam; Książczyk, Tomasz; Chojnicka, Joanna; Sliwinska, Elwira; Joachimiak, Andrzej J

    2015-06-01

    Rumex hastatulus is the North American endemic dioecious plant with heteromorphic sex chromosomes. It is differentiated into two chromosomal races: Texas (T) race characterised by a simple XX/XY sex chromosome system and North Carolina (NC) race with a polymorphic XX/XY1Y2 sex chromosome system. The gross karyotype morphology in NC race resembles the derived type, but chromosomal changes that occurred during its evolution are poorly understood. Our C-banding/DAPI and fluorescence in situ hybridization (FISH) experiments demonstrated that Y chromosomes of both races are enriched in DAPI-positive sequences and that the emergence of polymorphic sex chromosome system was accompanied by the break of ancestral Y chromosome and switch in the localization of 5S rDNA, from autosomes to sex chromosomes (X and Y2). Two contrasting domains were detected within North Carolina Y chromosomes: the older, highly heterochromatinised, inherited from the original Y chromosome and the younger, euchromatic, representing translocated autosomal material. The flow-cytometric DNA estimation showed ∼3.5 % genome downsizing in the North Carolina race. Our results are in contradiction to earlier reports on the lack of heterochromatin within Y chromosomes of this species and enable unambiguous identification of autosomes involved in the autosome-heterosome translocation, providing useful chromosome landmarks for further studies on the karyotype and sex chromosome differentiation in this species. PMID:25394583

  15. Occurrence of B chromosomes in Tetragonisca Latreille, 1811 (Hymenoptera, Apidae, Meliponini): A new contribution to the cytotaxonomy of the genus.

    Barth, Adriane; Fernandes, Anderson; Pompolo, Silvia das Graças; Costa, Marco Antônio

    2011-01-01

    Tetragonisca angustula and Tetragonisca fiebrigi have recently been listed as valid species. This study aimed to cytogenetically investigate both species, emphasizing the new registry of B chromosomes in the tribe Meliponini. We analyzed colonies of T. angustula and T. fiebrigi collected at Tangará da Serra, Mato Grosso, Brazil, through conventional Giemsa staining, C-banding, and base-specific fluorochrome staining (CMA(3)/DAPI). T. angustula showed 2n = 34 chromosomes in females and n = 17 in males, with karyotype formula 2K = 34A(M). T. fiebrigi showed numeric variation, with chromosome number varying from 2n = 34 to 2n = 36 in females and from n = 17 to n = 18 in males, with karyotype formula 2K = 32A(M)+2A(Mc) and 2K = 32A(M)+2A(Mc) + 1 or 2 B-chromosomes. The B chromosomes are heterochromatic. In T. fiebrigi, the CMA(3)/DAPI staining revealed four chromosomes with a CMA(3) positive band. All individuals from the same colony showed the same number of B chromosomes. T. angustula and T. fiebrigi showed karyotype divergence, principally due to the presence of B chromosomes, which are found only in T. fiebrigi. Our data corroborate the status of valid species for both T. angustula and T. fiebrigi, as recently proposed. PMID:21637547

  16. Occurrence of B chromosomes in Tetragonisca Latreille, 1811 (Hymenoptera, Apidae, Meliponini: a new contribution to the cytotaxonomy of the genus

    Adriane Barth

    2011-01-01

    Full Text Available Tetragonisca angustula and Tetragonisca fiebrigi have recently been listed as valid species. This study aimed to cytogenetically investigate both species, emphasizing the new registry of B chromosomes in the tribe Meliponini. We analyzed colonies of T. angustula and T. fiebrigi collected at Tangará da Serra, Mato Grosso, Brazil, through conventional Giemsa staining, C-banding, and base-specific fluorochrome staining (CMA3/DAPI. T. angustula showed 2n = 34 chromosomes in females and n = 17 in males, with karyotype formula 2K = 34A M. T. fiebrigi showed numeric variation, with chromosome number varying from 2n = 34 to 2n = 36 in females and from n = 17 to n=18in males, with karyotype formula 2K = 32A M+2A Mc and 2K = 32A M+2A Mc + 1 or 2 B-chromosomes. The B chromosomes are heterochromatic. In T. fiebrigi, the CMA3/DAPI staining revealed four chromosomes with a CMA3 positive band. All individuals from the same colony showed the same number of B chromosomes. T. angustula and T. fiebrigi showed karyotype divergence, principally due to the presence of B chromosomes, which are found only in T. fiebrigi. Our data corroborate the status of valid species for both T. angustula and T. fiebrigi, as recently proposed.

  17. The Molecular Cytogenetic Characterization of Pistachio (Pistacia vera L. Suggests the Arrest of Recombination in the Largest Heteropycnotic Pair HC1.

    Pedro J Sola-Campoy

    Full Text Available This paper represents the first molecular cytogenetic characterization of the strictly dioecious pistachio tree (Pistacia vera L.. The karyotype was characterized by fluorescent in situ hybridization (FISH with probes for 5S and 45S rDNAs, and the pistachio specific satellite DNAs PIVE-40, and PIVE-180, together with DAPI-staining. PIVE-180 has a monomeric unit of 176-178 bp and high sequence homology between family members; PIVE-40 has a 43 bp consensus monomeric unit, and is most likely arranged in higher order repeats (HORs of two units. The P. vera genome is highly heterochromatic, and prominent DAPI positive blocks are detected in most chromosomes. Despite the difficulty in classifying chromosomes according to morphology, 10 out of 15 pairs (2n = 30 could be distinguished by their unique banding patterns using a combination of FISH probes. Significantly, the largest pair, designated HC1, is strongly heteropycnotic, shows differential condensation, and has massive enrichment in PIVE-40 repeats. There are two types of HC1 chromosomes (type-I and type-II with differing PIVE-40 hybridization signal. Only type-I/II heterozygotes and type-I homozygotes individuals were found. We speculate that the differentiation between the two HC1 chromosomes is due to suppression of homologous recombination at meiosis, reinforced by the presence of PIVE-40 HORs and differences in PIVE-40 abundance. This would be compatible with a ZW sex-determination system in the pistachio tree.

  18. The Molecular Cytogenetic Characterization of Pistachio (Pistacia vera L.) Suggests the Arrest of Recombination in the Largest Heteropycnotic Pair HC1.

    Sola-Campoy, Pedro J; Robles, Francisca; Schwarzacher, Trude; Ruiz Rejón, Carmelo; de la Herrán, Roberto; Navajas-Pérez, Rafael

    2015-01-01

    This paper represents the first molecular cytogenetic characterization of the strictly dioecious pistachio tree (Pistacia vera L.). The karyotype was characterized by fluorescent in situ hybridization (FISH) with probes for 5S and 45S rDNAs, and the pistachio specific satellite DNAs PIVE-40, and PIVE-180, together with DAPI-staining. PIVE-180 has a monomeric unit of 176-178 bp and high sequence homology between family members; PIVE-40 has a 43 bp consensus monomeric unit, and is most likely arranged in higher order repeats (HORs) of two units. The P. vera genome is highly heterochromatic, and prominent DAPI positive blocks are detected in most chromosomes. Despite the difficulty in classifying chromosomes according to morphology, 10 out of 15 pairs (2n = 30) could be distinguished by their unique banding patterns using a combination of FISH probes. Significantly, the largest pair, designated HC1, is strongly heteropycnotic, shows differential condensation, and has massive enrichment in PIVE-40 repeats. There are two types of HC1 chromosomes (type-I and type-II) with differing PIVE-40 hybridization signal. Only type-I/II heterozygotes and type-I homozygotes individuals were found. We speculate that the differentiation between the two HC1 chromosomes is due to suppression of homologous recombination at meiosis, reinforced by the presence of PIVE-40 HORs and differences in PIVE-40 abundance. This would be compatible with a ZW sex-determination system in the pistachio tree. PMID:26633808

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

    Ebenézer C.S. Bernardes

    2013-01-01

    Full Text Available 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 chromosomes 1 and 3 and interstitial in the short arm of chromosome 8. Among endive accessions, FISH procedures revealed conserved position and number of 5S and 45S rDNA sites (two and three pairs, respectively, associated with the CMA-positive bands. Notwithstanding, polymorphisms were detected within chicory accessions regarding the number and the distribution of rDNA sites in relation to the most frequent karyotype (two pairs with 45S and one with 5S rDNA. The karyological markers developed allowed karyotypic differentiation between both species, uncovering peculiarities in the number and position of rDNA sites, which suggest chromosome rearrangements, such as translocations in chicory cultivars. The interspecific and intraspecific polymorphisms observed emphasize the potential of karyomorphological evaluations, helping our understanding of the relationships and evolution of the group.

  20. A New Method for Estimating Bacterial Abundances in Natural Samples using Sublimation

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

    2004-01-01

    We have developed a new method based on the sublimation of adenine from Escherichia coli to estimate bacterial cell counts in natural samples. To demonstrate this technique, several types of natural samples including beach sand, seawater, deep-sea sediment, and two soil samples from the Atacama Desert were heated to a temperature of 500 C for several seconds under reduced pressure. The sublimate was collected on a cold finger and the amount of adenine released from the samples then determined by high performance liquid chromatography (HPLC) with UV absorbance detection. Based on the total amount of adenine recovered from DNA and RNA in these samples, we estimated bacterial cell counts ranging from approx. l0(exp 5) to l0(exp 9) E. coli cell equivalents per gram. For most of these samples, the sublimation based cell counts were in agreement with total bacterial counts obtained by traditional DAPI staining. The simplicity and robustness of the sublimation technique compared to the DAPI staining method makes this approach particularly attractive for use by spacecraft instrumentation. NASA is currently planning to send a lander to Mars in 2009 in order to assess whether or not organic compounds, especially those that might be associated with life, are present in Martian surface samples. Based on our analyses of the Atacama Desert soil samples, several million bacterial cells per gam of Martian soil should be detectable using this sublimation technique.

  1. Karyotypes of two species of the genus Pimelodus (Siluriformes, Pimelodidae).

    Treco, Fernando Rodrigo; Dias, Ana Lúcia

    2009-01-01

    A cytogenetic study was conducted on two species of the genus Pimelodus that were collected from the Piquiri river, Paraná, Brazil: Pimelodus paranaensis and Pimelodus heraldoi. Both had a diploid number of 2n=56 chromosomes and a fundamental number (FN) of 104. In P. paranaensis, the karyotype consisted of 22m+22sm+4st+8a chromosomes, whereas the karyotype of P. heraldoi consisted of 18m+24sm+6st+8a. The AgNORs were localized in the terminal region of the long arm in one pair of subtelocentric chromosomes, pair 24 in P. paranaensis and pair 23 in P. heraldoi. The latter species showed size heteromorphism of these regions between the chromosome homologues. Heterochromatin was distributed mainly in the terminal regions in the two species. CMA3-positive staining was observed in some chromosomes, besides being associated with NORs, which were all DAPI-negative, in both species of Pimelodus. C-banding plus CMA3 and DAPI showed that most of the heterochromatic regions were rich in AT bases in P. paranaensis and P. heraldoi. PMID:19459460

  2. Effect of CPP/ACP on Initial Bioadhesion to Enamel and Dentin In Situ

    Susann Grychtol

    2014-01-01

    Full Text Available The present in situ study investigated the influence of a preparation containing CPP/ACP (caseinphosphopeptide-amorphous calcium phosphate (GC Tooth mousse on initial bacterial colonization of enamel and dentin. Therefore, pellicle formation was performed in situ on bovine enamel and dentin specimens fixed to individual upper jaw splints worn by 8 subjects. After 1 min of pellicle formation GC Tooth mousse was used according to manufacturer’s recommendations. Rinses with chlorhexidine served as positive controls. Specimens carried without any rinse served as negative controls. After 8 h overnight exposure of the splints, bacterial colonization was quantified by fluorescence microscopy (DAPI and BacLight live/dead staining. Additionally, the colony forming units (CFU were determined after desorption. Furthermore, the effects on Streptococcus mutans bacteria were tested in vitro (BacLight. There was no significant impact of CPP/ACP on initial bacterial colonization proved with DAPI and BacLight. Determination of CFU showed statistical significance for CPP/ACP to reduce bacterial adherence on enamel. The in vitro investigation indicated no antimicrobial effects for CPP/ACP on Streptococcus mutans suspension. Under the chosen conditions, CPP/ACP (GC Tooth mousse had no significant impact on initial biofilm formation on dental hard tissues. The tested preparation cannot be recommended for biofilm management.

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

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

    2013-09-01

    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 chromosomes 1 and 3 and interstitial in the short arm of chromosome 8. Among endive accessions, FISH procedures revealed conserved position and number of 5S and 45S rDNA sites (two and three pairs, respectively), associated with the CMA-positive bands. Notwithstanding, polymorphisms were detected within chicory accessions regarding the number and the distribution of rDNA sites in relation to the most frequent karyotype (two pairs with 45S and one with 5S rDNA). The karyological markers developed allowed karyotypic differentiation between both species, uncovering peculiarities in the number and position of rDNA sites, which suggest chromosome rearrangements, such as translocations in chicory cultivars. The interspecific and intraspecific polymorphisms observed emphasize the potential of karyomorphological evaluations, helping our understanding of the relationships and evolution of the group. PMID:24130443

  4. Cytogenetic characterization of three Balistoidea fish species from the Atlantic with inferences on chromosomal evolution in the families Monacanthidae and Balistidae

    de Lima, Lorena Corina Bezerra; Martinez, Pablo Ariel; Molina, Wagner Franco

    2011-01-01

    Abstract The Tetraodontiformes are the most derived group of teleostean fish. Among other apomorphies, they are characterized by a high degree of fusions or significant bone loss in the head and body. In the early phylogenetic proposals presented for this order, the families Balistidae and Monacanthidae have been unanimously considered to be closely related. Although they have moderate species diversity, they are scarcely known in cytogenetic aspect and chromosomal pattern comparisons between these groups have yet to be established. The species Cantherhines macrocerus (Hollard,1853), Cantherhines pullus (Ranzani, 1842) (Monacanthidae) and Melichthys niger (Bloch, 1786) (Balistidae) were cytogenetically analyzed using conventional (Ag-impregnation, C-banding, CMA3- and DAPI-fluorescence) and molecular (FISH with an 18S rDNA probe) cytogenetic protocols. The karyotypes of all three species were very similar possessing diploid chromosome numbers 2n = 40 and composed exclusively of acrocentric chromosomes. Single NOR-bearing pair as well as positive heterochromatic blocks at pericentromeric regions were identified in the karyotypes of the three species studied. NOR-bearing sites were positively labeled after Ag-impregnation, C-banding, CMA3-fluorescence and FISH with an 18S rDNA probe but were negative after DAPI-fluorescence. Such remarkable shared conspicuous chromosomal characters corroborate either close phylogenetic relationship of these families, previously established by morphological and molecular data, or rather conservative nature of karyotype differentiation processes. The later hypothesis, however, appears less probable due to centric or in tandem fusions documented for another Balistoidea species. PMID:24260619

  5. Isolation, culture and intraportal transplantation of rat marrow stromal cell

    Objective: To observe the tracing and evolution of marrow stromal cell (MSC) after intraportal transplantation into the liver of homogenous rats, and to provide experimental data for MSC differentiation to hepatocyte in vivo. Methods: The MSC was isolated from the leg bone marrow of adult SD rats, and purified by culture-expanded in vitro. Before transplantation, MSC was labeled with DAPI. Then 105 MSC were intraportally transplanted into the homogenous rat liver. Rats were killed at 2 hours and 1, 2, 3 and 4 weeks after transplantation. The cryosection samples of liver and lung were observed under fluorescence microscopy. Results: MSC in vitro culture had high ability of proliferation. Except 4 rats were dead because of abdominal bleeding or infection, other recipients were healthy until sacrificed. The implantation cells were detected by identifying the DAPI labeled MSC in the host livers, but not in the host lungs. Conclusion: Intraportal transplanted MSC could immigrate and survive in the host livers at least for 4 weeks. They could immigrate from the small branches of portal veins to hepatic parenchyma

  6. Cytogenetics of the Brazilian Bolitoglossa paraensis (Unterstein, 1930 salamanders (Caudata, Plethodontidae

    Jéssica Barata da Silva

    2014-09-01

    Full Text Available Plethodontid salamanders of genus Bolitoglossa constitute the largest and most diverse group of salamanders, including around 20% of living caudate species. Recent studies have indicated the occurrence of five recognized species in the Brazilian Amazon Rainforest. We present here the first cytogenetic data of a Brazilian salamander, which may prove to be a useful by contribution to the cytotaxonomy of the genus. Specimens were collected near the "type" locality (Utinga, Belém, PA, Brazil. Chromosomal preparations from duodenal epithelial cells and testes were subjected to Giemsa staining, C-banding and DAPI/CMA3 fluorochrome staining. All specimens showed a karyotype with 13 bi-armed chromosome pairs (2n = 26. Nucleolar Organizer Regions, evidenced by CMA3, were located distally on the long arm of pair 7 (7q. DAPI+ heterochromatin was predominantly centromeric, with some small pericentromeric bands. Although the C-banding patterns of other Bolitoglossa species are so far unknown, cytogenetic studies conducted in other Plethodontid salamanders have demonstrated that pericentromeric heterochromatin is a useful cytological marker for identifying interspecific homeologies. Species diversification is usually accompanied by chromosomal changes. Therefore, the cytogenetic characterization of Bolitoglossa populations from the middle and western Brazilian Amazon Basin could identify differences which may lead to the identification of new species.

  7. Roles of Fas signaling pathway in vitamin E succinate-induced apoptosis in human gastric cancer SGC-7901 cells

    Kun Wu; Yao Li; Yan Zhao; Yu-Juan Shan; Wei Xia; Wei-Ping Yu; Lan Zhao

    2002-01-01

    AIM: To investigate the roles of Fas signaling pathway in vitamin E succinate-induced apoptosis in human gastric cancer SGC-7901 cells.METHODS: Human gastric cancer SGC-7901 cells were treated with VES at 5, 10, 20 mg@L-1, succinic acid and vitamin E as vehicle control and condition media only as untreated (UT) control. Apoptotic morphology was observed by DAPI staining. Western blot analysis was applied to measure the expression of Fas, FADD and caspase-8 proteins. After the cells were transiently transfected with Fas and FADD antisense oligonucleotides, respectively, caspase-8 activity was determined by flurometric method.RESULTS: The morphologically apoptotic changes were observed after VES treatment by DAPI staining. 23.7 % and 89.6 % apoptosis occurred after 24 h and 48 h of 20 mg@L-1 VES treatment, respectively. The protein levels of Fas, FADD and caspase-8 were evidently increased in a dose-dependent manner after 24 h of VES treatment. The blockage of Fas by transfection with Fas antisense oligonucleotides obviously inhibited the expression of FADD protein. After SGC-7901 cells were transfected with Fas and FADD antisense oligonucleotides, caspase-8 activity was obviously decreased (P<0.01), whereas Fas blocked more than FADD.CONCLUSION: VES-induced apoptosis in human gastric cancer SGC-7901 cells involves Fas signaling pathway including the interaction of Fas, FADD and caspase-8.

  8. Naturally Ocurring Polyphosphate-accumulating Bacteria in Benthic Biofilms

    Locke, N. A.; Saia, S. M.; Walter, M. T.; Carrick, H. J.; Buda, A. R.; Regan, J. M.

    2014-12-01

    Polyphosphate accumulating organisms (PAOs), known to store excess phosphorus (P) as polyphosphate (poly-P), influence P transport in the environment. Enhanced biological phosphorus removal (EBPR) from wastewater has long served as a basis to study bacterial PAOs, yet little research has genetically identified similar organisms in natural settings. Aerobic/anaerobic cycles, used to select for PAOs in EBPR, can result from changing environmental conditions such as night/day cycles for benthic biofilms. Benthic biofilms from eight Pennsylvanian streams were studied for naturally-occurring bacterial PAOs similar to those typically found in EBPR systems. PAOs were confirmed in the benthic biofilms by a characteristic yellow fluorescent emission from DAPI staining. Cells containing yellow fluorescence were separated from the rest of the sample using a flow cytometer, resulting in a physically enriched culture of PAOs from the benthic biofilms. Amplicon-based metagenomic sequencing will reveal the phylogeny of bacteria responsible for poly-P accumulation in these benthic biofilms. Sequencing data will be used to develop fluorescent in-situ hybridization (FISH) probes, and hybridizations will be performed on DAPI-stained cells to confirm poly-P accumulation by targeted phylotypes. Identifying PAOs in natural settings is a critical step towards studying environments that support high concentrations of PAOs, serving as significant factors in the P cycle. PAOs can then be connected to P transport models to help understand and mitigate P pollution in agricultural watersheds.

  9. Description of the karyotype of Rhagomys rufescens Thomas, 1886 (Rodentia, Sigmodontinae from Southern Brazil Atlantic forest

    André Filipe Testoni

    2010-01-01

    Full Text Available Rhagomys rufescens (Rodentia: Sigmodontinae is an endemic species of the Atlantic forest from Southern and Southeastern Brazil. Some authors consider Rhagomys as part of the tribe Thomasomyini; but its phylogenetic relationships remain unclear. Chromosomal studies on eight specimens of Rhagomys rufescens revealed a diploid number of 2n = 36 and a number of autosome arms FN = 50. GTG, CBG and Ag-NOR banding and CMA3/DAPI staining were performed on metaphase chromosomes. Eight biarmed and nine acrocentric pairs were found in the karyotype of this species. The X and Y chromosomes were both acrocentric. Most of the autosomes and the sex chromosomes showed positive C-bands in the pericentromeric region. The X chromosome showed an additional heterochromatic block in the proximal region of the long arm. Nucleolus organizer regions (NORs were located in the pericentromeric region of three biarmed autosomes (pairs 4, 6 and 8 and in the telomeric region of the short arm of three acrocentrics (pairs 10, 12 and 17. CMA3/DAPI staining produced fluorescent signals in many autosomes, especially in pairs 4, 6, and 8. This study presents cytogenetic data of Rhagomys rufescens for the first time.

  10. Anti-proliferative effect of biogenic gold nanoparticles against breast cancer cell lines (MDA-MB-231 & MCF-7)

    K. S., Uma Suganya; Govindaraju, K.; Ganesh Kumar, V.; Prabhu, D.; Arulvasu, C.; Stalin Dhas, T.; Karthick, V.; Changmai, Niranjan

    2016-05-01

    Breast cancer is a major complication in women and numerous approaches are being developed to overcome this problem. In conventional treatments such as chemotherapy and radiotherapy the post side effects cause an unsuitable effect in treatment of cancer. Hence, it is essential to develop a novel strategy for the treatment of this disease. In the present investigation, a possible route for green synthesis of gold nanoparticles (AuNPs) using leaf extract of Mimosa pudica and its anticancer efficacy in the treatment of breast cancer cell lines is studied. The synthesized nanoparticles were found to be effective in killing cancer cells (MDA-MB-231 & MCF-7) which were studied using various anticancer assays (MTT assay, cell morphology determination, cell cycle analysis, comet assay, Annexin V-FITC/PI staining and DAPI staining). Cell morphological analysis showed the changes occurred in cancer cells during the treatment with AuNPs. Cell cycle analysis revealed apoptosis in G0/G1 to S phase. Similarly in Comet assay, there was an increase in tail length in treated cells in comparison with the control. Annexin V-FITC/PI staining assay showed prompt fluorescence in treated cells indicating the translocation of phosphatidylserine from the inner membrane. PI and DAPI staining showed the DNA damage in treated cells.

  11. Differentiation potential of bone marrow mesenchymal stem cells into retina in normal and laser-injured rat eye

    ZHANG Jie; SHAN Qing; MA Ping; JIANG Yanming; CHEN Peng; WEN Jingxia; ZHOU You; QIAN Huanwen; PEI Xuetao

    2004-01-01

    Bone marrow mesenchymal stem cells (MSCs) can develop into hematopoietic and mesenchymal lineages but have not been known to participate in the production of retina. Here we report that bone marrow mesenchymal stem cells, after being subretinally transplanted into normal or Nd: YAG laser-injured rat eye, can integrate into RPE layer, photoreceptor layer, bipolar cell layer and ganglion layer. DAPI-labeling detection was used to trace the origin of the repopulating cells. DAPI fluorescence was used to identify retina cells of bone marrow origin 10, 20, 35 and 50 days after transplantation. No formation of rosettes was found but some random cells were found at the end of the observation. MSCs-originated cells spread more widely in the injured retinas than in the normal ones. Immunohistochemical detection showed that though the cells could express neuronal nuclei (NeuN), neuron specific enolase (NSE), glial fibrillary acidic protein (GFAP) and cytokeratin (CK), the proteins expression in the injured transplantation group was abnormal in some region compared with that in the normal transplantation group. Electroretinogram (ERG) showed that ERG-b wave of the injured transplantation group is significantly higher than that of the two laser-injured control groups. These results suggest that a proportion of MSCs can differentiate into retina-like structure in vivo and the differentiation differs in normal and laser-injured retinas.

  12. The ability of two cooked food mutagens to induce aberrant crypt foci in mice

    Kristiansen, E.; Meyer, Otto A.; Thorup, I.

    1997-01-01

    azoxymethane (AOM) (5 mg/kg body weight) and 1,2-dimethylhydrazine dihydrochloride (DMH-2HCI) (20 mg/kg body weight), respectively, one dose a week for two weeks, Animals were killed after four and 10 weeks, After four weeks only the mice dosed with IQ and PhIP had aberrant crypt foci, A much higher number of...... induced a higher percentage of medium or large sized aberrant crypt foci than PhIP or IQ, The interpretation of the aberrant crypt foci as precursor lesions for colon cancer in the PhIP and IQ mice is difficult because PhIP and IQ have not been reported to be colonic carcinogens, If cooked food mutagens...... such as IQ or PhIP are to be used as initiators in the aberrant crypt foci test, the use of rats may be preferable....

  13. (R-4-[2-(Methylsulfanylpyrimidin-4-yl]-1-(tetrahydrofuran-3-yl-1H-pyrazol-5-amine

    Zhengyu Liu

    2009-04-01

    Full Text Available The title compound, C12H15N5OS, was obtained by reaction of 2-(2-(methylthiopyrimidin-4-yl-3-oxopropanenitrile with (tetrahydrofuran-3-ylhydrazine dihydrochloride, and the racemic product was subsequently separated by chiral chromatography (first peak; [α]D20 = +51.3°. The chiral center at the substituted atom of the tetrahydrofuranyl group has an R-configuration. The pyrimidine and pyrazolyl rings are almost coplanar, their mean planes forming a dihedral angle of 6.4 (1°. One of the H atoms of the amino group participates in an intramolecular hydrogen bond with the pyrimidine N atom in position 3. The second H atom is involved in an intermolecular hydrogen bond, which links the molecules into an infinite chain.

  14. Experimental and quantum mechanical studies on the ion-pair of levocetirizine and bromocresol green in aqueous solutions.

    Dena, Ahmed S Abo; Hassan, Walid M I

    2016-06-15

    In the present work, levocetirizine dihydrochloride (LEV) was found to interact with bromocresol green (BCG) via ion-pair formation. UV-vis and FTIR spectroscopy were used to validate the data obtained from quantum mechanical calculations (QMC). The electrostatic potential maps show that the reaction is preferred through the interaction of the sulfonic acid group of BCG and the quaternary ammonium group of LEV. The optimized geometry of the product shows that there are six different intermolecular hydrogen bonds between the studied molecules resulting from the ionic attraction between the oppositely charged groups. The UV-vis spectra suggest the formation of an ion-pair. This finding is contradicting with the previous charge-transfer hypothesis. PMID:27038582

  15. Stable Poly(methacrylic acid Brush Decorated Silica Nano-Particles by ARGET ATRP for Bioconjugation

    Marcello Iacono

    2015-08-01

    Full Text Available The synthesis of polymer brush decorated silica nano-particles is demonstrated by activator regeneration by electron transfer atom transfer radical polymerization (ARGET ATRP grafting of poly(tert-butyl methacrylate. ATRP initiator decorated silica nano-particles were obtained using a novel trimethylsiloxane derivatised ATRP initiator obtained by click chemistry. Comparison of de-grafted polymers with polymer obtained from a sacrificial initiator demonstrated good agreement up to 55% monomer conversion. Subsequent mild deprotection of the tert-butyl ester groups using phosphoric acid yielded highly colloidal and pH stable hydrophilic nano-particles comprising approximately 50% methacrylic acid groups. The successful bio-conjugation was achieved by immobilization of Horseradish Peroxidase to the polymer brush decorated nano-particles and the enzyme activity demonstrated in a conversion of o-phenylene diamine dihydrochloride assay.

  16. DIZZINESS OF ISCHEMIC GENESIS: DESCRIPTION OF VESTIBULAR DISORDERS AND POSSIBILITY OF THERAPY

    N. S. Alekseeva

    2015-12-01

    Full Text Available Aim. To reveal the peculiarities of peripheral and central vestibular disorders in patients with mild dizziness in initial and reversible cerebrovascular diseases and estimate the efficacy of dizziness therapy with betahistine dihydrochloride (Betaserc, Solvay Pharma.Material and methods: 40 patients (all women with dizziness were studied. Neurological and otoneurological examination was done; central hemodynamics and number of vascular indices were analyzed. Therapy with Betaserc in daily dose of 48 mg was held during 2 months.Results. Dysfunction of both peripheral and central parts of vestibular analyzer was revealed in all patients. Most patients complained on diminished hearing, buzzing in ears and head, imbalance while walking. All patients mentioned the performance impairment, easy fatigability, change of mood. Therapy with Betaserc improved the condition of 97% patients with dizziness, the intensity of associated acoustic disturbances considerably reduced.Conclusion. Betaserc is an effective drug for dizziness therapy in patients with initial and reversible cerebrovascular diseases.

  17. Effects of thyroid hormones on the bypasses of the antimycin A block in the bc1 complex of rat liver mitochondria.

    Horrum, M A; Tobin, R B; Ecklund, R E

    1991-07-15

    The effect of thyroid hormones on the electron flow through the bc1 complex of rat liver mitochondria was studied using two dye bypasses of the Antimycin A block of the bc1 complex by the method of Alexandre and Lehninger (Biochim. Biophys. Acta 767:120; 1984). Bypass respiration rates with both DCIP (2,6-dichlorophenolindophenol) and TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride) were elevated in the hyperthyroid rats and depressed in the hypothyroid groups compared to the euthyroid controls. T3 treatment of hypothyroid rats returned the bypass rates to control levels in 24 hours with the TMPD dye but not for the DCIP. This further demonstrates that different portions of the bc1 complex respond individually to the thyroid state. PMID:1648915

  18. Determination of cetirizine in tablets and compounded capsules: comparative study between CE and HPLC

    Lisiane Bajerski

    2010-01-01

    Full Text Available A capillary electrophoresis (CE method was developed and validated for determination of cetirizine dihydrochloride in tablets and compounded capsules. The electrophoretic separation was performed in an uncoated fused-silica capillary (40 cm x 50 μm i.d. using 20 mmol L-1 sodium tetraborate buffer (pH 9.3 as background electrolyte, a hydrodinamic sample injection at 50 mBar for 5 s, 20 KV applied voltage at 25 °C, and detection at 232 nm. The proposed method was compared with the high performance liquid chromatographic (HPLC method previously validated for this drug, and statistical analysis showed no significant difference between the techniques.

  19. Refined carbohydrate enhancement of aberrant crypt foci (ACF) in rat colon induced by the food-borne carcinogen 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ)

    Kristiansen, E.; Meyer, Otto A.; Thorup, I.

    1996-01-01

    ,2-dimethylhydrazine dihydrochloride (DMH) and azoxymethane (AOM), the use of a diet-related colon cancer initiator, such as the heterocyclic amine 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) formed during meat cooking, would probably give a more relevant insight into diet-related colon carcinogenesis. In the...... present study it is shown that a feeding regimen with continuous low IQ doses (0.03% in the diet) throughout a study period of 10 weeks has a significant effect on the induction of ACF in the colon of male F344 rats. In addition, the study illustrates that the incidence of the IQ-induced ACF can be...

  20. XPS study of PBO fiber surface modified by incorporation of hydroxyl polar groups in main chains

    Dihydroxy poly(p-phenylene benzobisoxazole) (DHPBO), a modified poly(p-phenylene benzoxazole) (PBO) polymer containing double hydroxyl groups in polymer chains, was synthesized by copolymerization of 4,6-diamino resorcinol dihydrochloride (DAR), purified terephthalic acid (TA) and 2,5-dihydroxyterephthalic acid (DHTA). DHPBO fibers were prepared by dry-jet wet-spinning method. The effects of hydroxyl polar groups on the surface elemental compositions of PBO fiber were investigated by X-ray photoelectron spectroscopy (XPS). The results show that the ratio of oxygen/carbon on the surface of DHPBO fibers is higher than that on the surface of PBO fibers, which indicates the content of polar groups on the surface of DHPBO fiber increase compared with PBO fiber.

  1. Experimental and quantum mechanical studies on the ion-pair of levocetirizine and bromocresol green in aqueous solutions

    Dena, Ahmed S. Abo; Hassan, Walid M. I.

    2016-06-01

    In the present work, levocetirizine dihydrochloride (LEV) was found to interact with bromocresol green (BCG) via ion-pair formation. UV-vis and FTIR spectroscopy were used to validate the data obtained from quantum mechanical calculations (QMC). The electrostatic potential maps show that the reaction is preferred through the interaction of the sulfonic acid group of BCG and the quaternary ammonium group of LEV. The optimized geometry of the product shows that there are six different intermolecular hydrogen bonds between the studied molecules resulting from the ionic attraction between the oppositely charged groups. The UV-vis spectra suggest the formation of an ion-pair. This finding is contradicting with the previous charge-transfer hypothesis.

  2. Evaluation of skin anti-aging potential of Citrus reticulata blanco peel

    Vinita D Apraj

    2016-01-01

    Abbreviation Used: ECM: Extracellular matrix, UV: Ultra violet, ROS: Reactive Oxygen Species, MMP: Matrix metalloproteinase, Chc: Clostridium histolyticum collagenase, DPPH: 2, 2-diphenyl-1-picrylhydrazyl, GC-MS: Gas Chromatography- Mass Spectroscopy, RT: Room Temperature, μg GAE/ mg: Microgram Gallic acid equivalent / milligram, W/V: Weight by Volume, μg QE/ mg: Microgram Quercetin equivalent / milligram, CR HAE: Hot Alcoholic Extract of Citrus reticulata Blanco, CR CAE: Cold Alcoholic Extract of Citrus reticulata Blanco, EC50: Half Maximal Effective Concentration, PMS NADH: Phenazine methosulfate nicotinamide adenine dinucleotide, NBT: Nitroblue tetrazolium, DMSO: Dimethyl sulfoxide, APS: Ammonium Persulphate, AAPH: 2,2 -azobis(2-amidino-propane dihydrochloride, TROLOX: (± 6-hydroxy-2,5,7,8-tetramethyl chromane-2-carboxylic acid, ORAC: Oxygen Radical Absorbance Capacity, FALGPA: N-[3-(2-Furyl acryloyl]-Leu-Gly-Pro-Ala, SANA: Succinyl-Ala-Ala-Ala-p-nitroanilide, Rf: Retardation Factor, MSD: Mass Selective Detector

  3. A LC-MS metabolomics approach to investigate the effect of raw apple intake in the rat plasma metabolome

    Rago, Daniela; Kristensen, Mette; Gürdeniz, Gözde;

    2013-01-01

    the postprandial to the fasting state and affect steroid metabolism by suppressing the plasma level of stress corticosteroids, certain mineralocorticoids and oxidised bile acid metabolites. Several new hypotheses regarding the cause of health effects from apple intake can be generated from this study for further......Fruit and vegetable consumption has been associated with several health benefits; however the mechanisms are largely unknown at the biochemical level. Our research aims to investigate whether plasma metabolome profiling can reflect biological effects after feeding rats with raw apple by using...... dihydrochloride (DMH) once a week. Plasma samples were taken at the end of the intervention and among all groups, about half the animals were 12 h fasted. An initial ANOVA-simultaneous component analysis with a three-factor or two-factor design was employed in order to isolate potential metabolic variations...

  4. Preparation of magnetic latexes functionalized with chloromethyl groups via emulsifier-free miniemulsion polymerization

    Faridi-Majidi, Reza [School of Chemistry, University College of Science, Tehran University, Tehran (Iran, Islamic Republic of)]. E-mail: refaridi@khayam.ut.ac.ir; Sharifi-Sanjani, Naser [School of Chemistry, University College of Science, Tehran University, Tehran (Iran, Islamic Republic of)

    2007-04-15

    Functionalized crosslinked polystyrene-co-divinylbenzene-co-chloromethylstyrene magnetic latex particles were prepared via emulsifier-free miniemulsion polymerization using 2, 2' azobis (2-amidinopropane) dihydrochloride (V-50) as an initiator and in the presence of magnetite nanoparticles in the monomers. Transmission electron microscopy (TEM) proved the presence of magnetite nanoparticles in polymer particles. Differential scanning calorimetery (DSC) analysis of the product showed an exothermic signal due to crosslinking of chains through electrophilic aromatic substitution of phenyl groups with chloromethyl groups in the presence of the dispersed Fe{sub 3}O{sub 4} as Lewis acid. This was proven by thermogravimetric analysis (TGA) via the loss of gaseous HCl. The results were also compared with those of magnetite-free miniemulsion polymerization using V-50.

  5. Formulation, optimization and evaluation of levocetirizine dihyrochloride oral thin strip

    J Gunjan Patel

    2012-01-01

    Full Text Available The aim of present research was to develop a fast releasing oral polymeric film, with good mechanical properties, instant disintegration and dissolution, producing an acceptable taste when placed on tongue. Solvent casting method was used to prepare oral films. Levocetirizine dihydrochloride, an antihistaminic was incorporated to relieve the symptoms of allergic rhinitis. The polymers selected were HPMC E 15 and PVA. Propylene glycol was the plasticizers used. Nine batches of films with drug were prepared using different combinations of polymers and plasticizer concentration. The resultant films were evaluated for weight variation, content uniformity, folding endurance, thickness, surface pH, in vitro disintegration and in vitro dissolution. The optimized films which disintegrated in less than 30 sec, releasing 85-98% of drug within 2 minutes. The percentage release was varying with concentration of plasticizer and polymer. The films made with HPMC: PVA (1:2 released 96% of drug in 1 min, which was the best release amongst all.

  6. Spectrophotometric determination of azathioprine in pharmaceutical formulations.

    Lakshmi, C S; Reddy, M N

    1998-12-01

    Four simple and sensitive visible spectrophotometric methods (A-D) have been described for the assay of azathioprine (ATP) either in pure form or in pharmaceutical formulations. Methods A and B are based on the oxidation of ATP with excess N-bromosuccinimide (NBS) or chloramine-T (CAT) and determining the consumed NBS or CAT with a decrease in colour intensity of celestine blue (CB) (method A) or gallocyanine (GC) (method B), respectively. Methods C and D are based on the diazotisation of reduced azathioprine (RATP) with excess nitrous acid and estimating either the consumed nitrous acid (HNO(2)) with cresyl fast violet acetate (CFVA) (method C) or by coupling reaction of the diazonium salt formed with N-1-naphthyl ethylene diamine dihydrochloride (NED) (method D). All of the variables have been optimized and the reactions presented. The concentration measurements are reproducible within a relative standard deviation of 1.0%. Recoveries are 99.2-100.3%. PMID:18967434

  7. Effect of propofol and thiopentone on free radical mediated oxidative stress of the erythrocyte

    Murphy, P G; Davies, Michael Jonathan; Columb, M O; Stratford, N

    1996-01-01

    Propofol has free radical scavenging properties similar to those of recognized phenol-based antioxidants. We have examined these properties in an in vitro model of radical-induced cellular injury, comparing its activity with that of thiopentone (which has also been shown to have radical scavenging...... activity). Haemolysis of human erythrocytes was induced using the azo compound 2,2'-azo-bis(2-amidinopropane) dihydrochloride (ABAP). This was achieved by incubating a 10% suspension of erythrocytes with ABAP 100 mmol litre-1 at 37 degrees C. For propofol, at concentrations of 12.5, 25 and 50 mumol litre-1......, with the highest concentrations in the propofol-containing flasks. The formation of methaemoglobin was preceded by the generation of ferrylhaemoglobin (a Fe4+ haemoglobin species). Further experiments examining oxidation of purified methaemoglobin to ferrylhaemoglobin by hydrogen peroxide suggested...

  8. Spacer conformation in biologically active molecules. Part 2. Structure and conformation of 4-[2-(diphenylmethylamino)ethyl]-1-(2-methoxyphenyl) piperazine and its diphenylmethoxy analog—potential 5-HT 1A receptor ligands

    Karolak-Wojciechowska, J.; Fruziński, A.; Czylkowski, R.; Paluchowska, M. H.; Mokrosz, M. J.

    2003-09-01

    As a part of studies on biologically active molecule structures with aliphatic linking chain, the structures of 4-[2-diphenylmethylamino)ethyl]-1-(2-methoxyphenyl)piperazine dihydrochloride ( 1) and 4-[2-diphenylmethoxy)ethyl]-1-(2-methoxyphenyl)piperazine fumarate ( 2) have been reported. In both compounds, four atomic non-all-carbons linking chains (N)C-C-X-C are present. The conformation of that linking spacer depends on the nature of the X-atom. The preferred conformation for chain with XNH has been found to be fully extended while for that with XO—the bend one. It was confirmed by conformational calculations (strain energy distribution and random search) and crystallographic data, including statistics from CCDC.

  9. Inhibition of free radical induced oxidative hemolysis of red blood cells by green tea polyphenols

    2000-01-01

    The in vitro oxidative hemolysis of human red blood cells (RBC) was used as a model to study the free radical induced damage of biological membranes and the inhibitory effect of natural antioxidants. The hemolysis was induced by a water-soluble free radical initiator 2,2′-azo(2- asmidinopropane)dihydrochloride (AAPH) and inhibited by the principal polyphenolic components extracted from green tea leaves, i.e. (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicat- echin gallate (ECG), (-)-epigallocatechin gallate (EGCG) and gallic acid (GA). Addition of AAPH at 37°C caused fast hemolysis after a short period of inhibition period, while addition of the green tea polyphenols efficiently suppressed the hemolysis in the activity sequence of EGCG>EGC>ECG≈EC>GA, demonstrating that these green tea polyphenols are effective antioxidants which could protect biological membranes from free radical induced oxidative damage.

  10. Studies on N-vinylformamide cross-linked copolymers

    Świder, Joanna; Tąta, Agnieszka; Sokołowska, Katarzyna; Witek, Ewa; Proniewicz, Edyta

    2015-12-01

    Copolymers of N-vinylformamide (NVF) cross-linked with three multifunctional monomers, including divinylbenzene (DVB), ethylene glycol dimethacrylate (EGDMA), and N,N‧-methylenebisacrylamide (MBA) were synthetized by a three-dimensional free radical polymerization in inverse suspension using 2,2‧-azobis(2-methylpropionamide) dihydrochloride (AIBA) as an initiator. Methyl silicon oil was used as the continuous phase during the polymerization processes. Fourier-transform adsorption infrared (FT-IR) spectra revealed the presence of silicone oil traces and suggested that silicone oil strongly interacted with the copolymers surface. Purification procedure allowed to completely remove the silicon oil traces from P(NVF-co-DVB) only. The morphology and the structure of the investigated copolymers were examined by optical microscopy, FT-IR, and FT-Raman (Fourier-transform Raman spectroscopy) methods.

  11. Preparation of cationic polyacrylamide by aqueous two-phase polymerization

    2010-05-01

    Full Text Available Cationic polyacrylamide (CPAM was synthesized by aqueous two-phase polymerization technique using acrylamide (AM and dimethylaminoethyl methacrylate methyl chloride (DMC as raw materials, aqueous polyethylene glycol 20000 (PEG 20000 solution as dispersant, 2,2′-azobis(2-amidinopropane dihydrochloride (V-50 as initiator and poly(dimethylaminoethyl methacrylate methyl chloride (PDMC as stabilizer. The polymer was characterized by infrared (IR spectroscopy, 1H-NMR spectrum and transmission electron microscopy (TEM. The copolymer composition was analyzed. The effect of monomers concentration, PEG 20000 concentration and stabilizer concentration on copolymer were investigated, respectively. The optimum reaction conditions for obtaining a stable CPAM aqueous two-phase system were monomers concentration 8~15%, PEG 20000 concentration 15~25%, and PDMC concentration 0.5~1.5%. Finally, the formation process of copolymer particles was investigated by optical microscope.

  12. 非对苯二胺染发剂合成的研究进展%Progress on synthesis of non-p-phenylenediamine hair dyes

    高莉红; 高海燕; 岳娟

    2012-01-01

    概述了3类非对苯二胺染发剂的作用原理、合成方法及其染发效果.其中植物色素染发剂详细介绍了散沫花色素、姜黄素、槲皮素和苏木精;苯胺类衍生物染发剂主要介绍了2,5 -二氨基苯乙醇、β-(2,4-二氨基苯氧基)-乙醇盐酸盐、N,N-双-(2-羟乙基)-对苯二胺;其他化学合成染发剂介绍了染色硅胶纳米粒子、含巯基染发剂和天然黑色素制备的染发剂.简单总结了各种合成路线的优缺点,并对染发剂的开发和应用前景进行了展望.%The dyeing mechanism, synthetic method, and dyeing effect of three kinds of non - p -phenylenediamine hair dyes were reviewed with their advantages and disadvantages described. Henna, curcumin,quercetin and hematoxylin were proposed as plant colorants for natural hair dye. 2,5 - Diamino -phenylethanol ,β -2,4 - diaminophenoxyethanol dihydrochloride, and N, N - bis ( 2 - hydroxyethyl) - p -phenylenediamine dihydrochloride were introduced as aniline derivative hair dyes. Other hair dyes, including colored silica nanoparticles, mercapto - modified dyes and natural melanin, were introduced too. Further, outlooks for the developing trends and application of hair dyes were prospected.

  13. Ropivacaine-Induced Contraction Is Attenuated by Both Endothelial Nitric Oxide and Voltage-Dependent Potassium Channels in Isolated Rat Aortae

    Seong-Ho Ok

    2013-01-01

    Full Text Available This study investigated endothelium-derived vasodilators and potassium channels involved in the modulation of ropivacaine-induced contraction. In endothelium-intact rat aortae, ropivacaine concentration-response curves were generated in the presence or absence of the following inhibitors: the nonspecific nitric oxide synthase (NOS inhibitor Nω-nitro-L-arginine methyl ester (L-NAME, the neuronal NOS inhibitor Nω-propyl-L-arginine hydrochloride, the inducible NOS inhibitor 1400W dihydrochloride, the nitric oxide-sensitive guanylyl cyclase (GC inhibitor ODQ, the NOS and GC inhibitor methylene blue, the phosphoinositide-3 kinase inhibitor wortmannin, the cytochrome p450 epoxygenase inhibitor fluconazole, the voltage-dependent potassium channel inhibitor 4-aminopyridine (4-AP, the calcium-activated potassium channel inhibitor tetraethylammonium (TEA, the inward-rectifying potassium channel inhibitor barium chloride, and the ATP-sensitive potassium channel inhibitor glibenclamide. The effect of ropivacaine on endothelial nitric oxide synthase (eNOS phosphorylation in human umbilical vein endothelial cells was examined by western blotting. Ropivacaine-induced contraction was weaker in endothelium-intact aortae than in endothelium-denuded aortae. L-NAME, ODQ, and methylene blue enhanced ropivacaine-induced contraction, whereas wortmannin, Nω-propyl-L-arginine hydrochloride, 1400W dihydrochloride, and fluconazole had no effect. 4-AP and TEA enhanced ropivacaine-induced contraction; however, barium chloride and glibenclamide had no effect. eNOS phosphorylation was induced by ropivacaine. These results suggest that ropivacaine-induced contraction is attenuated primarily by both endothelial nitric oxide and voltage-dependent potassium channels.

  14. Pretreatment With Inactivated Bacillus Calmette-Guerin Increases CD4+CD25+ Regulatory T Cell Function and Decreases Functional and Structural Effects of Asthma Induction in a Rat Asthma Model.

    Gong, Ping; Li, Yun; Tan, Yu-Pin; Li, Hong

    2016-04-01

    Bacillus Calmette-Guerin (BCG) has been shown to have therapeutic effects on asthma through CD4+CD25+ regulatory T cells (Tregs). We sought to assess pretreatment with inactivated BCG on CD4+CD25+ Tregs and its functional and structural effects in rat asthma model. The rat asthma model was established using ovalbumin (OVA) sensitization and challenge. Ten rats were pretreated with BCG prior to OVA and received continued BCG injections during OVA challenge (BCG+OVA group), 10 rats were treated with OVA alone (OVA group), and 10 rats were treated with saline (control group). After 9 weeks, histamine dihydrochloride effect on airway resistance was measured. Number of CD4+CD25+ Tregs was measured by flow cytometry, expression of Foxp3 and CTLA-4 mRNA was measured, and serum TGF-β levels were determined. Differential cell count in bronchoalveolar lavage fluid (BALF) was determined, and lung tissue was processed and stained with hematoxylin and eosin, Masson's trichrome, and alcine blue and periodic acid Schiff's reaction to evaluate inflammatory cell infiltration, collagen deposition, and presence of goblet cells, respectively. BCG treatment led to an increase in CD4+CD25+ Tregs, as well as an increase in Foxp3 and CTLA-4 expression and serum TGF-β levels. In addition, we observed a decrease in histamine dihydrochloride-induced airway resistance, a decrease in inflammatory leukocytes in BALF, and a decrease in airway remodeling indicators in BCG+OVA-treated rats compared with OVA-treated rats. Intradermally injected inactivated BCG has the potential to improve airway inflammation, airway resistance, and airway remodeling through a mechanism that may involve CD4+CD25+ Tregs. PMID:26495900

  15. Spatiotemporal control of degenerate multiphoton fluorescence microscopy with delay-tunable femtosecond pulse pairs

    Das, Dhiman; Bhattacharyya, Indrajit; Goswami, Debabrata

    2016-07-01

    Selective excitation of a particular fluorophore in an ensemble of different fluorophores with overlapping fluorescence spectra is shown to be dependent on the time delay of femtosecond pulse pairs in multiphoton fluorescence microscopy. In particular, the two-photon fluorescence behavior of the Texas Red and DAPI dye pair inside Bovine Pulmonary Artery Endothelial (BPAE) cells depends strongly on the center wavelength of the laser, as well as the delay between two identical laser pulses in one-color femtosecond pulse-pair excitation scheme. Thus, we present a novel design concept using pairs of femtosecond pulses at different central wavelengths and tunable pulse separations for controlling the image contrast between two spatially and spectrally overlapping fluorophores. This femtosecond pulse-pair technique is unique in utilizing the variation of dye dynamics inside biological cells as a contrast mode in microscopy of different fluorophores.

  16. Programmed cell death of Ulmus pumila L. seeds during aging

    Yulan ZHANG; Ming ZHANG; Fang LI; Xiaofeng WANG

    2008-01-01

    The programmed cell death (PCD) character-istics of Ulmus pumila L. seeds were investigated. The seeds were treated at a high temperature of 37℃ and 100% relative humidity for six days. DAPI (4'6-diami-dino-2-phenylindole) staining revealed that the aging treatment induced condensation and margination of chro-matin, as well as the formation of apoptotic bodies. DNA electrophoresis results of U. pumila seeds on an agarose gel showed a characteristic "ladder" pattern. Levels of electrolyte leakage of seed cells showed that membranes retained their integral form during almost the entire aging time. There was an immediate increase in the production rate of superoxide anion (O2-) and in the amount of hydrogen peroxide (H2O2), which remained at a μmol level. All of these common characteristics indicate that seed aging can be classified as PCD.

  17. COMMUNITY ANALYSIS OF AGGREGATED BACTERIA IN SOUTHERN LAKE BAIKAL%韩国贝加尔湖南部群集细菌的群落分析

    Ahn T. S.; Kim O. S.; Spiglazov L. P; Drucker V. V.; Hong S-H.

    2006-01-01

    This study focuses on the community structure of aggregated bacteria in Lake Baikal and relationships with free-living bacteria.Fluorescent in situ hybridization (FISH) methods were used in samples of bacteria taken in April, 2001. Bacterial counts of free-living ranged from 52.3 to 74.1% in free-living bacteria and from 39.6 to 66.7% in aggregated bacteria, respectively. Community composition of aggregated bacteria was very different from free-living bacteria, especially at 25m depth where highest phytoplankton numbers were observed. The vertical profile of aggregated bacteria community was very characteristic. Beta-Proteobacteria increased with depth down to 100m. At 250m, gamma-Proteobacteria was 44% of DAPI bound cells, while other groups were less than 1%. We conclude that community structures of free-living and aggregated bacteria were different, and they may sustain the ecosystem in independent ways.

  18. Cytogenetic characterization of two species of Frieseomelitta Ihering, 1912 (Hymenoptera, Apidae, Meliponini

    Antônio F. Carvalho

    2011-01-01

    Full Text Available The cytogenetic analysis of Frieseomelitta dispar and F. francoi revealed the chromosome numbers 2n = 30 and n = 15 and a karyotypic formula 2K = 4M+2Mt+4A+20A M. The number of chromosomes observed was consistent with those reported for other Frieseomelitta species. The occurrence of the Mt chromosome and other features of the karyotype formulae suggest a close relationship between F. dispar, F. francoi and F. varia. Nevertheless, it was possible to differentiate the karyotypes of the species by DAPI/CMA3 staining, which revealed GC-rich regions on two chromosome pairs of F. dispar: one acrocentric and one pseudoacrocentric. In F. francoi, the same kinds of regions were observed on a pair of metacentrics and on a pair of acrocentrics. Our analysis also confirmed the chromosome number conservation in Frieseomelitta and suggests that infrequent pericentric inversion could constitute a synapomorphy for the group including F. dispar, F. francoi, and F. varia.

  19. Phenotypic and molecular characterization of isolates of Staphylococcus spp. obtained from sheep milk Chapecó-SCCaracterização fenotípica e molecular de isolados de Staphylococcus spp. obtidos de leite de ovelhas do Município de Chapecó-SC

    José Francisco Manta Bragança

    2013-03-01

    Full Text Available This study aimed to determine the antimicrobial susceptibility profile and to assess the presence of mechanisms of antimicrobial resistance in Staphylococcus spp. (n=36 isolated from mastitis in sheep’s Chapecó-SC. The potential for biofilm production was determined by phenotypic tests of Congo Red Agar, DAPI and Gentian Violet and by PCR for the detection of icaD gene. To evaluate the antimicrobial resistance testing was performed disk diffusion and detection of resistance genes blaZ, mecA, ermA, ermB and ermC and msrA also performed by PCR.The pump test was conducted by efuxo crop growth Muller Hinton agar containing ethidium bromide. The results showed that 1 (2,78%, 36 (100% and 10 (27,78% isolates were considered to produce a biofilm on Congo Red Agar test, Gentian Violet and DAPI, respectively, while the gene icaD was observed in only 2 (5.55% isolates. The lowest percentage of sensitivity was observed for ampicillin (58.33% and penicillin (58.33%. All strains tested were negative for the mecA, ermA, ermB and ermC genes. However, the isolates were positive for other resistance genes, being the blaZ and the msrA, with percentages of positivity of 58.33% and 11.11% respectively. Only one sample was positive for efflux pump test.O presente trabalho teve como objetivos determinar o perfil de sensibilidade aos antimicrobianos e avaliar a presença de mecanismos de resistência antimicrobiana em Staphylococcus spp. (n=36 isolados de mastite em ovelhas do município de Chapecó-SC. O potencial para produção de biofilme foi determinado pelos testes fenotípicos de Agar Vermelho Congo, DAPI e Violeta de Genciana e por teste molecular pela técnica de PCR para a detecção do gene icaD. Para determinar o perfil de resistência aos antimicrobianos, foi realizado o teste de difusão em disco e detecção dos genes de resistência blaZ, mecA, erm (A, B e C e msrA. O teste da bomba de efuxo foi realizado através do crescimento das culturas em Agar

  20. Detection of DNA strand breaks in mammalian cells using the radioresistant bacterium PprA protein

    We have previously found that the PprA protein from Deinococcus radiodurans possesses ability to recognize DNA carrying strand breaks. In the present study, we attempted to visualize radiation-induced DNA strand breaks with PprA protein using immunofluorescence technique to elucidate the DNA damage response mechanism in mammalian cultured cells. As a result, colocalization of Cy2 and DAPI fluorescent signals was observed. This observation suggests that DNA strand breaks in the nucleus of CHO-K1 cells were effectively detected using the PprA protein. The amount of DNA strand breaks (integrated density of Cy2 fluorescent signals) was increased with the increase in the radiation dose. (author)

  1. Karyotypic description of the stingless bee Oxytrigona cf. flaveola (Hymenoptera, Apidae, Meliponina) of a colony from Tangará da Serra, Mato Grosso State, Brazil.

    Krinski, Diones; Fernandes, Anderson; Rocha, Marla Piumbini; Pompolo, Silvia das Graças

    2010-07-01

    The aim was to broaden knowledge on the cytogenetics of the subtribe Meliponina, by furnishing cytogenetic data as a contribution to the characterization of bees from the genus Oxytrigona. Individuals of the species Oxytrigona cf. flaveola, members of a colony from Tangará da Serra, Mato Grosso State, Brazil, were studied. The chromosome number was 2n = 34, distributed among four chromosomal morphologies, with the karyotype formula 8m+8sm+16st+2t. Size heteromorphism in the first metacentric pair, subsequently confirmed by sequential staining with fluorochrome (DA/DAPI/CMA(3) ), was apparent in all the examined individuals The nucleolar organizing regions (NORs) are possibly located in this metacentric chromosome pair. These data will contribute towards a better understanding of the genus Oxytrigona. Given that species in this group are threatened, the importance of their preservation and conservation can be shown in a sensible, concise fashion through studies such as this. PMID:21637423

  2. Karyotypic description of the stingless bee Oxytrigona cf. flaveola (Hymenoptera, Apidae, Meliponina of a colony from Tangará da Serra, Mato Grosso State, Brazil

    Diones Krinski

    2010-01-01

    Full Text Available The aim was to broaden knowledge on the cytogenetics of the subtribe Meliponina, by furnishing cytogenetic data as a contribution to the characterization of bees from the genus Oxytrigona. Individuals of the species Oxytrigona cf. flaveola, members of a colony from Tangará da Serra, Mato Grosso State, Brazil, were studied. The chromosome number was 2n = 34, distributed among four chromosomal morphologies, with the karyotype formula 8m+8sm+16st+2t. Size heteromorphism in the first metacentric pair, subsequently confirmed by sequential staining with fluorochrome (DA/DAPI/CMA3, was apparent in all the examined individuals The nucleolar organizing regions (NORs are possibly located in this metacentric chromosome pair. These data will contribute towards a better understanding of the genus Oxytrigona. Given that species in this group are threatened, the importance of their preservation and conservation can be shown in a sensible, concise fashion through studies such as this.

  3. Naphthoflavones as Antiproliferative Agents: Design, Synthesis and Biological Evaluation.

    Kumar, Dinesh; Singh, Onkar; Nepali, Kunal; Bedi, Pms; Qayum, Arem; Singh, Shashank; Jain, Subheet K

    2016-01-01

    The present study involves the design and synthesis of naphthoflavones as antiproliferative agents. The strategy presents naphthoflavones as hybrids of naphthyl based chalcones and flavones. A panel of human cancer cell lines were employed for the cytotoxicity studies. DK-13 exhibited significant cytoxicity against MiaPaCa-2 cell lines with IC50 value of 1.93 μM and 5.63 μM against MCF-7 cell lines. The compound DK-13 was found to induce apoptosis evidenced through phase contrast microscopy, DAPI staining, and mitochondrial membrane potential loss. The cell phase distribution studies indicated an increase from 11.26 % (control sample) to 55.19 % (sample treated with 20 μM compound DK-13) in the apoptotic population. PMID:26845133

  4. Determination of main phylogenetic bacterial groups in hyporheic sediments of a small lowland stream (Sitka, Czech Republic) using fluorescent in situ hybridization (FISH)

    Community structure of biofilm microbial communities of hyporheic sediments in the small lowland Sitka Stream (Czech Republic) was analysed using fluorescent in situ hybridization (FISH). FISH provided detection of about 91 % of total DAPI-stained bacterial cells. In general, most of the EUB338-detectable cells (domain Eubacteria) could be related to the four major phylogenetic groups used in this study (α-, β-, γ-Proteobacteria and Cytophaga-Flavobacterium). The microbial community structure of biofilm analysed was dominated by β-Proteobacteria (36,3 % of the total counts). In addition to the domain Eubacteria, members of the domain Archaebacteria were detected in hyporheic sediments (approximately 6 % of total bacterial counts). (authors)

  5. Cytogenetic study of two species of the family Pimelodidae (Siluriformes collected in lago Guaíba, Rio Grande do Sul, Brazil

    Fernando Rodrigo Treco

    2008-03-01

    Full Text Available A cytogenetic study was conducted on specimens of Parapimelodus nigribarbis and Pimelodus maculatus collected in the lago Guaíba drainage, Porto Alegre, Rio Grande do Sul, Brazil. The two species had a diploid number of 56 chromosomes, with P. nigribarbis showing a karyotype of 20m + 20sm + 4st + 12a with FN of 100, and P. maculatus showing a karyotype of 24m + 20sm + 6st + 6a with FN of 106. NORs were demonstrated in both species in only one pair of subtelocentric chromosomes, in the terminal region of the long arm, which was coincident with C- banding and CMA3 staining, while DAPI staining was negative in these regions. Parapimelodus nigribarbis had a greater number of heterochromatic bands than did P. maculatus, which were distributed mainly in the terminal regions, where the latter species showed an interstitial band on the short arm of the first metacentric pair. C-banding plus CMA3 demonstrated heterochromatin that was associated with GC-rich NORs in both P. nigribarbis and P. maculatus, although other fluorescent regions were also observed in the former species. With C-banding plus DAPI, various chromosomal regions were stained in the two species, along with interstitial staining in P. maculatus, indicating that heterochromatin contained a greater quantity of AT-rich regions.Exemplares de Parapimelodus nigribarbis e Pimelodus maculatus coletados na bacia do rio Guaíba, Porto Alegre, Rio Grande do Sul, foram analisados citogeneticamente. As duas espécies apresentaram um número diplóide de 56 cromossomos, com P. nigribarbis mostrando um cariótipo de 20m + 20sm + 4st + 12a, NF igual a 100; e P. maculatus com 24m + 20sm + 6st + 6a e NF igual 106. As NORs foram evidenciadas em apenas um par de cromossomos subtelocêntricos, na região terminal do braço longo, nas duas espécies estudadas, sendo coincidentes com a banda C e CMA3; o DAPI nestas regiões se mostrou negativo. Parapimelodus nigribarbis apresentou um maior número de bandas

  6. Modes of worker reproduction, reproductive dominance and brood cell construction in queenless honeybee (Apis mellifera L.) colonies

    Neumann, Peter; Randall Hepburn, H.; Radloff, Sarah

    2000-01-01

    Modes de reproduction des ouvrières, dominance de reproduction et construction des cellules de couvain dans des colonies orphelines d'Apis mellifera L. Le long d'un transect à travers la zone d'hybridation naturelle entre A. mellifera capensis, à reproduction thélytoque, et A. mellifera scutellata, à reproduction arrhénotoque, 70 colonies indigènes ont été récoltées (voir tableau pour les lieux). Toutes les reines et tous les rayons ont été ôtés et les abeilles adultes ont été transférées dan...

  7. Apoptosis in astrovirus-infected CaCo-2 cells

    Cell death processes during human astrovirus replication in CaCo-2 cells and their underlying mechanisms were investigated. Morphological and biochemical alterations typical of apoptosis were analyzed in infected cells using a combination of techniques, including DAPI staining, the sub-G0/G1 technique and the TUNEL assay. The onset of apoptosis was directly proportional to the virus multiplicity of infection. Transient expression experiments showed a direct link between astrovirus ORF1a encoded proteins and apoptosis induction. A computer analysis of the astrovirus genome revealed the presence of a death domain in the nonstructural protein p38 of unknown function, encoded in ORF1a. Apoptosis inhibition experiments suggested the involvement of caspase 8 in the apoptotic response, and led to a reduction in the infectivity of the virus progeny released to the supernatant. We conclude that apoptotic death of host cells seems necessary for efficient human astrovirus replication and particle maturation

  8. Cytological evidence for population-specific sex chromosome heteromorphism in Palaearctic green toads (Amphibia, Anura)

    G Odierna; G Aprea; T Capriglione; S Castellano; E Balletto

    2007-06-01

    A chromosome study was carried out on a number of European and Central Asiatic diploid green toad populations by means of standard and various other chromosome banding and staining methods (Ag-NOR-, Q-, CMA3-, late replicating [LR] banding pattern, C- and sequential C-banding + CMA3 + DAPI). This study revealed the remarkable karyological uniformity of specimens from all populations, with the only exception being specimens from a Moldavian population, where one chromosome pair was heteromorphic. Though similar in shape, size and with an identical heterochromatin distribution, the difference in the heteromorphic pair was due to a large inverted segment on its long arms. This heteromorphism was restricted to females, suggesting a female heterogametic sex chromosome system of ZZ/ZW type at a very early step of differentiation.

  9. First Evidence on the Role of Heavy Ion Irradiation of Meteorites and Formamide in the Origin of Biomolecules

    Saladino, Raffaele; Carota, Eleonora; Botta, Giorgia; Kapralov, Michail; Timoshenko, Gennady N.; Rozanov, Alexei; Krasavin, Eugene; Di Mauro, Ernesto

    2016-04-01

    Formamide (NH2CHO) has been irradiated in condensed phase at 273 K by 11B-boron beams in the presence of powdered meteorites of the chondrite and stony-iron types. Relative to the controls (no radiation or no catalysis), a variegate panel of compounds was observed, including purine and pyrimidine nucleobases (uracil, cytosine, adenine, and guanine), nucleobase analogues, heterocycles, and carboxylic acids involved in metabolic pathways. The presence of amino imidazole carbonitrile (AICN), 4,6-diamino purine (4,6-DAP) and 2,4-diamino pyrimidine (2,4-DAPy) among the observed products suggests the occurrence of an unified mechanism based on the generation of radical cyanide species (•CN). These observations contribute to outline plausible prebiotic scenarios involving 11B-boron as energy source.

  10. Investigation on biological properties of tacrolimus-loaded poly(1,3-trimethylene carbonate) in vitro

    Hou, Ruixia; Wu, Leigang; Wang, Jin; Huang, Nan

    2010-06-01

    The drug-eluting stents have been regarded as a milestone in inhibiting the restenosis of coronary arteries. However, adverse reactions caused by bare-metal stents and non-biodegradable polymer coatings may result in some clinical problems. In this study, a new tacrolimus-eluting stent coated with biodegradable poly(1,3-trimethylene carbonate) (PTMC) is developed. The structures are characterized by Fourier transform infrared (FTIR) analysis, and the wettability is measured by contact angle assay. The biological behaviors are evaluated by the in vitro platelets adhesion test, APTT test, the human umbilical cord artery smooth muscle cells (HUCASMCs), 4',6-diamidine-2-phenylindole (DAPI) and actin immunofluorescence staining, MTT colorimetric assay. These results show that after blending tacrolimus into PTMC, the anticoagulant behavior is improved, and the adhesion and proliferation of HUCASMCs on samples are inhibited. This work aims to find one kind of surface erosion biodegradable polymers that can be applied as drug-eluting stent coatings.

  11. Investigation on biological properties of tacrolimus-loaded poly(1,3-trimethylene carbonate) in vitro

    The drug-eluting stents have been regarded as a milestone in inhibiting the restenosis of coronary arteries. However, adverse reactions caused by bare-metal stents and non-biodegradable polymer coatings may result in some clinical problems. In this study, a new tacrolimus-eluting stent coated with biodegradable poly(1,3-trimethylene carbonate) (PTMC) is developed. The structures are characterized by Fourier transform infrared (FTIR) analysis, and the wettability is measured by contact angle assay. The biological behaviors are evaluated by the in vitro platelets adhesion test, APTT test, the human umbilical cord artery smooth muscle cells (HUCASMCs), 4',6-diamidine-2-phenylindole (DAPI) and actin immunofluorescence staining, MTT colorimetric assay. These results show that after blending tacrolimus into PTMC, the anticoagulant behavior is improved, and the adhesion and proliferation of HUCASMCs on samples are inhibited. This work aims to find one kind of surface erosion biodegradable polymers that can be applied as drug-eluting stent coatings.

  12. The gene for human erythrocyte protein 4. 2 maps to chromosome 15q15

    Najfeld, V. (Mount Sinai School of Medicine, NY (United States)); Ballard, S.G.; Menninger, J.; Ward, D.C. (Yale Univ., New Haven, CT (United States)); Bouhassira, E.E.; Schwartz, R.S.; Nagel, R.L.; Rybicki, A.C. (Albert Einstein Coll. of Medicine/Montefiore Medical Center, Bronx, NY (United States))

    1992-01-01

    Protein 4.2 (P4.2), one of the major components of the red-blood-cell membrane, is located on the interior surface, where it binds with high affinity to the cytoplasmic domain of band 3. Individuals whose red blood cells are deficient in P4.2 have osmotically fragile, abnormally shaped cells and moderate hemolytic anemia. cDNA clones from both the 5{prime} and the 3{prime} coding regions of the P4.2 gene were used to map its chromosomal location by fluorescence in situ hybridization. The probes, individually or in combination, gave specific hybridization signals on chromosome 15. The hybridization locus was identified by combining fluorescence images of the probe signals with fluorescence banding patterns generated by Alu-PCR (R-like) probe and by DAPI staining (G-like). The authors results demonstrate that the locus of the P4.2 gene is located within 15q15.

  13. Pseudomonas aeruginosa biofilm aggravates skin inflammatory response in BALB/c mice in a novel chronic wound model

    Trøstrup, Hannah; Thomsen, Kim; Christophersen, Lars J;

    2013-01-01

    Chronic wounds are presumed to persist in the inflammatory state, preventing healing. Emerging evidence indicates a clinical impact of bacterial biofilms in soft tissues, including Pseudomonas aeruginosa (PA) biofilms. To further investigate this, we developed a chronic PA biofilm wound infection...... model in C3H/HeN and BALB/c mice. The chronic wound was established by an injection of seaweed alginate-embedded P. aeruginosa PAO1 beneath a third-degree thermal lesion providing full thickness skin necrosis, as in human chronic wounds. Cultures revealed growth of PA, and both alginate with or without...... PAO1 generated a polymorphonuclear-dominated inflammation early after infection. However, both at days 4 and 7, there were a more acute polymorphonuclear-dominated and higher degree of inflammation in the PAO1 containing group (p <0.05). Furthermore, PNA-FISH and supplemented DAPI staining showed...

  14. A first generation cytogenetic ideogram for the Florida manatee (Trichechus manatus latirostris) based on multiple chromosome banding techniques

    Gray, B.A.; Zori, Roberto T.; McGuire, P.M.; Bonde, R.K.

    2002-01-01

    Detailed chromosome studies were conducted for the Florida manatee (Trichechus manatus latirostris) utilizing primary chromosome banding techniques (G- and Q-banding). Digital microscopic imaging methods were employed and a standard G-banded karyotype was constructed for both sexes. Based on chromosome banding patterns and measurements obtained in these studies, a standard karyotype and ideogram are proposed. Characterization of additional cytogenetic features of this species by supplemental chromosome banding techniques, C-banding (constitutive heterochromatin), Ag-NOR staining (nucleolar organizer regions), and DA/DAPI staining, was also performed. These studies provide detailed cytogenetic data for T. manatus latirostris, which could enhance future genetic mapping projects and interspecific and intraspecific genomic comparisons by techniques such as zoo-FISH.

  15. DNA analysis in three populations of African spinach (Basella spp.)

    African spinach (Basella spp.) is an important vegetable in West Africa, and was introduced by early colonialists. Its alien origin is supported by its narrow genetic variability. Flowcytometry and RAPD polymorphism were used to investigate genetic variation in three populations of Basella - 'Congo native', 'Cong domesticated', and an introduced cultivar, 'Sri Lanka' from Sri Lanka. Normal spinach (Spinacia oleracea) cv. 'Prince F1 Hybrid' was used to test sensitivity and to verify detection of genetic variation. Nuclei were isolated from young leaves of Basella, stained with DAPI and ethidium bromide, and ploidy level and total DNA content were determined by using a flowcytometer. The two sexually propagated populations, 'Cong domesticated' and 'Sri Lanka' showed very low amount of genetic variation as revealed by RAPD analysis; the third population 'Congo native' showed a limited amount of polymorphism. (author). 8 refs, 1 fig., 2 tabs

  16. The fluorescent detection of Paradiplozoon sp. (Monogenea: Diplozoidae attachment clamps' sclerites and integumental proteins : research communication

    S.J. Milne

    2006-09-01

    Full Text Available In the genus Paradiplozoon, few hard structures are present therefore most of the taxonomic studies have focused on the unique ultrastructure of the sclerites. Alcohol-fixed specimens were transferred to BSA-saline for 5 min. before staining indefinitely with 5 µl WGA-TRX, 5 µl SYTO 9 and 5 µl of Cal co fluor White M2R. Rhodamine, Fluorescein and DAPI bandpass filters on the microscope enabled selective light wavelength illumination of the three flourochromes by a mercury light source. This method provided an easy and rapid methodology to show the internal sclerites of attachment clamps. It is suitable for alcohol preserved specimens and may have additional applications in other helminth organisms.

  17. New staining methods for yeast like fungi under special consideration of human pathogenic fungi

    Paulitsch-Fuchs, Astrid; Treiber, Fritz; Grasser, Erik; Buzina, Walter; Rosker, Christian

    2010-11-01

    A new method for in-cellular staining of yeast like fungi with Oregon Green and SYTOX Green is presented enabling their detection as well as the observation of cellular details via confocal laser scanning microscopy. Fluorochromes play an important role in many scientific disciplines including medicine, cell biology and botany. For the visualisation of fungal cell walls Calcofluor White is the flourochrome of choice. The necessity of an UV laser for its excitation makes it unpracticable for daily use. Safranin O, DAPI, 2NBDG, Ethidium Bromide and Acridin-orange are commonly used stains for nuclei in fugal microscopy. The attention was given to the possibility of using the differences in staining patterns to distinguish certain pathogenic yeast species e.g. Candida albicans and Candida krusei. Our results show that high quality microscopy of yeast like organisms can readily be achieved by the use of two suitable fluorochromes.

  18. Fabrication of keratin-silica hydrogel for biomedical applications.

    Kakkar, Prachi; Madhan, Balaraman

    2016-09-01

    In the recent past, keratin has been fabricated into different forms of biomaterials like scaffold, gel, sponge, film etc. In lieu of the myriad advantages of the hydrogels for biomedical applications, a keratin-silica hydrogel was fabricated using tetraethyl orthosilicate (TEOS). Textural analysis shed light on the physical properties of the fabricated hydrogel, inturn enabling the optimization of the hydrogel. The optimized keratin-silica hydrogel was found to exhibit instant springiness, optimum hardness, with ease of spreadability. Moreover, the hydrogel showed excellent swelling with highly porous microarchitecture. MTT assay and DAPI staining revealed that keratin-silica hydrogel was biocompatible with fibroblast cells. Collectively, these properties make the fabricated keratin-silica hydrogel, a suitable dressing material for biomedical applications. PMID:27207052

  19. Studies on the interaction of the food colorant tartrazine with double stranded deoxyribonucleic acid.

    Basu, Anirban; Suresh Kumar, Gopinatha

    2016-05-01

    Interaction of the food additive tartrazine with double-stranded DNA was studied by spectroscopic and calorimetric techniques. Absorbance studies revealed that tartrazine exhibited hypochromism in the presence of DNA without any bathochromic effects. Minor groove displacement assay of DAPI and Hoechst 33258 suggested that tartrazine binds in the minor groove of DNA. The complexation was predominantly entropy driven with a smaller but favorable enthalpic contribution to the standard molar Gibbs energy. The equilibrium constant was evaluated to be (3.68 ± .08) × 10(4) M(-1) at 298.15 K. The negative standard molar heat capacity value along with an enthalpy-entropy compensation phenomenon proposed the involvement of dominant hydrophobic forces in the binding process. Tartrazine enhanced the thermal stability of DNA by 7.53 K under saturation conditions. PMID:26159358

  20. Advances in understanding paternally transmitted Chromosomal Abnormalities

    Marchetti, F; Sloter, E; Wyrobek, A J

    2001-03-01

    Multicolor FISH has been adapted for detecting the major types of chromosomal abnormalities in human sperm including aneuploidies for clinically-relevant chromosomes, chromosomal aberrations including breaks and rearrangements, and other numerical abnormalities. The various sperm FISH assays have been used to evaluate healthy men, men of advanced age, and men who have received mutagenic cancer therapy. The mouse has also been used as a model to investigate the mechanism of paternally transmitted genetic damage. Sperm FISH for the mouse has been used to detect chromosomally abnormal mouse sperm, while the PAINT/DAPI analysis of mouse zygotes has been used to evaluate the types of chromosomal defects that can be paternally transmitted to the embryo and their effects on embryonic development.

  1. Morphological and karyotypic differentiation in Caranx lugubris (Perciformes: Carangidae) in the St. Peter and St. Paul Archipelago, mid-Atlantic Ridge

    Jacobina, Uedson Pereira; Martinez, Pablo Ariel; Cioffi, Marcelo de Bello; Garcia, José; Bertollo, Luiz Antonio Carlos; Molina, Wagner Franco

    2014-03-01

    Isolated oceanic islands constitute interesting model systems for the study of colonization processes, as several climatic and oceanographic phenomena have played an important role in the history of the marine ichthyofauna. The present study describes the presence of two morphotypes of Caranx lugubris, in the St. Peter and St. Paul Archipelago located in the mid-Atlantic. Morphotypes were compared in regard to their morphological and cytogenetic patterns, using C-banding, Ag-NORs, staining with CMA3/DAPI fluorochromes and chromosome mapping by dual-color FISH analysis with 5S rDNA and 18S rDNA probes. We found differences in chromosome patterns and marked divergence in body patterns which suggest that different populations of the Atlantic or other provinces can be found in the Archipelago of St. Peter and St. Paul.

  2. Immunofluorescence analysis of the internal brain anatomy of Nereis diversicolor (Polychaeta, Annelida).

    Heuer, C M; Loesel, R

    2008-03-01

    Comparative analyses of neuroanatomical characters can make valuable contributions to the inference of phylogenetic relationships. Whereas investigations in this field are numerous for arthropods, in-depth studies on other protostomes are sparse. Here, we provide a survey of the internal neuroarchitecture of the brain of the aciculate ragworm Nereis diversicolor (Polychaeta, Annelida). Descriptions are based on confocal laser scanning microscope analyses of brain sections labeled with the nuclear marker DAPI and antibodies raised against FMRF-amide, serotonin, and histamine. Autofluorescence of the nervous tissue has been utilized to further elucidate the anatomical structures of the brain. The architecture of two major brain compartments, i.e., the paired mushroom bodies and the central optic neuropil, is described in detail. The findings are compared with existent literature on polychaete neuroanatomy and on arthropod neuroanatomy, and possible phylogenetic implications are outlined. PMID:18071754

  3. Cytogenetic characterization of Partamona cupira (Hymenoptera, Apidae by fluorochromes

    Jefferson de Brito Marthe

    2010-01-01

    Full Text Available Four colonies of the stingless bee Partamona cupira (Hymenoptera: Apidae were cytogenetically analyzed using conventional staining and the fluorochromes CMA3 e DAPI. The females have 2n = 34 chromosomes (2K=32+2. Some females, however, presented an additional large B acrocentric chromosome, to a total of 2n = 35. Chromosome B and the chromosomal pairs 2, 9 and 10 showed CMA3+ bands, indicating an excess of CG base-pairs. A clear association was verified between the P. helleri B chromosome SCAR marker and the presence of a B chromosome in P. cupira. The data obtained suggests that B chromosomes in P. helleri and P. cupira share a common origin.

  4. Phytophthora elicitor PB90 induced apoptosis in suspension cultures of tobacco

    JI Rui; ZHANG Zhengguang; WANG Yuanchao; ZHENG Xiaobo

    2005-01-01

    The protein elicitor PB90 secreted by Phytophthora boehmeriae is an efficient elicitor inducing the hypersensitive response and systemic acquired resistance in tobacco plants. Here, we observed cell death in suspension-cultured cells of Nicotiana tabacum BY-2 with PB90 treatment using Trypan blue staining method. And this cell death could be suppressed by cycloheximide, an inhibitor of proteins synthesis, which implies that PB90-induced cell death was an active cell death process requiring new protein synthesis. DAPI staining revealed that PB90 induce rapid chromatin condensation, margination, apoptotic bodies' formation and DNA laddering, further TUNEL assay also observed the specific breakage of 3′-OH ends. All of the above common morphological characteristics indicated that PB90 induced apoptosis in suspension cultures of tobacco, suggesting that hypersensitive response induced by PB90 is an apoptotic process.

  5. Evolutionary trends in the family Curimatidae (Characiformes): inferences from chromosome banding.

    Sampaio, Tatiane Ramos; Pires, Larissa Bettin; Venturelli, Natália Bortolazzi; Usso, Mariana Campaner; da Rosa, Renata; Dias, Ana Lúcia

    2016-01-01

    The family Curimatidae is a fish group usually considered chromosomally conserved in their diploid number. However, some studies show small changes in the karyotype microstructure, and the presence of B chromosomes, indicating a chromosomal diversification within the group, even if structural changes in the karyotypes are not visible. Few studies associate this trait with an evolutionary pattern within the family. This study aimed to characterize the karyotype, nucleolus organizer regions (NORs), and heterochromatin distribution of six species of Curimatidae of the genera Cyphocharax Fowler, 1906 and Steindachnerina Fowler, 1906: Cyphocharax voga (Hensel, 1870), Cyphocharax spilotus (Vari, 1987), Cyphocharax saladensis (Meinken, 1933), Cyphocharax modestus (Fernández-Yépez, 1948), Steindachnerina biornata (Braga et Azpelicueta, 1987) and Steindachnerina insculpta (Fernández-Yépez, 1948) and contribute data to a better understanding of the mechanisms involved in the chromosomal evolution of this group of fish. All specimens had 2n=54, m-sm, and B microchromosomes. Five species exhibited single NORs, except for Steindachnerina biornata, which showed a multiple pattern of ribosomal sites. NORs were chromomycin A3 positive (CMA3 (+)) and 4'-6-diamino-2-phenylindole (DAPI(-)) negative, exhibiting differences in the pair and chromosomal location of each individual of the species. FISH with 5S rDNA probe revealed sites in the pericentrometic position of a pair of chromosomes of five species. However, another site was detected on a metacentric chromosome of Cyphocharax spilotus. Heterochromatin distributed both in the pericentromeric and some terminal regions was revealed to be CMA3 (+)/DAPI(-). These data associated with the previously existing ones confirm that, although Curimatidae have a very conservative karyotype macrostructure, NORs and heterochromatin variability are caused by mechanisms of chromosome alterations, such as translocations and/or inversions

  6. Design of Portable Mass Spectrometers with Handheld Probes: Aspects of the Sampling and Miniature Pumping Systems

    Chen, Chien-Hsun; Chen, Tsung-Chi; Zhou, Xiaoyu; Kline-Schoder, Robert; Sorensen, Paul; Cooks, R. Graham; Ouyang, Zheng

    2015-02-01

    Miniature mass spectrometry analytical systems of backpack configuration fitted with sampling probes could potentially be of significant interest for in-field, real-time chemical analysis. In this study, various configurations were explored in which a long narrow tube was used to connect the turbo and backing pumps used to create and maintain vacuum. Also, for the first time we introduced two new types of pumps for miniature mass spectrometers, the Creare 130 g drag pump and Creare 350 g scroll backing pump. These pumps, along with another Creare 550 turbo pump and the commercially available Pfeiffer HiPace 10 turbo and KnF diaphragm backing pumps, were tested with the backpack configurations. The system performance, especially the scan time, was characterized when used with a discontinuous atmospheric pressure interface (DAPI) for ion introduction. The pumping performance in the pressure region above 1 mtorr is critical for DAPI operation. The 550 g turbo pump was shown to have a relatively higher pumping speed above 1 mtorr and gave a scan time of 300 ms, almost half the value obtained with the larger, heavier HiPace 10 often used with miniature mass spectrometers. The 350 g scroll pump was also found to be an improvement over the diaphragm pumps generally used as backing pumps. With a coaxial low temperature plasma ion source, direct analysis of low volatility compounds glass slides was demonstrated, including 1 ng DNP (2,4-Dinitrophenol) and 10 ng TNT (2,4,6-trinitrotoluene) with Creare 550 g turbo pump as well as 10 ng cocaine and 20 ng DNP with Creare 130 g drag pump.

  7. Evaluation of Arsenic Trioxide Potential for Lung Cancer Treatment: Assessment of Apoptotic Mechanisms and Oxidative Damage

    Walker, Alice M; Stevens, Jacqueline J; Ndebele, Kenneth; Tchounwou, Paul B

    2016-01-01

    Background Lung cancer is one of the most lethal and common cancers in the world, causing up to 3 million deaths annually. The chemotherapeutic drugs that have been used in treating lung cancer include cisplatin-pemetrexed, cisplastin-gencitabinoe, carboplatin-paclitaxel and crizotinib. Arsenic trioxide (ATO) has been used in the treatment of acute promyelocytic leukemia. However, its effects on lung cancer are not known. We hypothesize that ATO may also have a bioactivity against lung cancer, and its mechanisms of action may involve apoptosis, DNA damage and changes in stress-related proteins in lung cancer cells. Methods To test the above stated hypothesis, lung carcinoma (A549) cells were used as the test model. The effects of ATO were examined by performing 6-diamidine-2 phenylindole (DAPI) nuclear staining for morphological characterization of apoptosis, flow cytometry analysis for early apoptosis, and western blot analysis for stress-related proteins (Hsp70 and cfos) and apoptotic protein expressions. Also, the single cell gel electrophoresis (Comet) assay was used to evaluate the genotoxic effect. Results ATO-induced apoptosis was evidenced by chromatin condensation and formation of apoptotic bodies as revealed by DAPI nuclear staining. Cell shrinkage and membrane blebbing were observed at 4 and 6 µg/ml of ATO. Data from the western blot analysis revealed a significant dose-dependent increase (p < 0.05) in the Hsp 70, caspase 3 and p53 protein expression, and a significant (p < 0.05) decrease in the cfos, and bcl-2 protein expression at 4 and 6 µg/ml of ATO. There was a slight decrease in cytochrome c protein expression at 4 and 6 µg/ ml of ATO. Comet assay data revealed significant dose-dependent increases in the percentages of DNA damage, Comet tail lengths, and Comet tail moment. Conclusion Taken together our results indicate that ATO is cytotoxic to lung cancer cells and its bioactivity is associated with oxidative damage, changes in cellular

  8. Green-to-red photoconvertible fluorescent proteins: tracking cell and protein dynamics on standard wide-field mercury arc-based microscopes

    Buckheit Robert W

    2010-02-01

    Full Text Available Abstract Background Green fluorescent protein (GFP and other FP fusions have been extensively utilized to track protein dynamics in living cells. Recently, development of photoactivatable, photoswitchable and photoconvertible fluorescent proteins (PAFPs has made it possible to investigate the fate of discrete subpopulations of tagged proteins. Initial limitations to their use (due to their tetrameric nature were overcome when monomeric variants, such as Dendra, mEos, and mKikGR were cloned/engineered. Results Here, we report that by closing the field diaphragm, selective, precise and irreversible green-to-red photoconversion (330-380 nm illumination of discrete subcellular protein pools was achieved on a wide-field fluorescence microscope equipped with standard DAPI, Fluorescein, and Rhodamine filter sets and mercury arc illumination within 5-10 seconds. Use of a DAPI-filter cube with long-pass emission filter (LP420 allowed the observation and control of the photoconversion process in real time. Following photoconversion, living cells were imaged for up to 5 hours often without detectable phototoxicity or photobleaching. Conclusions We demonstrate the practicability of this technique using Dendra2 and mEos2 as monomeric, photoconvertible PAFP representatives fused to proteins with low (histone H2B, medium (gap junction channel protein connexin 43, and high (α-tubulin; clathrin light chain dynamic cellular mobility as examples. Comparable efficient, irreversible green-to-red photoconversion of selected portions of cell nuclei, gap junctions, microtubules and clathrin-coated vesicles was achieved. Tracking over time allowed elucidation of the dynamic live-cycle of these subcellular structures. The advantage of this technique is that it can be performed on a standard, relatively inexpensive wide-field fluorescence microscope with mercury arc illumination. Together with previously described laser scanning confocal microscope-based photoconversion

  9. Presence of lymphatics in a rat tendon lesion model.

    Tempfer, Herbert; Kaser-Eichberger, Alexandra; Korntner, Stefanie; Lehner, Christine; Kunkel, Nadja; Traweger, Andreas; Trost, Andrea; Strohmaier, Clemens; Bogner, Barbara; Runge, Christian; Bruckner, Daniela; Krefft, Karolina; Heindl, Ludwig M; Reitsamer, Herbert A; Schrödl, Falk

    2015-04-01

    Tendons lack sufficient blood supply and represent a bradytroph tissue with prolonged healing time under pathological conditions. While the role of lymphatics in wound/defect healing in tissues with regular blood supply is well investigated, its involvement in tendon defects is not clear. We here try to identify the role of the lymphatic system in a tendon lesion model with morphological methods. A rat Achilles tendon lesion model (n = 5) was created via surgical intervention. Two weeks after surgery, animals were killed and lesioned site removed and prepared for polarization microscopy (picrosirius red) and immunohistochemistry using the lymphatic markers PROX1, VEGFR3, CCL21, LYVE-1, PDPN, and the vascular marker CD31. Additionally, DAPI was applied. Untreated tendons served as controls, confocal laser-scanning microscopy was used for documentation. At the lesion site, polarization microscopy revealed a structural reintegration while immunohistochemistry detected band-like profiles immunoreactive for PDPN, VEGFR3, CCL21, LYVE1, and CD31, surrounding DAPI-positive nuclei. PROX1-positive nuclei were detected within the lesion forming lines and opposed to each other. These PROX1-positive nuclei were surrounded by LYVE-1- or VEGFR3-positive surfaces. Few CD31-positive profiles contained PROX1-positive nuclei, while the majority of CD31-positive profiles lacked PROX1-positive nuclei. VEGFR3-, PDPN-, and LYVE-1-positive profiles were numerous within the lesion site, but absent in control tissue. Within 2 weeks, a structural rearrangement takes place in this lesion model, with dense lymphatic supply. The role of lymphatics in tendon wound healing is unclear, and proposed model represents a good possibility to study healing dynamics and lymphangiogenesis in a tissue almost completely lacking lymphatics in physiological conditions. PMID:25371325

  10. Phenotypic consequences of a mosaic marker chromosome identified by fluorescence in situ hybridization (FISH) as being derived from chromosome 16

    Ray, J.H.; Zhou, X.; Pletcher, B.A. [Cornell Univ. Medical College, Manhasset, NY (United States)] [and others

    1994-09-01

    De novo marker chromosomes are detected in 1 in 2500 amniotic fluid samples and are associated with a 10-15% risk for phenotypic abnormality. FISH can be utilized as a research tool to identify the origins of marker chromosomes. The phenotypic consequences of a marker chromosome derived from the short arm of chromosome 16 are described. A 26-year-old woman underwent amniocentesis at 28 weeks gestation because of a prenatally diagnosed tetralogy of Fallot. Follow-up ultrasounds also showed ventriculomegaly and cleft lip and palate. 32 of 45 cells had the karyotype 47,XY,+mar; the remaining cells were 46,XY. The de novo marker chromosome was C-band positive and non-satellited and failed to stain with distamycin A/DAPI. At birth the ultrasound findings were confirmed and dysmorphic features and cryptorchidism were noted. Although a newborn blood sample contained only normal cells, mosaicism was confirmed in 2 skin biopsies. FISH using whole-chromosome painting and alpha-satellite DNA probes showed that the marker chromosome had originated from chromosome 16. As proximal 16q is distamycin A/DAPI positive, the marker is apparently derived from proximal 16p. At 15 months of age, this child is hypotonic, globally delayed and is gavage-fed. His physical examination is significant for microbrachycephaly, a round face, sparse scalp hair, ocular hypertelorism, exotropia, a flat, wide nasal bridge and tip, mild micrognathia, and tapered fingers with lymphedema of hands and feet. Inguinal hernias have been repaired. His features are consistent with those described for patients trisomic for most or all of the short arm of chromosome 16. Marker chromosomes derived from the short arm of chromosome 16 appear to have phenotypic consequences. As the origin of more marker chromosomes are identified using FISH, their karyotype/phenotype correlations will become more apparent, which will permit more accurate genetic counseling.

  11. Molecular cytogenetic characterisation and phylogenetic analysis of the seven cultivated Vigna species (Fabaceae).

    She, C-W; Jiang, X-H; Ou, L-J; Liu, J; Long, K-L; Zhang, L-H; Duan, W-T; Zhao, W; Hu, J-C

    2015-01-01

    The genomic organisation of the seven cultivated Vigna species, V. unguiculata, V. subterranea, V. angularis, V. umbellata, V. radiata, V. mungo and V. aconitifolia, was determined using sequential combined PI and DAPI (CPD) staining and dual-colour fluorescence in situ hybridisation (FISH) with 5S and 45S rDNA probes. For phylogenetic analyses, comparative genomic in situ hybridisation (cGISH) onto somatic chromosomes and sequence analysis of the internal transcribed spacer (ITS) of 45S rDNA were used. Quantitative karyotypes were established using chromosome measurements, fluorochrome bands and rDNA FISH signals. All species had symmetrical karyotypes composed of only metacentric or metacentric and submetacentric chromosomes. Distinct heterochromatin differentiation was revealed by CPD staining and DAPI counterstaining after FISH. The rDNA sites among all species differed in their number, location and size. cGISH of V. umbellata genomic DNA to the chromosomes of all species produced strong signals in all centromeric regions of V. umbellata and V. angularis, weak signals in all pericentromeric regions of V. aconitifolia, and CPD-banded proximal regions of V. mungo var. mungo. Molecular phylogenetic trees showed that V. angularis and V. umbellata were the closest relatives, and V. mungo and V. aconitifolia were relatively closely related; these species formed a group that was separated from another group comprising V. radiata, V. unguiculata ssp. sesquipedalis and V. subterranea. This result was consistent with the phylogenetic relationships inferred from the heterochromatin and cGISH patterns; thus, fluorochrome banding and cGISH are efficient tools for the phylogenetic analysis of Vigna species. PMID:24750425

  12. Extremely low frequency electromagnetic field sensitizes cisplatin-resistant human ovarian adenocarcinoma cells via P53 activation.

    Baharara, Javad; Hosseini, Nasrin; Farzin, Tayebe Ramezani

    2016-08-01

    In the following study, extremely low frequency electromagnetic fields (EL-EMF) radiation was used to restore sensitivity in the cisplatin-resistant A2780 ovarian cancer cells. For this purpose A2780 cells were treated with different doses of cisplatin and EL-EMF (50 Hz, 200 gauss, and 2 h) alone. Cytotoxicity was the measurement using MTT assay. After calculating IC50 for cisplatin (90 µg/ml) a lower concentration from IC50 (30 and 60 µg/ml) was used to be combined with EL-EMF. We compare the effects of each cisplatin, EL-EMF and combination groups using acridine orange-propidium iodide (AO/PI) and DAPI staining, caspase 3/9 activation assay and Annexin/PI assay. We also assessed changes in P53 and Matrix metalloproteinases 2 (MMPs) gene expression with semi-quantitative RT-PCR. Results indicated an EL-EMF-dependent proliferative decrease which was found <10 %, and occurred independently of cisplatin. The decreased proliferation rate for 30 and 60 µg/ml cisplatin was about 20 and 40 %, respectively, while for synergistic groups 30 and 60 µg/ml cisplatin with 2 h EL-EMF exposer, showed 47 and 71 % decrease in viability in rats. DAPI staining indicated that chromatin break down significantly increased in synergistic groups. Acridine orange staining also confirmed MTT assay results. Caspase activity significantly increased in the combined groups. Semi-quantitative RT-PCR showed that in synergistic groups of cisplatin and EL-EMF, expression of P53 was increased but the expression level of MPP-2 gene decreased. Results from this study showed that changes generated by the non-invasive EL-EMF can make resistant cells sensitive to cisplatin. PMID:26370097

  13. Real-time PCR assay is superior to other methods for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran.

    Molla Kazemiha, Vahid; Bonakdar, Shahin; Amanzadeh, Amir; Azari, Shahram; Memarnejadian, Arash; Shahbazi, Shirin; Shokrgozar, Mohammad Ali; Mahdian, Reza

    2016-08-01

    Mycoplasmas are the most important contaminants of cell cultures throughout the world. They are considered as a major problem in biological studies and biopharmaceutical economic issues. In this study, our aim was to find the best standard technique as a rapid method with high sensitivity, specificity and accuracy for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran. Thirty cell lines suspected to mycoplasma contamination were evaluated by five different techniques including microbial culture, indirect DNA DAPI staining, enzymatic mycoalert(®) assay, conventional PCR and real-time PCR. Five mycoplasma-contaminated cell lines were assigned as positive controls and five mycoplasma-free cell lines as negative controls. The enzymatic method was performed using the mycoalert(®) mycoplasma detection kit. Real-time PCR technique was conducted by PromoKine diagnostic kits. In the conventional PCR method, mycoplasma genus-specific primers were designed to analyze the sequences based on a fixed and common region on 16S ribosomal RNA with PCR product size of 425 bp. Mycoplasma contamination was observed in 60, 56.66, 53.33, 46.66 and 33.33 % of 30 different cell cultures by real-time PCR, PCR, enzymatic mycoalert(®), indirect DNA DAPI staining and microbial culture methods, respectively. The analysis of the results of the different methods showed that the real-time PCR assay was superior the other methods with the sensitivity, specificity, accuracy, predictive value of positive and negative results of 100 %. These values were 94.44, 100, 96.77, 100 and 92.85 % for the conventional PCR method, respectively. Therefore, this study showed that real-time PCR and PCR assays based on the common sequences in the 16S ribosomal RNA are reliable methods with high sensitivity, specificity and accuracy for detection of mycoplasma contamination in cell cultures and other biological products. PMID:25742733

  14. Selective cytotoxic effect of 1-O-undecylglycerol in human melanoma cells

    Marian Hernández-Colina

    2016-04-01

    Full Text Available Context: 1-O-alkylglycerols are ether-linked glycerols derived from shark liver oil and found in small amounts in human milk. Previous studies showed antineoplastic activity for this family of compounds, structurally related to alkylphospholipids, but the activity of linear chain synthetic alkylglycerols in cancer cell lines is less documented. Melanoma is a high incidence cancer, highly resistant to potential treatments. Finding new anti-cancer compounds to improve melanoma prognosis is a relevant research issue. Aims: To study the cytotoxic effect of 1-O-undecylglycerol in primary cultured normal fibroblasts and A375 human melanoma cell line. Methods: Cells were treated with different concentrations of 1-O-undecylglycerol and viability assessed by MTT assay. Morphological changes were visualized by DAPI and acridine orange-ethidium bromide staining. Mitochondrial membrane potential was evaluated, and gene expression of P53 and BcL-2 was semi-quantified. Results: 1-O-undecylglycerol decreased viability of A375 cells and exerted very low cytotoxicity on primary cultured normal fibroblasts. Necrosis appeared in A375 cells but not in fibroblasts, and no apoptotic changes were visualized in DAPI staining experiments. After 24 h fibroblasts and melanoma cells developed mitochondrial potential changes similar to valinomycin. The gene expression of P53 and BcL-2 decreased in treated cells. Conclusions: 1-O-undecylglycerol exhibited selective cytotoxic activity in A375 melanoma cells when compared with primary cultured fibroblast. Its toxicity is mediated by necrosis that may be related with mitochondrial events and decrease in P53 and BcL-2 expression. The results suggest that UDG could be a useful strategy to combine with other chemotherapeutic agents in melanoma treatment.

  15. Induction of Apoptosis by Green Synthesized Gold Nanoparticles Through Activation of Caspase-3 and 9 in Human Cervical Cancer Cells

    Baharara, Javad; Ramezani, Tayebe; Divsalar, Adeleh; Mousavi, Marzieh; Seyedarabi, Arefeh

    2016-01-01

    Background: Gold Nanoparticles (GNPs) are used in imaging and molecular diagnostic applications. As the development of a novel approach in the green synthesis of metal nanoparticles is of great importance and a necessity, a simple and safe method for the synthesis of GNPs using plant extracts of Zataria multiflora leaves was applied in this study and the results on GNPs’ anticancer activity against HeLa cells were reported. Methods: The GNPs were characterized by UV-visible spectroscopy, FTIR, TEM, DLS and Zeta-potential measurements. In addition, the cellular up-take of nanoparticles was investigated using Dark Field Microscopy (DFM). Induction of apoptosis by high dose of GNPs in HeLa cells was assessed by MTT assay, Acridin orange, DAPI staining, Annexin V/PI double-labeling flow cytometry and caspase activity assay. Results: UV-visible spectroscopy results showed a surface plasmon resonance band for GNPs at 530 nm. FTIR results demonstrated an interaction between plant extract and nanoparticles. TEM images revealed different shapes for GNPs and DLS results indicated that the GNPs range in size from 10 to 42 nm. The Zeta potential values of the synthesized GNPs were between 30 to 50 Mev, indicating the formation of stable particles. As evidenced by MTT assay, GNPs inhibit proliferation of HeLa cells in dose-dependent GNPs and cytotoxicity of GNPs in Bone Marrow Mesenchymal Stem Cell (BMSCs) was lower than cancerous cells. At nontoxic concentrations, the cellular up-take of the nanoparticles took place. Acridin orange and DAPI staining showed morphological changes in the cell’s nucleus due to apoptosis. Finally, caspase activity assay demonstrated HeLa cell’s apoptosis through caspase activation. Conclusion: The results showed that GNPs have the ability to induce apoptosis in HeLa cells. PMID:27141266

  16. Improving fluorescence diagnosis of cancer by SLIM

    Rück, Angelika; Dolp, Frank; Kinzler, Ingrid; Hauser, Carmen; Scalfi-Happ, Claudia

    2006-02-01

    Although during the last years, significant progress was made in cancer diagnosis, using either intrinsic or specially designed fluorophores, still problems exist, due to difficulties in spectral separation of highly overlapping probes or in lack of specificity. Many of the problems could be circumvented by focusing on time-resolved methods. In combination with spectral resolved detection (spectral fluorescence lifetime imaging, SLIM) highly sophisticated fluorescence lifetime imaging can be performed which might improve specificity of cell diagnosis. To record lifetime images (τ-mapping) with spectral resolution a setup was realized consisting of a laser scanning microscope equipped with a 16 channel array for time-correlated single photon counting (TCSPC) and a spectrograph in front of the array. A Ti:Saphir laser can be used for excitation or alternatively ps diode lasers. With this system the time- and spectral-resolved fluorescence characteristics of different fluorophores were investigated in solution and in cell culture. As an example, not only the mitochondria staining dye rhodamine 123 could be easily distinguished from DAPI, which intercalates into nucleic acids, but also different binding sites of DAPI. This was proved by the appearance of different lifetime components within different spectral channels. Another example is Photofrin, a photosensitizer which is approved for bladder cancer and for palliative lung and esophageal cancer in 20 countries, including the United States, Canada and many European countries. Photofrin is a complex mixture of different monomeric and aggregated porphyrins. The phototoxic efficiency during photodynamic therapy (PDT) seems to be correlated with the relative amounts of monomers and aggregates. With SLIM different lifetimes could be attributed to various, spectrally highly overlapping compounds. In addition, a detailed analysis of the autofluorescence by SLIM could explain changes of mitochondrial metabolism during

  17. Vitamins A, C, and E and selenium in the treatment of idiopathic sudden sensorineural hearing loss.

    Kaya, Hakan; Koç, Arzu Karaman; Sayın, İbrahim; Güneş, Selçuk; Altıntaş, Ahmet; Yeğin, Yakup; Kayhan, Fatma Tülin

    2015-05-01

    This study evaluated the effectiveness of vitamins A, C, and E, with selenium, in the treatment of idiopathic sudden sensorineural hearing loss (ISSNHL). This was a prospective, controlled study performed at a tertiary teaching and research hospital. Over a 32-month period, patients were treated with either our standard ISSNHL treatment regimen plus vitamins A, C, and E and selenium (ACE+ group) or with only our standard ISSNHL treatment regimen (ACE- group). The demographics, additional symptoms, mean initial and final hearing levels, mean hearing gain, and recovery data were compared between the two groups. The ACE+ group, consisting of 70 (55.5 %) patients, received vitamin A (natural beta-carotene, 26,000 IU), vitamin C (ascorbic acid, 200 mg), vitamin E (d-alpha-tocopherol, 200 IU), and selenium (50 μg) twice daily for 30 days in addition to our ISSNHL treatment regimen: methylprednisolone at an initial dose of 1 mg/kg body weight per day, tapered over 14 days; Rheomacrodex(®) [(10 g of dextran and 0.9 g of NaCl)/100 ml] 500 ml daily for 5 days; Vastarel(®) 20-mg tablet (20 mg of trimetazidine dihydrochloride) three times daily for 30 days; and ten 60-min hyperbaric oxygen (HBO) sessions (2.5 absolute atmospheres of 100 % O2), once daily, starting the day of hospitalization. The ACE- group comprised 56 (44.4 %) patients, who received only our ISSNHL treatment regimen. The mean hearing gains were 36.2 ± 20.3 dB in the ACE+ group and 27.1 ± 20.6 dB in the ACE- group. The mean hearing gain rates were significantly higher in the ACE+ group than in the ACE- group (p = 0.014). Treatment with vitamins A, C, and E and selenium was effective in ISSNHL patients undergoing treatment with methylprednisolone, dextran, trimetazidine dihydrochloride, and HBO, and might be more effective when the initial hearing level is below 46 dB. PMID:24519034

  18. Dual effect of beta-amyloid on α7 and α4β2 nicotinic receptors controlling the release of glutamate, aspartate and GABA in rat hippocampus.

    Elisa Mura

    Full Text Available BACKGROUND: We previously showed that beta-amyloid (Aβ, a peptide considered as relevant to Alzheimer's Disease, is able to act as a neuromodulator affecting neurotransmitter release in absence of evident sign of neurotoxicity in two different rat brain areas. In this paper we focused on the hippocampus, a brain area which is sensitive to Alzheimer's Disease pathology, evaluating the effect of Aβ (at different concentrations on the neurotransmitter release stimulated by the activation of pre-synaptic cholinergic nicotinic receptors (nAChRs, α4β2 and α7 subtypes. Particularly, we focused on some neurotransmitters that are usually involved in learning and memory: glutamate, aspartate and GABA. METHODOLOGY/FINDINGS: WE USED A DUAL APPROACH: in vivo experiments (microdialysis technique on freely moving rats in parallel to in vitro experiments (isolated nerve endings derived from rat hippocampus. Both in vivo and in vitro the administration of nicotine stimulated an overflow of aspartate, glutamate and GABA. This effect was greatly inhibited by the highest concentrations of Aβ considered (10 µM in vivo and 100 nM in vitro. In vivo administration of 100 nM Aβ (the lowest concentration considered potentiated the GABA overflow evoked by nicotine. All these effects were specific for Aβ and for nicotinic secretory stimuli. The in vitro administration of either choline or 5-Iodo-A-85380 dihydrochloride (α7 and α4β2 nAChRs selective agonists, respectively elicited the hippocampal release of aspartate, glutamate, and GABA. High Aβ concentrations (100 nM inhibited the overflow of all three neurotransmitters evoked by both choline and 5-Iodo-A-85380 dihydrochloride. On the contrary, low Aβ concentrations (1 nM and 100 pM selectively acted on α7 subtypes potentiating the choline-induced release of both aspartate and glutamate, but not the one of GABA. CONCLUSIONS/SIGNIFICANCE: The results reinforce the concept that Aβ has relevant

  19. Direct Determination of ECD in ECD Kit: A Solid Sample Quantitation Method for Active Pharmaceutical Ingredient in Drug Product

    Ming-Yu Chao

    2011-01-01

    Full Text Available Technetium-99m ethyl cysteinate dimer (Tc-99m-ECD is an essential imaging agent used in evaluating the regional cerebral blood flow in patients with cerebrovascular diseases. Determination of active pharmaceutical ingredient, that is, L-Cysteine, N, N′-1,2-ethanediylbis-, diethyl ester, dihydrochloride (ECD in ECD Kit is a relevant requirement for the pharmaceutical quality control in processes of mass fabrication. We here presented a direct solid sample determination method of ECD in ECD Kit without sample dissolution to avoid the rapid degradation of ECD. An elemental analyzer equipped with a nondispersive infrared detector and a calibration curve of coal standard was used for the quantitation of sulfur in ECD Kit. No significant matrix effect was found. The peak area of coal standard against the amount of sulfur was linear over the range of 0.03–0.10 mg, with a correlation coefficient ( of 0.9993. Method validation parameters were achieved to demonstrate the potential of this method.

  20. Enhanced down regulation of cortical ±-propranolol sensitive [3H]-DHA binding sites by co-administration of DMI and 5-HT1A partial agonist gepirone

    The putative interrelationship between the noradrenergic and serotonergic systems has been supported by numerous studies. Recently, Dudley et al. (1989) demonstrated significant down regulation of cortical β-adrenergic receptors by co-administration of desipramine (DMI), a norepinephrine uptake inhibitor, and the full 5-HT1A agonist 8-OH-DPAT. To this end, the effects of acute and chronic (4 and 14 day) administration of DMI, gepirone, a selective 5-HT1A post-synaptic partial agonist, as well as a combination of the two, on cortical (±)-propranolol sensitive [3H]-DHA binding sites were examined in rats. Down regulation was apparent after 4 and 14 day treatment with DMI. However, this was not the case with gepirone. Of particular importance is the demonstration of a greater magnitude of down regulation with co-administration of a greater magnitude of down regulation with co-administration of DMI and gepirone. These results suggests that alteration in rat cortical (±)-propranolol sensitive [3H]-DHA binding sites by noradrenergic uptake inhibitors can be further modulated by selective partial agonist activity at central 5-HT1A postsynaptic receptors. Further data on the co-administration of DMI and BMY 7378 (7,9-dioxo-8-[2-(4-o-methoxyphenylpiperazinyl)ethyl]-8-azaspiro[4,5]decane dihydrochloride), a weak partial agonist at postsynaptic 5-HT1A receptors, are also presented

  1. Isolation of autochthonous non-white rot fungi with potential for enzymatic upgrading of Venezuelan extra-heavy crude oil.

    Naranjo, Leopoldo; Urbina, Hector; De Sisto, Angela; Leon, Vladimir

    2007-03-01

    The increasing world demand for fuels makes it necessary to exploit the largest reserve of extra-heavy crude oil (EHCO) of the Orinoco Oil Belt from Venezuela. We propose the use of extracellular oxidative enzymes, in particular, lignin-degrading enzyme systems (LDS) of fungi, for enzymatic improvement of EHCO. Autochthonous non-white rot fungal strains able to use EHCO, and several polycyclic aromatic hydrocarbons (PAHs) as sole carbon source and energy, were isolated from EHCO-polluted soils and identified as belonging to the genera Fusarium, Penicillium , Trichoderma , Aspergillus , Neosartorya, Pseudallescheria, Cladosporium, Pestalotiopsis , Phoma and Paecillomyces. Phenotypic and biochemical assays revealed the ability of these filamentous fungi to synthesize extracellular oxidative enzymes, and suggested a relationship between the LDS and EHCO bioconversion. This work reports, for the first time, the use of o-phenylenediamine dihydrochloride (OPD) as substrate to measure extracellular ligninolytic peroxidases (ELP) in culture broths of filamentous fungi (Fusarium solani HP-1), and constitutes the first formal study of the fungal community associated with the EHCO of the Orinoco Oil Belt. PMID:18833334

  2. Ethosomes-based topical delivery system of antihistaminic drug for treatment of skin allergies.

    Goindi, Shishu; Dhatt, Bhavnita; Kaur, Amanpreet

    2014-01-01

    Cetirizine is indicated for the treatment of allergic conditions such as insect bites and stings, atopic and contact dermatitis, eczema, urticaria. This investigation deals with development of a novel ethosome-based topical formulation of cetirizine dihydrochloride for effective delivery. The optimised formulation consisting of drug, phospholipon 90 G™ and ethanol was characterised for drug content, entrapment efficiency, pH, vesicular size, spreadability and rheological behaviour. The ex vivo permeation studies through mice skin showed highest permeation flux (16.300 ± 0.300 µg/h/cm(2)) and skin retention (20.686 ± 0.517 µg/cm(2)) for cetirizine-loaded ethosomal vesicles as compared to conventional formulations. The in vivo pharmacodynamic evaluation of optimised formulation was assessed against oxazolone-induced atopic dermatitis (AD) in mice. The parameters evaluated were reduction in scratching score, erythema score, skin hyperplasia and dermal eosinophil count. Our results suggest that ethosomes are effective carriers for dermal delivery of antihistaminic drug, cetirizine, for the treatment of AD. PMID:24963956

  3. Compositional characteristics and antioxidant properties of fresh and processed sea cucumber (Cucumaria frondosa).

    Zhong, Ying; Khan, Muhammad Ahmad; Shahidi, Fereidoon

    2007-02-21

    The antioxidant activity of fresh and rehydrated sea cucumber (Cucumaria frondosa) samples with/without internal organs was evaluated for the first time. In addition, their proximate, amino acid, and fatty acid compositions were examined. Rehydrated sea cucumber samples in distilled water were prepared from oven-dried products. All samples contained 83-90% moisture, but showed a significant difference among groups in their protein and lipid contents. Glutamic acid was the predominant amino acid in sea cucumber, followed by glycine and aspartic acid. Essential amino acids such as leucine and lysine were also present at high levels. The trend for free amino acid was different from that of total amino acids and varied among groups. Lipids in sea cucumber were dominated by eicosapentaenoic acid (EPA, C20:5n-3), ranging from 43.2 to 56.7% of the total fatty acids. Docosahexaenoic acid (DHA, C22:6n-3) was present at a much lower concentration of 2.0-5.8%. All sea cucumber samples exhibited radical scavenging property against 2,2'-azobis(2-aminopropane) dihydrochloride (AAPH) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, with rehydrated samples, especially those with internal organs, possessing higher antioxidant activity than their fresh counterparts. No correlation existed between radical scavenging capacity and total phenolics content, suggesting that other components, in addition to phenolic compounds, contribute to the antioxidant activity of sea cucumber. PMID:17243707

  4. Calmodulin of the tropical sea cucumber: Gene structure, inducible expression and contribution to nitric oxide production and pathogen clearance during immune response.

    Chen, Ting; Ren, Chunhua; Li, Wuhu; Jiang, Xiao; Xia, Jianjun; Wong, Nai-Kei; Hu, Chaoqun

    2015-08-01

    Calmodulin (CaM) is an essential second messenger protein that transduces calcium signals by binding calcium ions (Ca(2+)) and modulating its interactions with various target proteins. In contrast to vertebrates, where CaM is well established as a cofactor for Ca(2+)-dependent physiological and cellular functions including host defense, there is a paucity of understanding on CaM in invertebrates (such as echinoderms) in response to immune challenge or microbial infections. In this study, we obtained and described the gene sequence of CaM from the tropical sea cucumber Stichopus monotuberculatus, a promising yet poorly characterized aquacultural species. mRNA expression of StmCaM could be detected in the intestine and coelomic fluid after Vibrio alginolyticus injection. Transcriptional and translational expression of StmCaM was inducible in nature, as evidenced by the expression patterns in primary coelomocytes following Vibrio challenge. This response could be mimicked by the Vibrio cells membrane components or lipopolysaccharides (LPS), and blocked by co-treatment of the LPS-neutralizing agent polymyxin B (PMB). Furthermore, inhibition of CaM activity by incubation with its inhibitor trifluoroperazine dihydrochloride (TFP) blunted the production of Vibrio-induced nitric oxide (NO) and augmented the survival of invading Vibrio in coelomocytes. Collectively, our study here supplied the first evidence for echinoderm CaM participation in innate immunity, and provided a functional link between CaM expression and antibacterial NO production in sea cucumber. PMID:25913576

  5. Multi properties of new schiff base and its mononuclear copper(II) complex: synthesis, characterization, liquid crystal properties and antioxidant activities

    A new Schiff base ligand, 4-(octadecyloxy)-N-(4-hexyloxyphenyl)-2-hydroxy-benzal imine, was synthesized by condensation of 4-octadecyloxy-2-hydroxybenzaldehyde with 4-hexyloxy aniline. This ligand have been characterized by UV-VIS, FTIR, H-NMR, C-NMR, mass spectra. Copper(II) complex of this ligand has been prepared by the interaction of copper(II) acetate dihydrate with ethanolic solution of the ligand. The copper(II) complex was confirmed by various spectroscopic studies like UV-VIS, FTIR and elemental analysis. The liquid crystalline behaviour of the ligand and copper(II) complex was determined by polarized light microscopy (PM) and differential scanning calorimetry (DSC). The antioxidant activities of the free Schiff base and its complex have been investigated with different tests such as 2,2-diphenyl-1-picrylhydrazyl (DPPH), superoxide, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and N,N-dimethyl-p-phenylenediamine dihydrochloride (DMPD ) radical scavenging activities. The results were compared with known antioxidants e.g. butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), trolox, -tocopherol or ascorbic acid. The ligand exhibited different levels of antioxidant activity in all tests. Therefore, some activities were pressurized in the its copper(II) complex. (author)

  6. Chromatographic assay to study the activity of multiple enzymes involved in the synthesis and metabolism of dopamine and serotonin.

    Morgan, Lindsay D; Baker, Hannah; Yeoman, Mark S; Patel, Bhavik Anil

    2012-03-21

    Serotonin and dopamine are crucial regulators of signalling in the peripheral and central nervous systems. We present an ex-vivo, isocratic chromatographic method that allows for the measurement of tyrosine, L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), tryptophan, 5-hydroxytryptophan (5-HTP), serotonin and 5-hydroxy-3-indoleacetic acid (5-HIAA) in a model central nervous (CNS) system, to study the role of key enzymes involved in the synthesis and metabolism of serotonin and dopamine. By utilising a sample splitting technique, we could test a single CNS sample at multiple time points under various pharmacological treatments. In, addition, we were able to conduct this assay by utilising the endogenous biochemical components of the CNS to study the synthesis and metabolism of serotonin and dopamine, negating the requirement of additional enzyme activators or stabilisers in the biological matrix. Finally we utilised NSD-1015, an aromatic amino acid decarboxylase enzyme inhibitor used to study the synthesis of dopamine and serotonin to monitor alterations in levels of key neurochemicals. 3-hydroxybenzylhydrazine dihydrochloride (NSD-1015) was able to reduce levels of serotonin and dopamine, whilst elevating precursors L-DOPA and 5-HTP. PMID:22290325

  7. Anti-apoptotic effects of novel phenolic antioxidant isolated from the Pacific oyster (Crassostrea gigas) on cultured human hepatocytes under oxidative stress.

    Fuda, Hirotoshi; Watanabe, Mitsugu; Hui, Shu-Ping; Joko, Sae; Okabe, Hiroaki; Jin, Shigeki; Takeda, Seiji; Miki, Emiko; Watanabe, Takayuki; Chiba, Hitoshi

    2015-06-01

    The antioxidant, and hepatoprotective properties of 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), a natural phenolic antioxidant isolated from the Pacific oyster, were defined using cultured human hepatocyte-derived cells (C3A). DHMBA showed no cytotoxicity at 62.5-500μM, as well as chlorogenic acid (CGA), vitamin C, and vitamin E. However, butylated hydroxytoluene, eicosapentaenoic acid, docosahexaenoic acid and catechin reduced cell viability. In the presence of the prooxidant 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), DHMBA at 125-500μM improved cell viability, whereas CGA had no effect. DNA ladder formation and flow-cytometric studies indicated that DHMBA inhibited AAPH-induced apoptosis and necrosis. CGA was ineffective. Thus, DHMBA is a novel, potent antioxidant, effectively protecting cultured hepatocytes from apoptosis and necrosis caused by oxidative stress. Additionally, the concentration of DHMBA was determined by mass spectrometry to be 24.4μmol/kg wet oyster meat, and three polyphenols (gentisic acid, daidzein, and matairesinol) were newly identified in the oyster extracts. PMID:25624228

  8. Inhibition of Oxidation of Aqueous Emulsions of Omega-3 Fatty Acids and Fish Oil by Phloretin and Phloridzin

    Afsana Yasmin

    2010-01-01

    Full Text Available The antioxidant properties of two apple dihydrochalcones, namely phloretin and phloridzin, were evaluated and compared with those of α-tocopherol and butylated hydroxytoluene (BHT. The effects were studied in an oil-in-water emulsion system containing methyl linolenate (ML, methyl eicosapentaenoate (MEPA, and methyl docosahexaenoate (MDHA in which oxidation was initiated by the peroxyl radical generator 2,2-azobis(2-amidinopropane dihydrochloride (AAPH and in fish oil where oxidation was initiated thermally. In the emulsion system, phloretin (1 and 5 mM completely inhibited the oxidation of ML tested as evidenced by the thiobarbituric acid reactive substances (TBARS assay. Under the same conditions, phloridzin was less effective than phloretin, but still more effective than α-tocopherol. Both phloretin and phloridzin molecules had a marginal inhibitory effect against oxidation of fish oil induced by heating at 70 °C for 3 hours, when compared to BHT. These results indicate that phloretin and phloridzin have the potential to suppress lipid oxidation in polyunsaturated fatty acid (PUFA containing foods.

  9. Facile "one-pot" synthesis of poly(methacrylic acid)-based hybrid monolith via thiol-ene click reaction for hydrophilic interaction chromatography.

    Lv, Xumei; Tan, Wangming; Chen, Ye; Chen, Yingzhuang; Ma, Ming; Chen, Bo; Yao, Shouzhuo

    2016-07-01

    A novel sol-gel "one-pot" approach in tandem with a radical-mediated thiol-ene reaction for the synthesis of a methacrylic acid-based hybrid monolith was developed. The polymerization monomers, tetramethoxysilane (TMOS) and 3-mercaptopropyl trimethoxysilane (MPTS), were hydrolyzed in high-concentration methacrylic acid solution that also served as a hydrophilic functional monomer. The resulting solution was then mixed with initiator (2, 2'-azobis (2-methylpropionamide) dihydrochloride) and porogen (urea, polyethylene glycol 20,000) in a capillary column and polymerized in water bath. The column had a uniform porous structure and a good permeability. The evaluation of the monolith was performed by separation of small molecules including nucleosides, phenols, amides, bases and Triton X-100. The calibration curves for uridine, inosine, adenosine and cytidine were determined. All the calibration curves exhibited good linear regressions (R(2)≥0.995) within the test ranges of 0.5-40μg/mL for four nucleosides. Additionaliy, atypical hydrophilic mechanism was proved by elution order from low to high according to polarity retention time increased with increases in the content of the organic solvent in the mobile phase. Further studies indicated that hydrogen bond and electrostatic interactions existed between the polar analytes and the stationary phase. This was the mechanism of retention. The excellent separation of the BSA digest showed good hydrophility of the column and indicated the potential in separation of complex biological samples. PMID:27264742

  10. Programmable flow system for automation of oxygen radical absorbance capacity assay using pyrogallol red for estimation of antioxidant reactivity.

    Ramos, Inês I; Gregório, Bruno J R; Barreiros, Luísa; Magalhães, Luís M; Tóth, Ildikó V; Reis, Salette; Lima, José L F C; Segundo, Marcela A

    2016-04-01

    An automated oxygen radical absorbance capacity (ORAC) method based on programmable flow injection analysis was developed for the assessment of antioxidant reactivity. The method relies on real time spectrophotometric monitoring (540 nm) of pyrogallol red (PGR) bleaching mediated by peroxyl radicals in the presence of antioxidant compounds within the first minute of reaction, providing information about their initial reactivity against this type of radicals. The ORAC-PGR assay under programmable flow format affords a strict control of reaction conditions namely reagent mixing, temperature and reaction timing, which are critical parameters for in situ generation of peroxyl radical from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). The influence of reagent concentrations and programmable flow conditions on reaction development was studied, with application of 37.5 µM of PGR and 125 mM of AAPH in the flow cell, guaranteeing first order kinetics towards peroxyl radicals and pseudo-zero order towards PGR. Peroxyl-scavenging reactivity of antioxidants, bioactive compounds and phenolic-rich beverages was estimated employing the proposed methodology. Recovery assays using synthetic saliva provided values of 90 ± 5% for reduced glutathione. Detection limit calculated using the standard antioxidant compound Trolox was 8 μM. RSD values were high sampling frequency (29 h(-1)), low operating costs and low generation of waste. PMID:26838448

  11. Modification of AOAC method 973.31 for determination of nitrite in cured meats.

    Mohamed, Ashraf A; Mubarak, Ahmed T; Fawy, Khaled F; El-Shahat, Mohamed F

    2008-01-01

    A modification of AOAC Method 973.31 is proposed to improve the extraction efficiency of nitrite from cured meat samples and its subsequent quantification based on the diazotization-coupling reaction of sulfanilamide with N-(1-naphthyl)ethylenediamine dihydrochloride (NED). The various experimental parameters were thoroughly investigated. A 5 g meat sample was mixed with 400 mL water; the pH of the mixture was adjusted to 5.5 +/- 0.3 and allowed to stand for 2 h on a water bath at 80 degrees C, with occasional shaking for the complete extraction of nitrite. After quantitative filtration, an aliquot was mixed with chloroacetic-chloroacetate buffer, pH 1.80 +/- 0.05, sulfanilamide, and NED, and the absorbance of the resulting azodye was recorded at 540 nm against water as a reference. Following the recommended procedure, a linear calibration graph was obtained for up to 0.8 microg/mL NO2(-), with a correlation coefficient of 0.9996 and a detection limit (based on the 3 Sb-criterion) of 5.6 ng/mL NO2(-). The proposed method was conveniently applied to various cured meat samples and was validated by comparison with the original AOAC method and by recovery experiments that gave quantitative results (94-98%) with convenient reproducibility. Statistical analysis of the analytical data could not detect any systematic error and revealed the high accuracy and precision of the proposed method. PMID:18727542

  12. Use of liposomes to evaluate the role of membrane interactions on antioxidant activity

    In this study the possibility of using liposomes as membrane mimetic systems was evaluated to estimate the antioxidant properties of oxicams and establish a relationship between the interactions of the drugs with the membrane and their consequent antioxidant activity. Different experiments were performed covering the study of the protective effect of oxicams in lipid peroxidation induced by the peroxyl radical (ROO·) derived from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and using two fluorescence probes with distinct lipophilic properties. Lipid peroxidation using the hydrophilic probe fluorescein was evaluated in lipid and aqueous media. Lipid systems labelled with the fluorescent probe diphenylhexatriene propionic acid (DPH-PA) were used to assess the effects of the drugs on membrane peroxidation simultaneously by fluorescence intensity decay and changes in membrane fluidity by steady-state anisotropy measurements. The use of different probes and liposomes as membrane mimetic systems allowed to conclude that membrane lipoperoxidation is related not only to the scavenging characteristics of the antioxidants but also to their ability to interact with the lipid bilayers

  13. Different effects of short- and long-chained fructans on large intestinal physiology and carcinogen-induced aberrant crypt foci in rats.

    Poulsen, Morten; Mølck, Anne-Marie; Jacobsen, Bodil Lund

    2002-01-01

    Inulin-type fructans, which are nondigestible carbohydrates, have been shown to modulate the number of induced preneoplastic lesions in the colon as well as the colonic microflora in laboratory animals. The present study was designed to investigate the effect of a short- and long-chained inulin-type fructan on 1,2-dimethylhydrazine dihydrochloride-induced aberrant crypt foci (ACF) in the rat colon. In addition, the present study investigated the influence of chain length, dietary level (5% or 15%), and duration of feeding (5 or 10 wk) on the following intestinal parameters supposed to be involved in the development of ACF: microflora, short-chain fatty acids, pH, and cell proliferation. A 3-wk pretreatment period with both fructans was included. Feeding the long-chained fructan (5% or 15%) significantly inhibited the numbers of small and total ACF after 5 and 10 wk. The short-chained fructan (15%) inhibited the number of small and total ACF after 5 and 10 wk but significantly increased the numbers of medium and large ACF after 10 wk. In conclusion, the effect on ACF outcome was influenced by the chain length of the fructans. PMID:12416260

  14. Development of novel fluorescent probe 3-perylene diphenylphosphine for determination of lipid hydroperoxide with fluorescent image analysis

    A novel fluorescent probe 3-perylene diphenylphosphine (3-PeDPP) was synthesized for the direct analysis of lipid hydroperoxides. The structure of 3-PeDPP was identified by the spectroscopic data, FAB-MS, 1H NMR, and 13C NMR. The reactivities of 3-PeDPP with lipid hydroperoxides were investigated in chloroform/MeOH homogeneous solutions and PC liposome model systems oxidized by either 2,2'-azobis(2-amidinopropane)dihydrochloride and photosensitized oxidation. The fluorescence intensity derived from 3-perylene diphenylphosphineoxide (3-PeDPPO) increased proportionally with amount of hydroperoxides produced in homogeneous solutions and liposome model systems. 3-PeDPP was easily incorporated into mouse myeloma SP2 cells and thin tissue section for dynamic membrane lipid peroxidation studies. Linear correlations between fluorescence intensity and amount of hydroperoxides in the cell membrane and tissue sections were obtained. The fluorescence intensity from 2-dimensional image analysis was also well correlated with lipid hydroperoxide level in these models. Thus, the novel probe 3-PeDPP is useful for the direct determination of lipid hydroperoxides in biological materials

  15. Purification and some properties of ubiquinol oxidase from obligately chemolithotrophic iron-oxidizing bacterium, Thiobacillus ferrooxidans NASF-1.

    Kamimura, K; Fujii, S; Sugio, T

    2001-01-01

    Ubiquinol-oxidizing activity was detected in an acidophilic chemolithotrophic iron-oxidizing bacterium, T. ferrooxidans. The ubiquinol oxidase was purified 79-fold from plasma membranes of T. ferrooxidans NASF-1 cells. The purified oxidase is composed of two polypeptides with apparent molecular masses of 32,600 and 50,100 Da, as measured by gel electrophoresis in the presence of sodium dodecyl sulfate. The absorption spectrum of the reduced enzyme at room temperature showed big peaks at 530 and 563, and a small broad peak at 635 nm, indicating the involvement of cytochromes b and d. Characteristic peaks of cytochromes a and c were not observed in the spectrum at around 600 and 550 nm, respectively. This enzyme combined with CO, and its CO-reduced minus reduced difference spectrum showed peaks at 409 nm and 563 nm and a trough at 431 nm. These results indicated that the oxidase contained cytochrome b, but the involvement of cytochrome d was not clear. The enzyme catalyzed the oxidations of ubiquinol-2 and reduced N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride. The ubiquinol oxidase activity was activated by the addition of albumin and lecithin to the reaction mixture and inhibited by the respiratory inhibitors KCN, HQNO, NaN3, and antimycin A1, although the enzyme was relatively resistant to KCN, and the divalent cation, Zn2+, compared with ubiquinol oxidases of E. coli. PMID:11272847

  16. Photoinitiated Polymerization of Cationic Acrylamide in Aqueous Solution: Synthesis, Characterization, and Sludge Dewatering Performance

    Huaili Zheng

    2014-01-01

    Full Text Available A copolymer of acrylamide (AM with acryloyloxyethyl trimethyl ammonium chloride (DAC as the cationic monomer was synthesized under the irradiation of high-pressure mercury lamp with 2,2-azobis(2-amidinopropane dihydrochloride (V-50 as the photoinitiator. The compositions of the photoinduced copolymer were characterized by Fourier transform infrared spectra (FTIR, ultraviolet spectra (UV, and scanning electron microscope (SEM. The effects of 6 important factors, that is, photo-initiators concentration, monomers concentration, CO(NH22 (urea concentrations, pH value, mass ratio of AM to DAC, and irradiation time on the molecular weight and dissolving time, were investigated. The optimal reaction conditions were that the photo-initiators concentration was 0.3%, monomers concentration was 30 wt.%, irradiation time was 60 min, urea concentration was 0.4%, pH value was 5.0, and mass ratio of AM to DAC was 6 : 4. Its flocculation properties were evaluated with activated sludge using jar test. The zeta potential of supernatant at different cationic monomer contents was simultaneously measured. The results demonstrated the superiority of the copolymer over the commercial polyacrylamide as a flocculant.

  17. Antioxidant status of erythrocytes and their response to oxidative challenge in humans with argemone oil poisoning

    Oxidative damage of biomolecules and antioxidant status in erythrocytes of humans from an outbreak of argemone oil (AO) poisoning in Kannauj (India) and AO intoxicated experimental animals was investigated. Erythrocytes of the dropsy patients and AO treated rats were found to be more susceptible to 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) induced peroxidative stress. Significant decrease in RBC glutathione (GSH) levels (46, 63%) with concomitant enhancement in oxidized glutathione (172, 154%) levels was noticed in patients and AO intoxicated animals. Further, depletion of glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G-6-PDH) and glutathione-S-transferase (GST) (42-52%) was observed in dropsy patients. Oxidation of erythrocyte membrane lipids and proteins was increased (120-144%) in patients and AO treated animals (112-137%) along with 8-OHdG levels in whole blood (180%) of dropsy patients. A significant reduction in α-tocopherol content (68%) was noticed in erythrocytes of dropsy patients and hepatic, plasma and RBCs of AO treated rats (59-70%) thereby indicating the diminished antioxidant potential to scavenge free radicals or the limited transport of α-tocopherol from liver to RBCs leading to enhanced oxidation of lipids and proteins in erythrocytes. These studies implicate an important role of erythrocyte degradation in production of anemia and breathlessness in epidemic dropsy

  18. Hyptis pectinata: redox protection and orofacial antinociception.

    Paixão, M S; Melo, M S; Oliveira, M G B; Santana, M T; Lima, A C B; Damascena, N P; Dias, A S; Araujo, B S; Estevam, C S; Botelho, M A; Quintans, L J

    2013-09-01

    Hyptis pectinata L. Poit, known as 'sambacaitá', is used in Brazil to treat inflammatory and painful disorders. In this study, the antioxidant and orofacial antinociceptive properties of the aqueous extract of H. pectinata leaves (AEPH) were assessed using in vitro and in vivo models. Thus, AEPH reduced the 2,2-diphenyl-1-picrylhydrazyl radical up to 72.10% with an EC₅₀ of 14.56 µg/ml. It also inhibited 40.80% of the lipoperoxidation induced by 2'-azobis (2-amidinopropane) dihydrochloride in the thiobarbituric acid-reactive substances assay. The orofacial antinociceptive activity was evaluated in mice pre-treated with AEPH (100, 200 and 400 mg/kg, p.o.) and morphine (5 mg/kg, i.p.), which received afterwards formalin- (20 µl, 2% solution, s.c.), glutamate- (40 µl, 25 mM, s.c.) and capsaicin- (20 µl, 2.5 µg, s.c.) to induce orofacial nociception. AEPH at all doses reduced (p naloxone (1.5 mg/kg, i.p.), an opioid antagonist. AEPH significantly inhibited mice face rubbing for capsaicin (23-69%, p related to opioid receptors. PMID:23132789

  19. Synthesis and structural studies of amino amide salts derived from 2-(aminomethyl)benzimidazole and α-amino acids

    Avila-Montiel, Concepción; Tapia-Benavides, Antonio R.; Falcón-León, Martha; Ariza-Castolo, Armando; Tlahuext, Hugo; Tlahuextl, Margarita

    2015-11-01

    2-{[(Ammoniumacetyl)amino]methyl}-1H-benzimidazol-3-ium dichloride 4, 2-{[(2-ammoniumpropanoyl)amino]methyl}-1H-benzimidazol-3-ium dichloride 5, and 2-{[(2-ammonium-3-phenylpropanoyl)amino]methyl}-1H-benzimidazol-3-ium dichloride 6 amino amides were synthesized via condensation of 2AMBZ dihydrochloride with the corresponding amino acid. Compounds 7-12 were obtained by replacing chloride ions (in salts 4-6) with nitrate or tetrachlorozincate ions. The results of X-ray diffraction crystallographic studies indicated that the geometries, charges and sizes of the anions are essential for the formation of the strong hydrogen bond interactions of compounds 4, 5, 9-12. Moreover, in most cases, the presence of water and solvent molecules stabilizes the supramolecular structures of these compounds. Nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy indicated that the presence of chloride or tetrachlorozincate anions increases the acidity of the benzimidazolic and amide groups more significantly than the presence of nitrate anions. However, Quantum Theory of Atoms in Molecules (QTAIM) computations of the crystal structures demonstrate that amino amides interact more strongly with NO3- than with Cl- and ZnCl42- anions; this difference explains the spectroscopic results.

  20. Activation of metabotropic glutamate receptor 7 in spinal cord inhibits pain and hyperalgesia in a novel formalin model in sheep.

    Dolan, Sharron; Gunn, Mark Donald; Crossan, Claire; Nolan, Andrea Mary

    2011-09-01

    This study set out to characterize the contribution of group III metabotropic glutamate receptor 7 activation to nociceptive behaviour and mechanical hypersensitivity in a novel formalin test in sheep. The mGlu receptor 7 allosteric agonist, N,N'-dibenzhydrylethane-1,2-diamine dihydrochloride (AMN082; 2-20 mM), the nonselective group III mGlu receptor agonist L-(+)-2-amino-4-phosphonobutyric acid (0.2-20 mM) and drug vehicle were injected intrathecally into naive subjects (n=7 per group), or 5 min preformalin (3%; 0.2 ml)/saline injection (intradermal), into the lower forelimb of adult female sheep (n=5-7 per group). Forelimb withdrawal thresholds to noxious mechanical stimulation and pain behaviours (time spent nonweight bearing or flinching) were assessed for up to 180 min. Formalin induced a characteristic biphasic pain-behaviour response and mechanical hyperalgesia between 1-5 and 30-120 min postinjection. Treatment with AMN082, but not L-(+)-2-amino-4-phosphonobutyric acid significantly inhibited both early and late phase formalin-induced hyperalgesia and pain behaviours. AMN082 also induced a rapid but short lasting analgesia in naive subjects. These data suggest that enhancing endogenous metabotropic glutamate receptor 7 activity in spinal cord, using the novel allosteric modulator, AMN082, blocks pain and hyperalgesia, and may be of therapeutic benefit for the treatment of inflammatory pain. PMID:21597362

  1. Refined carbohydrate enhancement of aberrant crypt foci (ACF) in rat colon induced by the food-borne carcinogen 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ)

    Kristiansen, E.; Meyer, Otto A.; Thorup, I.

    1996-01-01

    The aberrant crypt foci (ACF) bioassay has been used extensively to study the early effects of different dietary components on the colonic mucosa of laboratory rodents. ACF are proposed to represent preneoplastic lesions of colon cancer. Compared to the normally used initiators 1,2-dimethylhydraz......The aberrant crypt foci (ACF) bioassay has been used extensively to study the early effects of different dietary components on the colonic mucosa of laboratory rodents. ACF are proposed to represent preneoplastic lesions of colon cancer. Compared to the normally used initiators 1......,2-dimethylhydrazine dihydrochloride (DMH) and azoxymethane (AOM), the use of a diet-related colon cancer initiator, such as the heterocyclic amine 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) formed during meat cooking, would probably give a more relevant insight into diet-related colon carcinogenesis. In the...... modulated by the amount of refined carbohydrates in the diet. Rats given a high sucrose/dextrin diet showed a significantly higher number of ACF compared to rats given a diet high in starches. The effect on tumor outcome will await the termination of a ongoing parallel study....

  2. Novel Combination of Prebiotics Galacto-Oligosaccharides and Inulin-Inhibited Aberrant Crypt Foci Formation and Biomarkers of Colon Cancer in Wistar Rats.

    Qamar, Tahir Rasool; Syed, Fatima; Nasir, Muhammad; Rehman, Habib; Zahid, Muhammad Nauman; Liu, Rui Hai; Iqbal, Sanaullah

    2016-01-01

    The selectivity and beneficial effects of prebiotics are mainly dependent on composition and glycosidic linkage among monosaccharide units. This is the first study to use prebiotic galacto-oligosaccharides (GOS) that contains β-1,6 and β-1,3 glycosidic linkages and the novel combination of GOS and inulin in cancer prevention. The objective of the present study is to explore the role of novel GOS and inulin against various biomarkers of colorectal cancer (CRC) and the incidence of aberrant crypt foci (ACF) in a 1,2-dimethyl hydrazine dihydrochloride (DMH)-induced rodent model. Prebiotic treatments of combined GOS and inulin (57 mg each), as well as individual doses (GOS: 76-151 mg; inulin 114 mg), were given to DMH-treated animals for 16 weeks. Our data reveal the significant preventive effect of the GOS and inulin combination against the development of CRC. It was observed that inhibition of ACF formation (55.8%) was significantly (p ≤ 0.05) higher using the GOS and inulin combination than GOS (41.4%) and inulin (51.2%) treatments alone. This combination also rendered better results on short-chain fatty acids (SCFA) and bacterial enzymatic activities. Dose-dependent effects of prebiotic treatments were also observed on cecum and fecal bacterial enzymes and on SCFA. Thus, this study demonstrated that novel combination of GOS and inulin exhibited stronger preventive activity than their individual treatments alone, and can be a promising strategy for CRC chemoprevention. PMID:27490566

  3. Development, validation and comparison of NIR and Raman methods for the identification and assay of poor-quality oral quinine drops.

    Mbinze, J K; Sacré, P-Y; Yemoa, A; Mavar Tayey Mbay, J; Habyalimana, V; Kalenda, N; Hubert, Ph; Marini, R D; Ziemons, E

    2015-01-01

    Poor quality antimalarial drugs are one of the public's major health problems in Africa. The depth of this problem may be explained in part by the lack of effective enforcement and the lack of efficient local drug analysis laboratories. To tackle part of this issue, two spectroscopic methods with the ability to detect and to quantify quinine dihydrochloride in children's oral drops formulations were developed and validated. Raman and near infrared (NIR) spectroscopy were selected for the drug analysis due to their low cost, non-destructive and rapid characteristics. Both of the methods developed were successfully validated using the total error approach in the range of 50-150% of the target concentration (20%W/V) within the 10% acceptance limits. Samples collected on the Congolese pharmaceutical market were analyzed by both techniques to detect potentially substandard drugs. After a comparison of the analytical performance of both methods, it has been decided to implement the method based on NIR spectroscopy to perform the routine analysis of quinine oral drop samples in the Quality Control Laboratory of Drugs at the University of Kinshasa (DRC). PMID:25828509

  4. Flow injection analysis of nitrate and nitrite in commercial baby foods.

    Chetty, Adrian A; Prasad, Surendra

    2016-04-15

    Commercial baby foods are an easy alternative to home-made meals especially for working parents in a nuclear family therefore it is imperative to determine the nitrate and nitrite content in commercially available baby foods varieties marketed in Fiji. A total of 108 baby food samples were analyzed for nitrate and nitrite using our standardized flow injection analysis (FIA) technique with colorimetric detection technique employing sulfanilamide and N-(1-naphthyl)ethylenediamine dihydrochloride as color reagents where the samples throughput was 38 h(-1). The commercial baby food varieties chosen comprised of vegetables, cereals, fruits and milk. The study shows that the nitrate content of the baby foods studied ranges from 2.10 to 220.67 mg kg(-1) whereas the nitrite content ranges from 0.44 to 3.67 mg kg(-1). Typical recoveries of spiked nitrate residues ranged from 92% to 106%. The study shows that the average nitrate content of commercially available baby foods in Fiji descends below the maximum level proposed by the European Union Legislation. PMID:26616981

  5. Polyimide Nanocomposites Prepared from High-Temperature, Reduced Charge Organoclays

    Delozier, D. M.; Orwoll, R. A.; Cahoon, J. F.; Ladislaw, J. S.; Smith, J. G., Jr.; Connell, J. W.

    2003-01-01

    Montmorillonite clays modified with the dihydrochloride salt of 1,3-bis(3-aminophenoxy)benzene (APB) were used in the preparation of polyimide/organoclay hybrid films. Organoclays with varying surface charge based upon APB were prepared and examined for their dispersion behavior in the polymer matrix. High molecular weight poly(amide acid) solutions were prepared in the presence of the organoclays. Films were cast and subsequently heated to 300C to cause imidization. The resulting nanocomposite films, containing 3 wt% of organoclay, were characterized by transmission electron microscopy and X-ray diffraction. The clay's cation exchange capacity (CEC) played a key role in determining the extent of dispersion in the polyimide matrix. Considerable dispersion was observed in some of the nanocomposite films. The most effective organoclay was found to have a CEC of 0.70 meq/g. Nanocomposite films prepared with 3-8 wt% of this organoclay were characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), and thin-film tensile testing. High levels of clay dispersion could be achieved even at the higher clay loadings. Results from mechanical testing revealed that while the moduli of the nanocomposites increased with increasing clay loadings, both strength and elongation decreased.

  6. Effects of linoleic acid position in phosphatidylcholines and cholesterol addition on their rates of peroxidation in unilamellar liposomes.

    Mazari, Azzedine; Iwamoto, Satoshi; Yamauchi, Ryo

    2010-01-01

    Unilamellar liposomes of phosphatidylcholines (PCs), 1-palmitoyl-2-linoleoyl-3-sn-PC (PLPC), 1-linoleoyl-2-palmitoyl-3-sn-PC (LPPC), and a 1:1 mixture of 1,2-dilinoleoyl-3-sn-PC and 1,2-dipalmitoyl-3-sn-PC (DLPC/DPPC), were peroxidized by the addition of a water-soluble 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and of a lipid-soluble 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeOAMVN). LPPC liposomes showed the lowest oxidizability and kinetic chain-length values on AAPH-initiated peroxidation. On MeOAMVN-initiated peroxidation, PLPC liposomes with their lower peroxidation kinetic values were more stable than LPPC or DLPC/DPPC liposomes. The incorporation of cholesterol into the liposomes induced dose-dependent inhibition of PLPC and of LPPC peroxidation, while its effect was less important for the DLPC/DPPC liposomes. Our results indicate that the sn-position of unsaturated acyl chains and the cholesterol content are important modulating factors in the oxidizability of membrane phospholipids. PMID:20460733

  7. Co-occurrence of the cyanotoxins BMAA, DABA and anatoxin-a in Nebraska reservoirs, fish, and aquatic plants.

    Al-Sammak, Maitham Ahmed; Hoagland, Kyle D; Cassada, David; Snow, Daniel D

    2014-02-01

    Several groups of microorganisms are capable of producing toxins in aquatic environments. Cyanobacteria are prevalent blue green algae in freshwater systems, and many species produce cyanotoxins which include a variety of chemical irritants, hepatotoxins and neurotoxins. Production and occurrence of potent neurotoxic cyanotoxins β-N-methylamino-L-alanine (BMAA), 2,4-diaminobutyric acid dihydrochloride (DABA), and anatoxin-a are especially critical with environmental implications to public and animal health. Biomagnification, though not well understood in aquatic systems, is potentially relevant to both human and animal health effects. Because little is known regarding their presence in fresh water, we investigated the occurrence and potential for bioaccumulation of cyanotoxins in several Nebraska reservoirs. Collection and analysis of 387 environmental and biological samples (water, fish, and aquatic plant) provided a snapshot of their occurrence. A sensitive detection method was developed using solid phase extraction (SPE) in combination with high pressure liquid chromatography-fluorescence detection (HPLC/FD) with confirmation by liquid chromatography-tandem mass spectrometry (LC/MS/MS). HPLC/FD detection limits ranged from 5 to 7 µg/L and LC/MS/MS detection limits were BMAA in about 18.1%, DABA in 17.1%, and anatoxin-a in 11.9%. PMID:24476710

  8. Fabrication and characteristics of microcantilever-based biosensor for detection of the protein-ligand binding

    This paper describes a proposal for a microcantilever-based biosensor that can be used in investigating the adsorption characteristics of protein-ligand binding on a silicon nitride/gold coated surface. We have detected streptavidin-ligand binding using this microcantilever detection system. The microcantilevers can be mass-produced by a conventional surface micromachining technique. This technique has advantages of cost efficiency, simplicity, and the ability to be fabricated in an array. A transparent fluid cell system, where a gold coated microcantilever was mounted for the injection of bio-molecular solution, was fabricated using polydimethylsiloxane (PDMS) and fused silica glass. The microcantilever was deflected as a result of the difference of surface stress caused by the formation of the self-assembly monolayers (SAMs) of biomolecules on the gold coated side of the microcantilever. The sequential specific interactions of cystamine dihydrochloride/ glutaraldehyde/streptavidin were detected by both optical and electrical methods. We confirmed that the deflections were induced by biomolecular adsorption on the gold coated microcantilever. This study proved to be applicable to real-time monitoring of biological interactions such as specific DNA sequences, proteins, and so on

  9. Kinetic-spectrophotometric determination of molybdenum(VI) and tungsten(VI) in mixtures

    A simple kinetic-spectrophotometric method is described for the determination of molybdenum(VI) and tungsten(VI) in mixtures, without prior separation. The method is based on the catalytic effect of molybdenum(VI) and tungsten(VI) on the 2,4-diaminophenol dihydrochloride (DAP) by hydrogen peroxide in acidic medium. The reaction was followed spectrophotometrically by measuring the rate of change in absorbance with time at 500 nm. A partial inhibition in the catalytic activity of each catalyst, when the other one is present, at all ratios of Mo(VI):W(VI) mixtures studied was observed. On the other hand, the catalytic activity of tungsten(VI) dropped to zero whilst that of molybdenum(VI) decreased slightly, in the presence of citrate ions. Two sets of experiments were carried out, the first in the absence and the other in the presence of citrate, and the resolution of Mo(VI)/W(VI) mixtures was achieved by solving two simultaneous equations. Various molar ratios of Mo(VI):W(VI), at the 10-6 M level, from 0.2:1 to 5:1 can be determined with satisfactory precision and accuracy. The selectivity of the method was investigated and the method was applied successfully to the determination of molybdenum and tungsten in each other's presence in steel. (authors)

  10. Development of cancer-targeting radionanoparticles

    Here we report the first formulation of porphyrin coated albumin nanoparticles that are coated by porphyrin which is known to be cancer-tropic agent and used for photodynamic therapy for the cancer. Albumin nanoparticles were prepared by solvent extraction method. Mean particle size of the prepares nanoparticles was dependent on the addition rate of ethanol and ranged from 100 to 200 nm. Hematoporphyrin IX dihydrochloride (HP) were chosen for surface modification of albumin nanoparticle. The amino residue of surface albumin nanoparticles (ep, from lysine) was linked with carboxyl group of HP. HP coated albumin nanoparticles were purified by ultracentrifugation and characterized by modulated differential calorimetry (MDSC), x-ray diffractometry, field-emission scanning electron microsope (FE-SEM), transmission electron microsopope (TEM). HP modified albumin nanoparticles showed a good chelation efficiency (more that 95 %) against 99mTc, being applicable to cancer targeting radio-nanoparticles. The uptake of HP modified albumin nanoparticles in CT-26 and A549 cancer cell line was greater than that of normal albumin anaopraticles, proving that HP modified albumin nanoparticles is valuable for the cancer targeting radio isotope nanoparticles

  11. Development of cancer-targeting radionanoparticles

    Sim, C. G.; Yang, S. G.; Jin, H. E. [Seoul National University, Seoul (Korea, Republic of)

    2007-06-15

    Here we report the first formulation of porphyrin coated albumin nanoparticles that are coated by porphyrin which is known to be cancer-tropic agent and used for photodynamic therapy for the cancer. Albumin nanoparticles were prepared by solvent extraction method. Mean particle size of the prepares nanoparticles was dependent on the addition rate of ethanol and ranged from 100 to 200 nm. Hematoporphyrin IX dihydrochloride (HP) were chosen for surface modification of albumin nanoparticle. The amino residue of surface albumin nanoparticles (ep, from lysine) was linked with carboxyl group of HP. HP coated albumin nanoparticles were purified by ultracentrifugation and characterized by modulated differential calorimetry (MDSC), x-ray diffractometry, field-emission scanning electron microsope (FE-SEM), transmission electron microsopope (TEM). HP modified albumin nanoparticles showed a good chelation efficiency (more that 95 %) against {sup 99m}Tc, being applicable to cancer targeting radio-nanoparticles. The uptake of HP modified albumin nanoparticles in CT-26 and A549 cancer cell line was greater than that of normal albumin anaopraticles, proving that HP modified albumin nanoparticles is valuable for the cancer targeting radio isotope nanoparticles.

  12. [Wound closure after irrigation with Octenisept® without possibility for drainage].

    Högele, A M; Neu, J

    2011-01-01

    A 39-year-old patient suffered a stab wound of the right thenar prominence after an accident with a screwdriver. In the first hospital the deep wound was irrigated with octenidine dihydrochloride/2-phenoxyethanol and closed by suture. During the further course pressure pain and numbness of the right thenar and swelling of the right hand occurred. Three weeks after the accident an operative revision of the wound in a second hospital was performed. The intraoperative findings showed inflammation and necrosis of the right m. abductor pollicis brevis, but no infection with pus.The patient accused the first hospital of irrigating the tissue of his right hand with Octenisept®. The expert option of the Arbitration Board identified improper care in the first hospital with insufficient excision of the wound and incorrect use of the Octenisept® solution. Against the explicit advice of the manufacturing company the wound had been sutured without the possibility of drainage for the Octenisept® solution. PMID:21229225

  13. Importance of Porins for Biocide Efficacy against Mycobacterium smegmatis▿

    Frenzel, Elrike; Schmidt, Stefan; Niederweis, Michael; Steinhauer, Katrin

    2011-01-01

    Mycobacteria are among the microorganisms least susceptible to biocides but cause devastating diseases, such as tuberculosis, and increasingly opportunistic infections. The exceptional resistance of mycobacteria to toxic solutes is due to an unusual outer membrane, which acts as an efficient permeability barrier, in synergy with other resistance mechanisms. Porins are channel-forming proteins in the outer membrane of mycobacteria. In this study we used the alamarBlue assay to show that the deletion of Msp porins in isogenic mutants increased the resistance of Mycobacterium smegmatis to isothiazolinones (methylchloroisothiazolinone [MCI]/methylisothiazolinone [MI] and octylisothiazolinone [2-n-octyl-4-isothiazolin-3-one; OIT]), formaldehyde-releasing biocides {hexahydrotriazine [1,3,5-tris (2-hydroxyethyl)-hexahydrotriazine; HHT] and methylenbisoxazolidine [N,N′-methylene-bis-5-(methyloxazolidine); MBO]}, and the lipophilic biocides polyhexamethylene biguanide and octenidine dihydrochloride 2- to 16-fold. Furthermore, the susceptibility of the porin triple mutant against a complex disinfectant was decreased 8-fold compared to wild-type (wt) M. smegmatis. Efficacy testing in the quantitative suspension test EN 14348 revealed 100-fold improved survival of the porin mutant in the presence of this biocide. These findings underline the importance of porins for the susceptibility of M. smegmatis to biocides. PMID:21398489

  14. Importance of porins for biocide efficacy against Mycobacterium smegmatis.

    Frenzel, Elrike; Schmidt, Stefan; Niederweis, Michael; Steinhauer, Katrin

    2011-05-01

    Mycobacteria are among the microorganisms least susceptible to biocides but cause devastating diseases, such as tuberculosis, and increasingly opportunistic infections. The exceptional resistance of mycobacteria to toxic solutes is due to an unusual outer membrane, which acts as an efficient permeability barrier, in synergy with other resistance mechanisms. Porins are channel-forming proteins in the outer membrane of mycobacteria. In this study we used the alamarBlue assay to show that the deletion of Msp porins in isogenic mutants increased the resistance of Mycobacterium smegmatis to isothiazolinones (methylchloroisothiazolinone [MCI]/methylisothiazolinone [MI] and octylisothiazolinone [2-n-octyl-4-isothiazolin-3-one; OIT]), formaldehyde-releasing biocides {hexahydrotriazine [1,3,5-tris (2-hydroxyethyl)-hexahydrotriazine; HHT] and methylenbisoxazolidine [N,N'-methylene-bis-5-(methyloxazolidine); MBO]}, and the lipophilic biocides polyhexamethylene biguanide and octenidine dihydrochloride 2- to 16-fold. Furthermore, the susceptibility of the porin triple mutant against a complex disinfectant was decreased 8-fold compared to wild-type (wt) M. smegmatis. Efficacy testing in the quantitative suspension test EN 14348 revealed 100-fold improved survival of the porin mutant in the presence of this biocide. These findings underline the importance of porins for the susceptibility of M. smegmatis to biocides. PMID:21398489

  15. Oxidative stress and alterations in actin cytoskeleton trigger glutathione efflux in Saccharomyces cerevisiae.

    Bradamante, Silvia; Villa, Alessandro; Versari, Silvia; Barenghi, Livia; Orlandi, Ivan; Vai, Marina

    2010-12-01

    A marked deficiency in glutathione (GSH), the most abundant antioxidant in living systems, plays a major role in aging and the pathogenesis of diseases ranging from neurological disorders to early atherosclerosis and the impairment of various immunological functions. In an attempt to shed light on GSH homeostasis, we carried out the space experiment SCORE (Saccharomyces cerevisiae oxidative stress response evaluation) during the FOTON-M3 mission. Microgravity and hyperoxic conditions induced an enormous extracellular release of GSH from S. cerevisiae cells (≈40% w/dw), changed the distribution of the buds, and activated the high osmolarity glycerol (HOG) and cell integrity/PKC pathways, as well as protein carbonylation. The results from the single spaceflight experiment were validated by a complete set of experiments under conditions of simulated microgravity and indicate that cytoskeletal alterations are mainly responsible for the observed effects. The results of ground experiments in which we induced cytoskeletal modifications by means of treatment with dihydrocytochalasin B (DHCB), a potent inhibitor of actin polymerisation, or (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632), a selective ROCK (Rho-associated coiled-coil forming protein serine/threonine kinase) inhibitor, confirmed the role of actin in GSH efflux. We also found that the GSH release can be inhibited using the potent chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). PMID:20708643

  16. Modifications of histamine receptor signaling affect bone mechanical properties in rats.

    Folwarczna, Joanna; Janas, Aleksandra; Pytlik, Maria; Śliwiński, Leszek; Wiercigroch, Marek; Brzęczek, Anna

    2014-02-01

    Histamine receptors are expressed on bone cells and histamine may be involved in regulation of bone metabolism. The aim of the present study was to investigate the effects of loratadine (an H(1) receptor antagonist), ranitidine (an H(2) receptor antagonist) and betahistine (an H(3) receptor antagonist and H(1) receptor agonist) on bone mechanical properties in rats. Loratadine (5 mg/kg/day, po), ranitidine (50 mg/kg/day, po), or betahistine dihydrochloride (5 mg/kg/day, po), were administered for 4 weeks to non-ovariectomized and bilaterally ovariectomized (estrogen-deficient) 3-month-old rats, and their effects were compared with appropriate controls. Serum levels of bone turnover markers, bone mineralization and mechanical properties of the proximal tibial metaphysis, femoral diaphysis and femoral neck were studied. In rats with normal estrogen level, administration of loratadine slightly favorably affected mechanical properties of compact bone, significantly increasing the strength of the femoral neck (p < 0.05), and tending to increase the strength of the femoral diaphysis. Ranitidine did not significantly affect the investigated parameters, and betahistine decreased the strength of the tibial metaphysis (cancellous bone, p < 0.01). There were no significant effects of the drugs on serum bone turnover markers. In estrogen-deficient rats, the drugs did not significantly affect the investigated skeletal parameters. In conclusion, the effects of histamine H(1), H(2) and H(3) receptor antagonists on the skeletal system in rats were differential and dependent on estrogen status. PMID:24905313

  17. A regioselective synthesis of some new pyrazol-1'-ylpyrazolo[1,5-a]pyrimidines in aqueous medium and their evaluation as antimicrobial agents.

    Aggarwal, Ranjana; Sumran, Garima; Garg, Neelam; Aggarwal, Ashok

    2011-07-01

    An efficient and environmental benign regioselective synthesis of some new pyrazol-1'-ylpyrazolo[1,5-a]pyrimidines (7b-h) has been accomplished via treatment of 3(5)-amino-5(3)-hydrazinopyrazole dihydrochloride (5) with several unsymmetrical 1,3-diketones (6b-h) using water as a solvent without any catalysts or additives. The structure of 7b-h was established on the basis of rigorous analysis of (1)H, (13)C NMR, IR spectral data and MS. Eight compounds (7a-h) were screened for their antibacterial activity against two gram-positive and two gram-negative bacteria and compounds (7a, b, d and e) for antifungal activity against four phytopathogenic fungi. Compounds 7c and 7e manifest rather broad antibacterial activity than standard antibiotics. One lead compound, 7a (10mg/ml and 200mg/ml) exhibited equipotent or more potent antifungal activity against all tested microorganisms than standard drug. PMID:21558044

  18. Arginase 2 deficiency reduces hyperoxia-mediated retinal neurodegeneration through the regulation of polyamine metabolism.

    Narayanan, S P; Xu, Z; Putluri, N; Sreekumar, A; Lemtalsi, T; Caldwell, R W; Caldwell, R B

    2014-01-01

    Hyperoxia treatment has been known to induce neuronal and glial death in the developing central nervous system. Retinopathy of prematurity (ROP) is a devastating disease in premature infants and a major cause of childhood vision impairment. Studies indicate that, in addition to vascular injury, retinal neurons are also affected in ROP. Using an oxygen-induced retinopathy (OIR) mouse model for ROP, we have previously shown that deletion of the arginase 2 (A2) significantly reduced neuro-glial injury and improved retinal function. In the current study, we investigated the mechanism of A2 deficiency-mediated neuroprotection in the OIR retina. Hyperoxia treatment has been known to induce neuronal death in neonates. During the hyperoxia phase of OIR, a significant increase in the number of apoptotic cells was observed in the wild-type (WT) OIR retina compared with A2-deficient OIR. Mass spectrometric analysis showed alterations in polyamine metabolism in WT OIR retina. Further, increased expression level of spermine oxidase was observed in WT OIR retina, suggesting increased oxidation of polyamines in OIR retina. These changes were minimal in A2-deficient OIR retina. Treatment using the polyamine oxidase inhibitor, N, N'-bis (2, 3-butadienyl)-1, 4-butanediamine dihydrochloride, significantly improved neuronal survival during OIR treatment. Our data suggest that retinal arginase is involved in the hyperoxia-induced neuronal degeneration in the OIR model, through the regulation of polyamine metabolism. PMID:24556690

  19. Reductive spectrophotometry of divalent tin sensitization on soda lime glass

    Bejugam, Vinith; Wei, Xingfei; Roper, D. Keith

    2016-07-01

    Rapid and facile evaluation of tin (II) sensitization could lead to improved understanding of metal deposition in electroless (EL) plating. This report used a balanced redox reaction between 3,3‧,5,5‧-tetramethylbenzidine dihydrochloride (TMB-HCL) and N-bromosuccinimide (NBS) to evaluate effects of sensitization conditions (i.e., sensitization time, analyte concentration, aqueous immersion, and acid content) on the accumulated mass of surface-associated divalent tin ion. The accumulated mass of tin (II) increased as the sensitization time increased up to 30 s in proportion to aqueous tin (II) chloride concentrations between 2.6 and 26 mM at a trifluoroacetic acid (TFA) content of 68 mM. The average mass peaked at 7.3 nanomoles (nmol) per cm2 after a 5 s aqueous immersion post-sensitization, and then decreased with increasing aqueous immersion post-sensitization. The total average tin (II) + tin (IV) accumulated on soda lime glass measured by inductively coupled plasma optical emission spectrometry (ICP-OES) was 17% higher at 30 s sensitization, suggesting a fraction of the tin (II) present may have oxidized to tin (IV). These results indicated that in situ spectrophotometric evaluation of tin (II) could support development of EL plating for electronics, catalysis, and solar cells.

  20. SYNTHESIS AND PROPERTIES OF POLY-1,3,5-TRIAZINES

    WANG Yulan; LU Fengcai

    1984-01-01

    Two new polytriazines: poly[2-methyl-4, 6-(4,' 4"-diphenylene)-1, 3, 5-triazine] (Ⅰ) and poly[2-phenyl-4, 6-(4', 4"-diphenylene)-1, 3, 5-triazine] (Ⅱ) were synthesized from the solution condensation of biphenyl-4, 4'-diamidine dihydrochloride with acetic anhydride and biphenyl-4, 4'-diamidine with benzaldehyde respectively. These two polymers were characterized by TGA, DTA, elemental analysis and IR spectroscopy. They exhibited good thermal oxidative stability as shown by the fact that the powders of these polymers suffered 5.4 %, 2.4 % weight loss after isothermal aging in air at 300℃ for 200 hours. The decomposition temperature of (Ⅱ) was 583℃ in air and 590℃ in N2. These linear poly-1, 3, 5-triazines were soluble in concentrated sulfuric acid, phosphoric acid and trifluoroacetic acid whereas the crosslinked poly-1, 3, 5-triazines reported in the literature were insoluble and infusible.It is interesting that these polymers can form complexes with metal halides as determined by X-ray photoelectron spectroscopy (XPS). The polymer metal complex (Ⅲ). PdCl2 possesses catalytic activity for hydrogenation.

  1. Natural phenylpropanoids protect endothelial cells against oxidized LDL-induced cytotoxicity.

    Martin-Nizard, Françoise; Sahpaz, Sevser; Furman, Christophe; Fruchart, Jean-Charles; Duriez, Patrick; Bailleul, François

    2003-03-01

    There is increasing evidence that oxidized low-density lipoproteins (Ox-LDL) might be involved in the pathogenesis of atherosclerosis and it has been reported that polyphenols inhibit LDL peroxidation and atherosclerosis. Minimally oxidized LDL (mOx-LDL) induce cytotoxicity in cultured bovine aortic endothelial cells (BAEC). The goal of this study was to test the protective effect of five natural polyphenols isolated from the aerial parts of Marrubium vulgare L. against mOx-LDL-induced cytotoxicity in BAEC. Four phenylpropanoid glycosides (acteoside 1, forsythoside B 2, arenarioside 3, ballotetroside 4) and one non-glycosidic derivative (caffeoyl-l-malic acid 5) were tested. These compounds inhibited both copper (Cu 2+)- and 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced in vitro LDL oxidation and preserved the morphological aspects of BAEC during their incubation with mOx-LDL. Furthermore, they reduced the accumulation of aldehydes in the cultured medium during the incubation of BAEC with mOx-LDL and prevented cellular LDH leakage during this period. These data suggest that natural phenylpropanoids inhibit mOx-LDL-induced cellular toxicity and that inhibition of lipid peroxidation could be a key mechanism in the cytoprotective effect of these molecules. PMID:12677522

  2. Cd(II) complexes with different nuclearity and dimensionality based on 3-hydrazino-4-amino-1,2,4-triazole

    Xu, Cai-Xia; Zhang, Jian-Guo; Yin, Xin; Jin, Xin; Li, Tong; Zhang, Tong-Lai; Zhou, Zun-Ning

    2015-03-01

    A series of zero- to two-dimensional Cd(II) coordination compounds have been synthesized by the reaction of Cd(II) salts and 3-hydrazino-4-amino-1,2,4-triazole di-hydrochloride (HATr·2HCl). [CdCl2(HATr)2] (1) and [Cd2Cl4(HATr)2(H2O)2] (2) have discrete mononuclear and binuclear structures, respectively. [Cd(HATr)2(ClO4)2]n (3) presents polymeric 1-D chain and [Cd2(NO3)2Cl2(HATr)2]n (4) shows 2-D frameworks. All Cd(II) ions exhibit distorted octahedral configurations in 1-3, whilst both hexa and heptacoordinated Cd(II) are formed in 4. The HATr ligands adopt chelating coordinated mode in 1, while tri-dentate bridging-chelating mode in 2-4. The chloride ion is a mono-coordinated ligand in 1 and 2, but it bridges two adjacent metal ions in 4. Furthermore, thermal behaviors have been investigated and the results reveal that all complexes have good thermal stability. The impact sensitivity test indicates that complex 3 is sensitive to impact stimuli.

  3. Antioxidant Activity and Acetylcholinesterase Inhibition of Grape Skin Anthocyanin (GSA

    Mehnaz Pervin

    2014-07-01

    Full Text Available We aimed to investigate the antioxidant and acetylcholinesterase inhibitory activities of the anthocyanin rich extract of grape skin. Grape skin anthocyanin (GSA neutralized free radicals in different test systems, such as 2,-2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS and 2,2-diphenyl-1-picrylhydrazyl (DPPH assays, to form complexes with Fe2+ preventing 2,2'-azobis(2-amidinopropane dihydrochloride (AAPH-induced erythrocyte hemolysis and oxidative DNA damage. Moreover, GSA decreased reactive oxygen species (ROS generation in isolated mitochondria thus inhibiting 2',-7'-dichlorofluorescin (DCFH oxidation. In an in vivo study, female BALB/c mice were administered GSA, at 12.5, 25, and 50 mg per kg per day orally for 30 consecutive days. Herein, we demonstrate that GSA administration significantly elevated the level of antioxidant enzymes in mice sera, livers, and brains. Furthermore, GSA inhibited acetylcholinesterase (AChE in the in vitro assay with an IC50 value of 363.61 µg/mL. Therefore, GSA could be an excellent source of antioxidants and its inhibition of cholinesterase is of interest with regard to neurodegenerative disorders such as Alzheimer’s disease.

  4. Kinetic and Mechanistic Studies of Oxidation of an Antiallergic Drug with Bromamine-T in Acid and Alkaline Media

    Cetrizine dihydrochloride (CTZH) is widely used as an anti-allergic drug. Sodium N-bromo-p-toluenesulfonamide or bromamine-T (BAT) is the bromine analogue of chloramine-T (CAT) and is found to be a better oxidizing agent than CAT. In the present research, the kinetics of oxidation of CTZH with BAT in acid and alkaline media was studied at 313 K. The experimental rate laws obtained are: -d[BAT]/dt = k[BAT] [CTZH]0.80[H+]-0.48 in acid medium and -d[BAT]/dt = k[BAT][CTZH]0.48[OH-]0.52[PTS]-0.40 in alkaline medium where PTS is p-toluenesulfonamide. Activation parameters and reaction constants were evaluated. The solvent isotope effect was studied using D2O. The dielectric effect is positive. The stoichiometry of the reaction was found to be 1:1 and the oxidation products were identified as 4-chlorobenzophenone and (2-piperazin-1-yl-ethoxy)-acetic acid in both media. The rate of oxidation of CTZH is faster in acid medium. Suitable mechanisms and related rate laws have been worked out

  5. Optimized and validated spectrophotometric methods for the determination of hydroxyzine hydrochloride in pharmaceuticals and urine using iodine and picric acid

    Rajendraprasad Nagaraju

    2011-01-01

    Full Text Available Two simple, rapid, cost-effective and sensitive spectrophotometric procedures are proposed for the determination of hydroxyzine dihydrochloride (HDH in pharmaceuticals and in spiked human urine. The methods are based on the charge transfer complexation reaction of the drug with either iodine (I2 as a σ-acceptor (method A in dichloromethane or picric acid (PA as a π-acceptor (method B in chloroform. The coloured products exhibit absorption maxima at 380 and 400 nm for I2 and PA, respectively. The Beer Law was obeyed over the concentration ranges of 1.25-15 and 3.75-45 μg mL-1 for method A and method B, respectively. The molar absorptivity values, Sandell sensitivities, limits of detection (LOD and quantification (LOQ are reported. The accuracy and precision of the methods were evaluated on intra-day and inter-day basis. The proposed methods were successfully applied for the determination of HDH in tablets and spiked human urine.

  6. Development and Validation of Spectrophotometric Methods for Estimating Sulfamethoxazole in Pharmaceutical Preparations

    Sangita Sharma

    2010-07-01

    Full Text Available Four simple, sensitive, accurate, and rapid visible spectrophotometric methods (A, B, C and D have been developed for the estimation of sulfamethoxazole in pharmaceutical Preparation. They are based on the diazotization of sulfamethoxazole with sodium nitrite and hydrochloric acid followed by coupling with N-(1-naphthyl ethylenediamine dihydrochloride (Method A to form pink coloured chromogen, diphenylamine (Method B to form pink coloured chromogen, β-napthol (in alkaline medium (Method C to form a orange yellow coloured chromogen and Resorcinol (in alkaline medium (Method D to form orange red coloured chromogen and exhibiting absorption maxima λmax at 536 nm, 516 nm, 477 nm and 502 nm respectively. The coloured chromogens formed are stable for more than 2 h. Beer's law was obeyed in the concentration range of 4 -12 µg mL-1 in method A , 2 – 10 µg mL-1 in method B, 5 – 25 µg mL-1 in method C and 1 – 5 µg mL-1 in method D respectively. The Results of the four analysis have been validated statistically and by recovery studies. The results obtained in the proposed methods are in good agreements with labeled amounts, when marketed pharmaceutical preparations are analyzed.

  7. Water-compatible dummy molecularly imprinted resin prepared in aqueous solution for green miniaturized solid-phase extraction of plant growth regulators.

    Wang, Mingyu; Chang, Xiaochen; Wu, Xingyu; Yan, Hongyuan; Qiao, Fengxia

    2016-08-01

    A water-compatible dummy molecularly imprinted resin (MIR) was synthesized in water using melamine, urea, and formaldehyde as hydrophilic monomers of co-polycondensation. A triblock copolymer (PEO-PPO-PEO, P123) was used as porogen to dredge the network structure of MIR, and N-(1-naphthyl) ethylenediamine dihydrochloride, which has similar shape and size to the target analytes, was the dummy template of molecular imprinting. The obtained MIR was used as the adsorbent in a green miniaturized solid-phase extraction (MIR⬜mini-SPE) of plant growth regulators, and there was no organic solvent used in the entire MIR⬜mini-SPE procedure. The calibration linearity of MIR⬜mini-SPE⬜HPLC method was obtained in a range 5⬜250ngmL(↙1) for IAA, IPA, IBA, and NAA with correlation coefficient (r) Ⱕ0.9998. Recoveries at three spike levels are in the range of 87.6⬜100.0% for coconut juice with relative standard deviations Ⱔ8.1%. The MIR⬜mini-SPE method possesses the advantages of environmental friendliness, simple operation, and high efficiency, so it is potential to apply the green pretreatment strategy to extraction of trace analytes in aqueous samples. PMID:27378249

  8. Screen Printed Carbon Electrode Based Electrochemical Immunosensor for the Detection of Dengue NS1 Antigen

    Om Parkash

    2014-11-01

    Full Text Available An electrochemical immunosensor modified with the streptavidin/biotin system on screen printed carbon electrodes (SPCEs for the detection of the dengue NS1 antigen was developed in this study. Monoclonal anti-NS1 capture antibody was immobilized on streptavidin-modified SPCEs to increase the sensitivity of the assay. Subsequently, a direct sandwich enzyme linked immunosorbent assay (ELISA format was developed and optimized. An anti-NS1 detection antibody conjugated with horseradish peroxidase enzyme (HRP and 3,3,5,5'-tetramethybezidine dihydrochloride (TMB/H2O2 was used as an enzyme mediator. Electrochemical detection was conducted using the chronoamperometric technique, and electrochemical responses were generated at −200 mV reduction potential. The calibration curve of the immunosensor showed a linear response between 0.5 µg/mL and 2 µg/mL and a detection limit of 0.03 µg/mL. Incorporation of a streptavidin/biotin system resulted in a well-oriented antibody immobilization of the capture antibody and consequently enhanced the sensitivity of the assay. In conclusion, this immunosensor is a promising technology for the rapid and convenient detection of acute dengue infection in real serum samples.

  9. Requirement of mitoses for the reversal of X-inactivation in cell hybrids between murine embryonal carcinoma cells and normal female thymocytes

    By means of a 5-bromodeoxyuridine (BrdU) incorporation and acridine orange fluorescence staining method the authors studied reactivation of the inactivated X chromosome (Xi) in newly formed cell hybrids between the near-diploid HPRT-deficient OTF9-63 murine embryonal carcinoma cell (ECC) with an XO sex chromosome constitution and the normal female mouse thymocyte. Synchronization of the late replicating S chromosome in such hybrid cells, indicative of reactivation, was found for the first time on Day 3, and the frequency of reactivation was attained 90% on Day 5. Inhibition of cell cycle progression either by methylglyoxal bis(guanylhydrazone) dihydrochloride, an inhibitor of polyamine metabolism, or by isoleucine-deficient medium after cell fusion delayed reactivation of the Xi, which implied that the number of cell division cycles traversed by individual cells rather than the length of time after cell fusion is critical for the reactivation. Double-labeling experiments using [3H]thymidine and BrdU indicated that hybrid cells had undergone three or four mitoses before reactivation of the Xi. Most probably reactivation of the Xi is consequent to reversion of the thymocyte genome to an undifferentiated state under the influence of OTF9 genome. DNA demethylation or dilution of Xi-specific factors by mitoses may be involved in this process

  10. Experimental antimelanoma agents: The synthesis of 4-S-cysteaminyl-[U-14C]phenol

    The detection and treatment of malignant melanomas remains a major challenge in oncology. The use of biochemical intermediates in this research has led to an interest in 4-S-cysteaminyl phenol. The title compound, an inhibitor of tyrosinase which localizes in melanotic melanomas, has now been radiolabelled via the reaction of [U-14C]phenol with cystamine dihydrochloride in 47% HBr, to afford 4-S-cysteaminyl-[U-14C]phenol (4-S-[U-14C]CAP) in 63,2% radiochemical yield. The recovered product had a specific activity of 12.4 mCi/mmol, with radiochemical purity greater than 98%. A small quantity of the structural isomer, 2-S-cysteaminyl-[U-14C]phenol, with identical specific activity, was recovered in 4% radiochemical yield. 4-S-[U-14]CAP will be used to delineate the biodistribution of this agent in experimental models of melanoma, using whole-body and microscopic autoradiography techniques. (orig.)

  11. Effects of the antituberculous drug ethambutol on zinc balance, distribution, and turnover: short-term studies modeling chronic toxicity

    King, A.B.

    1987-01-01

    Alterations in Zn metabolism have been reported in both tuberculous patients and experimental animals receiving ethambutol (d-2-2'-ethylenediimino-di-1-butanol dihydrochloride) (EMB), and these changes have been associated with ocular side effects of EMB. EMB has chelating properties but is not likely to chelate Zn at physiologic pH. However, its acid metabolite is a stronger chelator. This research addressed whether EMB affects the absorption and disposition of dietary Zn, and whether effects of EMB on Zn are modified by (a) marginal Zn intake of (b) drugs that may induce metabolism of EMB. Weanling male Sprague-Dawley rats fed an AIN-76A diet received daily by gavage either deionized water or EMB doses of 400-1600 mg/kg bw. in a preliminary, 15-day dose-response study and 400-600 mg/kg in three subsequent 15- to 30-day studies. Apparent absorption and biological turnover of Zn were measured by /sup 65/Zn balance and retention in rats fed adequate (49 ppm) or marginal (11 ppm) Zn. Effects of EMB were similar in both dietary groups. EMB treatment produced alopecia and reduced feed intake, feed efficiency, weight gain, and serum Zn, but showed no effect on hepatic, renal, or femoral Zn concentrations. Absorption, turnover, and urinary excretion of Zn were increased in rats fed EMB.

  12. Effects of Crataegus microphylla on vascular dysfunction in streptozotocin-induced diabetic rats.

    Topal, Gökçe; Koç, Ebru; Karaca, Cetin; Altuğ, Tuncay; Ergin, Bülent; Demirci, Cihan; Melikoğlu, Gülay; Meriçli, Ali H; Kucur, Mine; Ozdemir, Osman; Uydeş Doğan, B Sönmez

    2013-03-01

    Vascular dysfunction plays a key role in the pathogenesis of diabetic vascular disease. In this study, we aimed to investigate whether chronic in vivo treatment of Crataegus microphylla (CM) extract in diabetic rats induced with streptozotocin (STZ, intraperitoneal, 65 mg/kg) preserves vascular function and to evaluate whether the reduction of inducible nitric oxide synthase (iNOS), proinflammatory cytokines, and lipid peroxidation mediates its mechanisms of action. Starting at 4 weeks of diabetes, CM extract (100 mg/kg) was administrated to diabetic rats for 4 weeks. In aortic rings, relaxation to acetylcholine and vasoreactivity to noradrenaline were impaired, whereas aortic iNOS expression and plasma tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), total nitrite-nitrate, and malondialdehite levels were increased in diabetic rats compared with controls. Chronic CM treatment significantly corrected all the above abnormalities in diabetic rats. In comparison, pretreatment of the aorta of diabetic rats with N-[3(aminomethyl) benzyl]-acetamidine, dihydrochloride (10(-5)  M), a selective inhibitor of iNOS, produced a similar recovery in vascular reactivity. These results suggest that chronic in vivo treatment of CM preserves endothelium-dependent relaxation and vascular contraction in STZ-induced diabetes, possibly by reducing iNOS expression in the aorta and by decreasing plasma levels of TNF-α and IL-6 and by preventing lipid peroxidation. PMID:22585450

  13. Self-Assembly of Porphyrin J-Aggregates

    Snitka, Valentinas; Rackaitis, Mindaugas; Navickaite, Gintare

    2006-03-01

    The porphyrin nanotubes were built by ionic self-assembly of two oppositely charged porphyrins in aqueous solution. The porphyrins in the acid aqueous solution self-assemble into J-aggregates, wheels or other structures. The electrostatic forces between these porphyrin blocks contribute to the formation of porphyrin aggregates in the form of nanotubes, enhance the structural stability of these nanostructures. The nanotubes were composed mixing aqueous solutions of the two porphyrins - anionic Meso-tetra(4- sulfonatophrnyl)porhine dihydrochloride (TPPS4) and cationic Meso-tetra(4-pyridyl)porphine (T4MPyP). The porphyrin nanotubes obtained are hollow structures with the length of 300 nm and diameter 50 nm. Photocatalytic porphyrins are used to reduce metal complexes from aqueous solution and to control the deposition of Au from AuHCl4 and Au nanoparticles colloid solutions onto porphyrin nanotubes. Porphyrin nanotubes are shown to reduce metal complexes and deposit the metal selectively onto the inner or outer surface of the tubes, leading to nanotube-metal composite structures.

  14. Antimicrobial Evaluation of Diterpenes from Copaifera langsdorffii Oleoresin Against Periodontal Anaerobic Bacteria

    Rodrigo C. S. Veneziani

    2011-11-01

    Full Text Available The antimicrobial activity of four labdane-type diterpenes isolated from the oleoresin of Copaifera langsdorffii as well as of two commercially available diterpenes (sclareol and manool was investigated against a representative panel of microorganisms responsible for periodontitis. Among all the evaluated compounds, (−-copalic acid (CA was the most active, displaying a very promising MIC value (3.1 µg mL−1; 10.2 µM against the key pathogen (Porphyromonas gingivalis involved in this infectious disease. Moreover, CA did not exhibit cytotoxicity when tested in human fibroblasts. Time-kill curve assays performed with CA against P. gingivalis revealed that this compound only inhibited the growth of the inoculums in the first 12 h (bacteriostatic effect. However, its bactericidal effect was clearly noted thereafter (between 12 and 24 h. It was also possible to verify an additive effect when CA and chlorhexidine dihydrochloride (CHD, positive control were associated at their MBC values. The time curve profile resulting from this combination showed that this association needed only six hours for the bactericidal effect to be noted. In summary, CA has shown to be an important metabolite for the control of periodontal diseases. Moreover, the use of standardized extracts based on copaiba oleoresin with high CA contents can be an important strategy in the development of novel oral care products.

  15. Phorbol ester-mediated desensitization of histamine Hl receptors on a cultured smooth muscle cell line

    The present study was undertaken in order to examine the effect of protein kinase C (PKC) on histamine Hl receptors, (HlR) present on the smooth muscle cell line, DDT1MF-2. [3H]-pyrilamine binding revealed that specific [3H]-pyrilamine binding sites were reduced be pretreatment with 12-O-tetra-decanoylphorbol-13-acetate (TPA), an activator of PKC, but not the Kd. The TPA analogue, 4α phorbol 12,13-didecanoate, which does not activate PKC, failed to induce down-regulation of HlR. TPA-induced down regulation of HlR was inhibited by pretreatment with 1-(5-Isoquinilinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), a PKC inhibitor, in a dose dependent manner. The H-7 analogue, H-8, which is a less potent inhibitor of PKC, but a potent inhibitor of cyclic nucleotide dependent protein kinase, had no effect on HlR. Moreover, treatment with TPA inhibited histamine-induced increases in [Ca2+]/sub i/ in cells loaded with the fluorescent indicator, indo-1. These data suggest that HlR in DDT1MF-2 cells were functionally regulated by PKC

  16. Preparation of Mesoporous Carbons from Acrylonitrile-methyl Methacrylate Copolymer/Silica Nanocomposites Synthesized by in-situ Emulsion Polymerization

    BAO Yongzhong; ZHAO Wenting; HUANG Zhiming

    2013-01-01

    Acrylonitrile-methyl methacrylate (AN-MMA) copolymer/silica nanocomposites were synthesized by in-situ emulsion polymerization initiated by 2,2′-azobis(2-amidinopropane) dihydrochloride absorbed onto colloidal silica particles,and the mesoporous carbon materials were prepared through carbonization of the obtained AN-MMA copolymer/silica nanocomposites,followed by HF etching.Thermogravimetric analysis of AN-MMA copolymer/silica nanocomposites showed that the carbon yield of copolymer was slightly decreased as silica particle incorporated.N2 adsorption-desorption,scan electron microscopy (SEM) and transmission electron microscopy (TEM) were used to characterize the structure and morphology of the mesoporous carbon materials.Both SEM and TEM results showed that disordered mesopores were formed in the obtained carbon material mainly through templating effect of silica nanoparticles.The diameter of mesopores was mainly distributed in the range from 5 nm to 15 nm.The mean pore diameter and total pore volume of the material increased as the mass fraction of silica in the nanocomposites increased from 0 to 24.93%.The significant increase of the mean pore diameter and the decrease of surface area for the carbon material prepared from the nanocomposite with 24.93% silica were caused by partial aggregation of silica nanoparticles in the polymer matrix.

  17. Synthesis and Characterization of Mercuric Bromide-Phenothiazine Complexes

    Vidisha A. Alwani

    2014-12-01

    Full Text Available N-alkylphenothiazines (NAPTZs are biologically active heterocyclic compounds that find extensive applications in the field of medicine. In the pharmaceutical industry, they are used as psychotherapeutic, antiemetic, and antihistaminic drugs. In this study, complexation reactions of mercuric bromide with NAPTZs as principal ligands have been investigated in MeOH medium. Five mercuric bromide complexes of the NAPTZ ligands namely, chlorpromazine hydrochloride (CP.HCl, promethazine hydrochloride (PM.HCl, ethopropazine hydrochloride (EP.HCl, trifluoperazine dihydrochloride (TF.2HCl and thioridazine hydrochloride (TR.HCl have been synthesized. These complexes were subjected to elemental analysis, solubility, molar conductance and magnetic susceptibility, U.V-Vis, I.R, and NMR spectroscopy. The molecular formulations of the complexes have been found to be: [HgBr2(CP2].4H2O; [HgBr2(PM2].2H2O; [HgBr2(EP2]; [HgBr2(TF2].2H2O and [HgBr2(TR2]. Tentative molecular structures have been proposed and presented.

  18. Polyurethane-based scaffolds for myocardial tissue engineering.

    Chiono, Valeria; Mozetic, Pamela; Boffito, Monica; Sartori, Susanna; Gioffredi, Emilia; Silvestri, Antonella; Rainer, Alberto; Giannitelli, Sara Maria; Trombetta, Marcella; Nurzynska, Daria; Di Meglio, Franca; Castaldo, Clotilde; Miraglia, Rita; Montagnani, Stefania; Ciardelli, Gianluca

    2014-02-01

    Bi-layered scaffolds with a 0°/90° lay-down pattern were prepared by melt-extrusion additive manufacturing (AM) using a poly(ester urethane) (PU) synthesized from poly(ε-caprolactone) diol, 1,4-butandiisocyanate and l-lysine ethyl ester dihydrochloride chain extender. Rheological analysis and differential scanning calorimetry of the starting material showed that compression moulded PU films were in the molten state at a higher temperature than 155°C. The AM processing temperature was set at 155°C after verifying the absence of PU thermal degradation phenomena by isothermal thermogravimetry analysis and rheological characterization performed at 165°C. Scaffolds highly reproduced computer-aided design geometry and showed an elastomeric-like behaviour which is promising for applications in myocardial regeneration. PU scaffolds supported the adhesion and spreading of human cardiac progenitor cells (CPCs), whereas they did not stimulate CPC proliferation after 1-14 days culture time. In the future, scaffold surface functionalization with bioactive peptides/proteins will be performed to specifically guide CPC behaviour. PMID:24501673

  19. Taste Masked Orally Disintegrating Pellets of Antihistaminic and Mucolytic Drug: Formulation, Characterization, and In Vivo Studies in Human.

    Taj, Yasmeen; Pai, Roopa S; Kusum Devi, V; Singh, Gurinder

    2014-01-01

    The main aim of the present study was to evaluate the potential of orally disintegrating pellets (ODPs) as an approach for taste masking of bitter drugs, namely, Ambroxol hydrochloride (A-HCl) and Cetirizine dihydrochloride (C-DHCl). Pellets were prepared by extrusion/spheronization with Eudragit EPO, kyron T-134, Kyron T-314, mannitol, sorbitol, MCC (Avicel PH-101), sucralose, chocolate flavor, and 5% xanthum gum. The prepared pellets were characterized for percentage yield, drug content, particle size, in vitro drug release, and in vivo evaluation on humans for taste, mouth feel, and in vivo disintegration time. The results revealed that the average size of pellets was influenced greatly by the percentage of binder and extrusion speed. The optimized ODPs disintegrated in less than 20 s and showed more than 98% of drugs in ODPs dissolved within 15 min. Taste perception study was carried out on human volunteers to evaluate the taste masking ability of ODPs for taste, mouth feel, and in vivo disintegration time. Crystalline state evaluation of drugs in the optimized ODPs was conducted for X-ray powder diffraction. In conclusion, the study confirmed that ODPs can be utilized as an alternative approach for effective taste masking and rapid disintegration in the oral cavity. PMID:27379290

  20. Influence of Different Diets on Development of DMH-Induced Aberrant Crypt Foci and Colon Tumor Incidence in Wistar Rats

    Kristiansen, E.; Thorup, I.; Meyer, Otto A.

    1995-01-01

    week for to or 20 weeks. Throughout the study the animals were fed I) semisynthetic casein-based control diet, 2) control diet with 20% lard, 3) control diet with 20% lard and 20% dietary fiber, or 4) control diet where most of the carbohydrate pool was substituted with sucrose and dextrin. The...... significant increase (p less than or equal to 0.05) in the total number of ACF and number of small and medium ACF. Adding lard to the standard diet did not cause an increase in ACF, bur if the dietary fiber was added to the high-fat diet, a statistically significant reduction (p less than or equal to 0.05) in......The present study was undertaken to investigate certain dietary factors known to affect the development of colon cancer for their ability to modulate aberrant crypt foci (ACI;). Male Wistar rats were initiated with oral noses of dimethylhydrazine dihydrochloride (DMH-2HCl, 20 mg/kg body wt) once a...

  1. Dimethylhydrazine: a reliable positive control for the short sampling time in the UDS assay in vivo.

    Brendler-Schwaab, Susanne Y; Völkner, Wolfgang; Fautz, Rolf; Herbold, Bernd A

    2002-09-26

    In the first international guideline addressing the unscheduled DNA synthesis (UDS) assay in vivo (OECD guideline no. 486, adopted July 1997) only the genotoxic liver carcinogen N-nitrosodimethylamine (NDMA) is proposed as positive control for the short sampling time. Since NDMA is extremely volatile, alternative positive controls should be identified to facilitate handling and reduce exposure risk during routine testing. At Bayer AG and at RCC-CCR GmbH, the genotoxic but non-volatile dimethylhydrazine (DMH; as dihydrochloride) was used instead as positive control in livers of Wistar rats and to a limited extent of NRMI mice after 2-4h exposure. As shown by the data presented in this paper DMH induced a positive result in a total of 21 UDS in vivo studies over a period of 7 years. A negative result was never seen for DMH. Due to these results DMH was proven to be a suitable and reliable positive control in the UDS assay in vivo. Consequently, DMH should be considered as positive control for the short sampling time in the next issue of OECD guideline no. 486. PMID:12297144

  2. Detection of the Inflammation Biomarker C-Reactive Protein in Serum Samples: Towards an Optimal Biosensor Formula

    Wellington M. Fakanya

    2014-10-01

    Full Text Available The development of an electrochemical immunosensor for the biomarker, C-reactive protein (CRP, is reported in this work. CRP has been used to assess inflammation and is also used in a multi-biomarker system as a predictive biomarker for cardiovascular disease risk. A gold-based working electrode sensor was developed, and the types of electrode printing inks and ink curing techniques were then optimized. The electrodes with the best performance parameters were then employed for the construction of an immunosensor for CRP by immobilizing anti-human CRP antibody on the working electrode surface. A sandwich enzyme-linked immunosorbent assay (ELISA was then constructed after sample addition by using anti-human CRP antibody labelled with horseradish peroxidase (HRP. The signal was generated by the addition of a mediator/substrate system comprised of 3,3,5',5'-Tetramethylbenzidine dihydrochloride (TMB and hydrogen peroxide (H2O2. Measurements were conducted using chronoamperometry at −200 mV against an integrated Ag/AgCl reference electrode. A CRP limit of detection (LOD of 2.2 ng·mL−1 was achieved in spiked serum samples, and performance agreement was obtained with reference to a commercial ELISA kit. The developed CRP immunosensor was able to detect a diagnostically relevant range of the biomarker in serum without the need for signal amplification using nanoparticles, paving the way for future development on a cardiac panel electrochemical point-of-care diagnostic device.

  3. In vitro free radical scavenging activity of platinum nanoparticles

    Watanabe, Aki; Kajita, Masashi; Kim, Juewon; Kanayama, Atsuhiro; Miyamoto, Yusei [Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Bioscience Building 402, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562 (Japan); Takahashi, Kyoko; Mashino, Tadahiko, E-mail: yusei74@k.u-tokyo.ac.j [Department of Pharmaceutical Sciences, Faculty of Pharmacy, Keio University, 1-5-30 Shibakoen, Minato, Tokyo 105-8512 (Japan)

    2009-11-11

    A polyacrylic acid (PAA)-protected platinum nanoparticle species (PAA-Pt) was prepared by alcohol reduction of hexachloroplatinate. The PAA-Pt nanoparticles were well dispersed and homogeneous in size with an average diameter of 2.0 {+-} 0.4 nm (n = 200). We used electron spin resonance to quantify the residual peroxyl radical AOO. generated from 2,2-azobis (2-aminopropane) dihydrochloride (AAPH) by thermal decomposition in the presence of O{sub 2} and a spectrophotometric method to quantify the residual 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. PAA-Pt scavenged these two radicals in a dose-dependent manner. Platinum was the functional component. PAA-Pt reduced the rate of oxygen consumption required for linoleic acid peroxidation initiated by AOO. generated from AAPH, indicating inhibition of the propagation of linolate peroxidation. A thiobarbituric acid test also revealed dose-dependent inhibition of the linolate peroxidation by PAA-Pt. Fifty micromolar platinum, as PAA-Pt, completely quenched 250 {mu}M DPPH radical for 5 min. Even when twice diluted in half, the PAA-Pt still quenched 100% of the 250 {mu}M DPPH radical. The scavenging activity of PAA-Pt is durable. These observations suggest that PAA-Pt is an efficient scavenger of free radicals.

  4. In vitro free radical scavenging activity of platinum nanoparticles

    A polyacrylic acid (PAA)-protected platinum nanoparticle species (PAA-Pt) was prepared by alcohol reduction of hexachloroplatinate. The PAA-Pt nanoparticles were well dispersed and homogeneous in size with an average diameter of 2.0 ± 0.4 nm (n = 200). We used electron spin resonance to quantify the residual peroxyl radical AOO. generated from 2,2-azobis (2-aminopropane) dihydrochloride (AAPH) by thermal decomposition in the presence of O2 and a spectrophotometric method to quantify the residual 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. PAA-Pt scavenged these two radicals in a dose-dependent manner. Platinum was the functional component. PAA-Pt reduced the rate of oxygen consumption required for linoleic acid peroxidation initiated by AOO. generated from AAPH, indicating inhibition of the propagation of linolate peroxidation. A thiobarbituric acid test also revealed dose-dependent inhibition of the linolate peroxidation by PAA-Pt. Fifty micromolar platinum, as PAA-Pt, completely quenched 250 μM DPPH radical for 5 min. Even when twice diluted in half, the PAA-Pt still quenched 100% of the 250 μM DPPH radical. The scavenging activity of PAA-Pt is durable. These observations suggest that PAA-Pt is an efficient scavenger of free radicals.

  5. In vitro free radical scavenging activity of platinum nanoparticles

    Watanabe, Aki; Kajita, Masashi; Kim, Juewon; Kanayama, Atsuhiro; Takahashi, Kyoko; Mashino, Tadahiko; Miyamoto, Yusei

    2009-11-01

    A polyacrylic acid (PAA)-protected platinum nanoparticle species (PAA-Pt) was prepared by alcohol reduction of hexachloroplatinate. The PAA-Pt nanoparticles were well dispersed and homogeneous in size with an average diameter of 2.0 ± 0.4 nm (n = 200). We used electron spin resonance to quantify the residual peroxyl radical (\\mathrm {AOO}^{\\bullet } ) generated from 2,2-azobis (2-aminopropane) dihydrochloride (AAPH) by thermal decomposition in the presence of O2 and a spectrophotometric method to quantify the residual 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. PAA-Pt scavenged these two radicals in a dose-dependent manner. Platinum was the functional component. PAA-Pt reduced the rate of oxygen consumption required for linoleic acid peroxidation initiated by \\mathrm {AOO}^{\\bullet } generated from AAPH, indicating inhibition of the propagation of linolate peroxidation. A thiobarbituric acid test also revealed dose-dependent inhibition of the linolate peroxidation by PAA-Pt. Fifty micromolar platinum, as PAA-Pt, completely quenched 250 µM DPPH radical for 5 min. Even when twice diluted in half, the PAA-Pt still quenched 100% of the 250 µM DPPH radical. The scavenging activity of PAA-Pt is durable. These observations suggest that PAA-Pt is an efficient scavenger of free radicals.

  6. 不同浓度苦丁茶对TCA8113人舌鳞癌细胞的体外抗癌效果%Anticancer effect of different concentrations of llex kudingcha C.J.Tseng in TCA8113 human tongue squamous carcinoma cells in vitro

    赵欣; 王强; 骞宇; 庞谅

    2013-01-01

    One Ilex kudingcha C.J. Tseng product was sold for the evaluation of in vitro anticancer effect in TCA8113 human tongue squamous carcinoma cells.In MTT assay,apoptosis analysis by DAPI staining and RT-PCR, Western blot gene expressions were employed to check their anticancer effect. Using MTT assay to check the in vitro cancer cell growth inhibitory effect,at the concentration of 200μg/mL,//ex kudingcha C.J. Tseng (75% inhibition rates) showed the best inhibitory effect on the growth of TCA8113 human tongue squamous carcinoma cells. DAPI staining showed that 20Cμg/mL of Ilex kudingcha C.J. Tseng induced apoptosis on TCA8113 cells. llex kudingcha C.J. Tseng (200μg/ml_) significantly induced apoptosis ,up-regulated mRNA and protein expressions of Bax,caspase-3 and caspase-9,and down-regulated Bcl-2 expression.compared with 100 and 50μg/mU of Ilex kudingcha C.J. Tseng. These results suggested that Ilex kudingcha C.J. Tseng showed good cancer preventive effect.%对一种市售苦丁茶进行TCA8113人舌鳞癌细胞体外抗癌效果评价 通过MTT(3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐)比色法实验、DAPI(4’,6-二眯基-2-苯基吲哚)荧光染色分析、RT-PCR(逆转录聚合酶链式反应)和Western blot分析,研究其抗癌效果通过MTT法测定苦丁茶对癌细胞体外生长抑制效果时发现,200μg/mL浓度的苦丁茶(75%癌细胞生长抑制率)对TCA8113人舌鳞癌细胞生长有相对最强的抑制效果 DAPI染色分析显示,与100、50μg/mL苦丁茶相比,使用200μg/mL浓度的苦丁茶,对TCA8113细胞有较强的诱其凋亡的能力 在RT-PCR和Western blot分析结果中,凋亡因子Bax(B细胞淋巴瘤/白血病基因伴随蛋白x)、caspase-3(半胱天冬氨酸酶-3)、caspase-9(半胱天冬氧酸酶-9)的mRNA和蛋白质表达得到增强,Bcl-2(B细胞淋巴瘤/白血病基因-2)表达减弱 综合分析得出,一定浓度的苦丁茶具有良好的防癌抗癌效果.

  7. A multiattribute utility evaluation of different methods for the detection of enteric protozoa causing diarrhea in AIDS patients

    Sundar Shyam

    2010-01-01

    Full Text Available Abstract Background Enteric protozoa and sporozoa have emerged as important opportunistic parasites and can cause fatal infections in AIDS patients. The line of treatment being different for them necessitates an accurate and prompt identification of these to avoid empirical treatment. In this study which is the first of its kind from India we did a comprehensive evaluation of different techniques, comparing them on the basis of the attributes like yield, cost, time taken, expertise and infrastructure. For the first time combination of Calcoflour White and DAPI, a nuclear stain, were used to identify Microsporidia spp. Thus, a diagnostic protocol was devised for rapid, sensitive and cost effective identification of the opportunistic enteric protozoa. Results The organisms isolated from the stool samples of the cases (450 HIV patients were predominantly Cryptosporidium spp., Microsporidia spp. and Cyclospora spp. Interestingly, the control group (200 relatives of the patients who were HIV negative showed a high incidence (21% of Cryptosporidium spp. We found a significant increase in the sensitivity of microscopy in detecting Cryptosporidium spp. and Cyclospora spp. after formol ether concentration. Kinyoun's staining was better compared to Modified safranin staining for Cryptosporidium spp. identification. Although ELISA had a sensitivity of 93.25% and specificity of 97% for Cryptosporidium spp. detection, we ranked Kinyoun's staining better than ELISA because it is not affordable to most of our patients. For detecting Cyclospora cayetanensis, autoflourescence was the easiest and most cost effective method followed by Safranin technique. Combination of Calcoflour White stain and DAPI gave good results for the identification of Microsporidia spp. We assessed the above techniques and graded the attributes in the following descending order: cost effectiveness, sensitivity, ease of use and interpretation, time taken for the procedure and batch

  8. Discordance in HER2 gene amplification in circulating and disseminated tumor cells in patients with operable breast cancer

    Human epidermal growth factor receptor 2 (HER2) gene amplification in circulating tumor cells (CTCs) and disseminated tumor cells (DTCs) might be useful for modifying Herceptin therapy in breast cancer. In the process of investigating the utility of a microfluidic platform for detecting HER2 gene amplification in these cells, we observed novel results on discordance of HER2 status. Peripheral blood (8.5 mL) and bone marrow (BM) (7.5–10 mL) were collected prospectively from patients with clinical stages I–IV breast cancer. Mononuclear cells were recovered, stained with cytokeratin (CK), CD45, and DAPI, and processed through microfluidic channels for fluorescence in situ hybridization (FISH). A ratio of HER2:CEP17 >2 in any CK+/CD45 or CK−/CD45 cell was regarded as positive for HER2 gene amplification. Peripheral blood from 95 patients and BM from 78 patients were studied. We found CK+/CD45−/DAPI+ CTCs in 27.3% of patients. We evaluated HER2 gene amplification by FISH in 88 blood and 78 BM specimens and found HER2+ CTCs in 1 of 9 (11.1%) and HER2+ DTCs (27.2%) in 3 of 11 patients with HER2+ primary tumor. Among patients with a HER2− primary tumor, 5 of 79 had HER2+ CTCs (6.3%) and 14 of 67 had HER2+ DTCs (20.8%). The overall rate of discordance in HER2 status was 15% between primary tumor and CTCs and 28.2% between primary tumor and DTCs. HER2 was amplified in CTCs and DTCs in a portion of both HER2+ and HER2− primary tumors. HER2 discordance was more frequent for DTCs. The clinical implications of evaluating HER2 status in CTCs and DTCs in breast cancer needs to be established in prospective clinical trials. The cell enrichment and extraction microfluidic technology provides a sensitive platform for evaluation of HER2 gene amplification in CTCs and DTCs

  9. Replication of somatic micronuclei in bovine enucleated oocytes

    Canel Natalia

    2012-11-01

    Full Text Available Abstract Background Microcell-mediated chromosome transfer (MMCT was developed to introduce a low number of chromosomes into a host cell. We have designed a novel technique combining part of MMCT with somatic cell nuclear transfer, which consists of injecting a somatic micronucleus into an enucleated oocyte, and inducing its cellular machinery to replicate such micronucleus. It would allow the isolation and manipulation of a single or a low number of somatic chromosomes. Methods Micronuclei from adult bovine fibroblasts were produced by incubation in 0.05 μg/ml demecolcine for 46 h followed by 2 mg/ml mitomycin for 2 h. Cells were finally treated with 10 μg/ml cytochalasin B for 1 h. In vitro matured bovine oocytes were mechanically enucleated and intracytoplasmatically injected with one somatic micronucleus, which had been previously exposed [Micronucleus- injected (+] or not [Micronucleus- injected (−] to a transgene (50 ng/μl pCX-EGFP during 5 min. Enucleated oocytes [Enucleated (+] and parthenogenetic [Parthenogenetic (+] controls were injected into the cytoplasm with less than 10 pl of PVP containing 50 ng/μl pCX-EGFP. A non-injected parthenogenetic control [Parthenogenetic (−] was also included. Two hours after injection, oocytes and reconstituted embryos were activated by incubation in 5 μM ionomycin for 4 min + 1.9 mM 6-DMAP for 3 h. Cleavage stage and egfp expression were evaluated. DNA replication was confirmed by DAPI staining. On day 2, Micronucleus- injected (−, Parthenogenetic (− and in vitro fertilized (IVF embryos were karyotyped. Differences among treatments were determined by Fisher′s exact test (p≤0.05. Results All the experimental groups underwent the first cell divisions. Interestingly, a low number of Micronucleus-injected embryos showed egfp expression. DAPI staining confirmed replication of micronuclei in most of the evaluated embryos. Karyotype analysis revealed that all Micronucleus-injected embryos had

  10. 不同荧光染料对苜蓿原生质体染色效果的研究%Effects of Different Fluorescent Dyes on Staining Alfalfa Protoplast

    李玉珠; 师尚礼; 贺延玉

    2012-01-01

    The effects of five fluorescent dyes (FDA, Rhodamine B, Rhodamine 6G, Acridine Orange and DAPI) on staining protoplasts obtained from callus of Madicago sativa ' Gannong No. 4' were investigated in order to test the viability of protoplasts, count nuclei and survey processes of protoplast fusion. Result showed that FDA, Rhodamine B, Rhodamine 6G and Acridine Orange dye and mark protoplasts of alfalfa clearly, which could identify parental protoplasts from fusion clusters. FDA is an optimal dye for testing the viability of protoplasts. Acridine Orange and DAPI can dye specific nucleus of protoplasts, which are then used to count and survey nuclei in cell fusion. Acridine Orange can also test the viability of fusion cells. These studies suggest a basis for exploring cell fusion conditions and culturing fusion cells by identifying heterocaryon and testing their viability.%以紫花苜蓿品种甘农4号(Madicago sativa L.‘Gannong No.4’)愈伤组织酶解的原生质体为材料,采用FDA、罗丹明B、罗丹明6G、吖啶橙和DAPI共5种荧光染料对原生质体进行染色效果比较,以期为原生质体活力测定、细胞核计数及原生质体融合过程的观察提供参考.结果表明:FDA、罗丹明B、罗丹明6G和吖啶橙均可使紫花苜蓿原生质体清晰染色,可用于细胞融合时亲本原生质体的标识.FDA是检测原生质体活力的理想染料;吖啶橙和DAPI可对细胞核进行特异性染色,可用于融合时细胞核的观察和计数,前者还可用于检测融合细胞的活力.通过对异源融合体的鉴别及其活力的测定,为摸索融合条件和融合细胞的培养提供了理论依据.

  11. Cytogenetic data on six leafcutter ants of the genus Acromyrmex Mayr, 1865 (Hymenoptera, Formicidae, Myrmicinae): insights into chromosome evolution and taxonomic implications

    Barros, Luísa Antônia Campos; de Aguiar, Hilton Jeferson Alves Cardoso; Mariano, Cléa dos Santos Ferreira; Andrade-Souza, Vanderly; Costa, Marco Antonio; Delabie, Jacques Hubert Charles; Pompolo, Silvia das Graças

    2016-01-01

    Abstract Cytogenetic data for the genus Acromyrmex Mayr, 1865 are available, to date, for a few species from Brazil and Uruguay, which have uniform chromosome numbers (2n = 38). The recent cytogenetic data of Acromyrmex striatus (Roger, 1863), including its banding patterns, showed a distinct karyotype (2n = 22), similar to earlier studied Atta Fabricius, 1804 species. Karyological data are still scarce for the leafcutter ants and many gaps are still present for a proper understanding of this group. Therefore, this study aimed at increasing cytogenetic knowledge of the genus through the characterization of other six species: Acromyrmex balzani (Emery, 1890), Acromyrmex coronatus Fabricius, 1804, Acromyrmex disciger (Mayr, 1887), Acromyrmex echinatior (Forel, 1899), Acromyrmex niger (Smith, 1858) and Acromyrmex rugosus (Smith, 1858), all of which were collected in Minas Gerais – Brazil, except for Acromyrmex echinatior which was collected in Barro Colorado – Panama. The number and morphology of the chromosomes were studied and the following banding techniques were applied: C-banding, fluorochromes CMA3 and DAPI, as well as the detection of 45S rDNA using FISH technique. All the six species had the same chromosome number observed for already studied species, i.e. 2n = 38. Acromyrmex balzani had a different karyotype compared with other species mainly due to the first metacentric pair. The heterochromatin distribution also showed interspecific variation. Nevertheless, all the studied species had a pair of bands in the short arm of the first subtelocentric pair. The fluorochrome CMA3 visualized bands in the short arm of the first subtelocentric pair for all the six species, while Acromyrmex rugosus and Acromyrmex niger also demonstrated in the other chromosomes. The AT-rich regions with differential staining using DAPI were not observed. 45S ribosomal genes were identified by FISH in the short arm of the first subtelocentric pair in Acromyrmex coronatus, Acromyrmex

  12. In Vitro Antiproliferative Effect of Arthrocnemum indicum Extracts on Caco-2 Cancer Cells through Cell Cycle Control and Related Phenol LC-TOF-MS Identification

    Mondher Boulaaba

    2013-01-01

    Full Text Available This study aimed to determinate phenolic contents and antioxidant activities of the halophyte Arthrocnemum indicum shoot extracts. Moreover, the anticancer effect of this plant on human colon cancer cells and the likely underlying mechanisms were also investigated, and the major phenols were identified by LC-ESI-TOF-MS. Results showed that shoot extracts had an antiproliferative effect of about 55% as compared to the control and were characterised by substantial total polyphenol content (19 mg GAE/g DW and high antioxidant activity (IC50=40 μg/mL for DPPH test. DAPI staining revealed that these extracts decrease DNA synthesis and reduce the proliferation of Caco-2 cells which were stopped at the G2/M phase. The changes in the cell-cycle-associated proteins (cyclin B1, p38, Erk1/2, Chk1, and Chk2 correlate with the changes in cell cycle distribution. Eight phenolic compounds were also identified. In conclusion, A. indicum showed interesting antioxidant capacities associated with a significant antiproliferative effect explained by a cell cycle blocking at the G2/M phase. Taken together, these data suggest that A. indicum could be a promising candidate species as a source of anticancer molecules.

  13. Distribution of micro-organisms along a transect in the South-East Pacific Ocean (BIOSOPE cruise) from epifluorescence microscopy

    Masquelier, S.; Vaulot, D.

    2007-08-01

    The distribution of selected groups of micro-organisms was analyzed along a South-East Pacific Ocean transect sampled during the BIOSOPE cruise in 2004. The transect could be divided into four regions of contrasted trophic status: a high Nutrient Low Chlorophyll (HNLC) region (mesotrophic) near the equator, the South-East Pacific Ocean gyre (hyper-oligotrophic), the transition region between the gyre and the coast of South America (moderately oligotrophic), and the Chile upwelling (eutrophic). The abundance of phycoerythrin containing picocyanobacteria, autotrophic and heterotrophic eukaryotes in different size ranges, dinoflagellates, and ciliates was determined by epifluorescence microscopy after DAPI staining. All populations reached a maximum in the Chile upwelling and a minimum near the centre of the gyre. Picocyanobacteria reached a maximum abundance of 70×10³ cell mL-1. In the HNLC zone, up to 50% of picocyanobacteria formed colonies. Autotrophic eukaryote and dinoflagellate abundance reached 24.5×10³ and 200 cell mL-1, respectively. We observed a shift in the size distribution of autotrophic eukaryotes from 2-5 μm in eutrophic and mesotrophic regions to less than 2 μm in the central region. The contribution of autotrophic eukaryotes to total eukaryotes was the lowest in the central gyre. Maximum concentration of ciliates (18 cell ml-1) also occurred in the Chile upwelling, but, in contrast to the other groups, their abundance was very low in the HNLC zone and near the Marquesas Islands.

  14. Direct and Sensitive Detection of CWA Simulants by Active Capillary Plasma Ionization Coupled to a Handheld Ion Trap Mass Spectrometer

    Wolf, Jan-Christoph; Etter, Raphael; Schaer, Martin; Siegenthaler, Peter; Zenobi, Renato

    2016-07-01

    An active capillary plasma ionization (ACI) source was coupled to a handheld mass spectrometer (Mini 10.5; Aston Labs, West Lafayette, IN, USA) and applied to the direct gas-phase detection and quantification of chemical warfare agent (CWA) related chemicals. Complementing the discontinuous atmospheric pressure interface (DAPI) of the Mini 10.5 mass spectrometer with an additional membrane pump, a quasi-continuous sample introduction through the ACI source was achieved. Nerve agent simulants (three dialkyl alkylphosphonates, a dialkyl phosporamidate, and the pesticide dichlorvos) were detected at low gas-phase concentrations with limits of detection ranging from 1.0 μg/m3 to 6.3 μg/m3. Our results demonstrate a sensitivity enhancement for portable MS-instrumentation by using an ACI source, enabling direct, quantitative measurements of volatile organic compounds. Due to its high sensitivity, selectivity, low power consumption (<80 W) and weight (<13 kg), this instrumentation has the potential for direct on-site CWA detection as required by military or civil protection.

  15. Fluid phase biopsy for detection and characterization of circulating endothelial cells in myocardial infarction

    Elevated levels of circulating endothelial cells (CECs) occur in response to various pathological conditions including myocardial infarction (MI). Here, we adapted a fluid phase biopsy technology platform that successfully detects circulating tumor cells in the blood of cancer patients (HD-CTC assay), to create a high-definition circulating endothelial cell (HD-CEC) assay for the detection and characterization of CECs. Peripheral blood samples were collected from 79 MI patients, 25 healthy controls and six patients undergoing vascular surgery (VS). CECs were defined by positive staining for DAPI, CD146 and von Willebrand Factor and negative staining for CD45. In addition, CECs exhibited distinct morphological features that enable differentiation from surrounding white blood cells. CECs were found both as individual cells and as aggregates. CEC numbers were higher in MI patients compared with healthy controls. VS patients had lower CEC counts when compared with MI patients but were not different from healthy controls. Both HD-CEC and CellSearch® assays could discriminate MI patients from healthy controls with comparable accuracy but the HD-CEC assay exhibited higher specificity while maintaining high sensitivity. Our HD-CEC assay may be used as a robust diagnostic biomarker in MI patients. (paper)

  16. Development of a biological dosimeter for translocation scoring based on two-color fluorescence in situ hybridization of chromosome subsets

    Recently fluorescence in situ hybridization protocols have been developed which allow the paining of individual chromosomes using DNA-libraries from sorted human chromosomes. This approach has the particular advantage that radiation induced chromosome translocations can be easily detected, if chromosomes of distinctly different colors take part in the translocation event. To enhance the sensitivity of this approach two metaphase chromosome subsets A and B (A: chromosome 1, 2, 4, 8, 16; B: 3, 5, 9, 10, 13) were simultaneously painted in green and red color. Counterstaining of the chromosomes with DAPI resulted in a third subset which exhibited blue fluorescence only. Green-red, green-blue and red-blue translocation chromosomes could be easily detected after irradiation of lymphocyte cultures with 137Cs-γ-rays. Analyses of painted chromosomes can be combined with conventional GTG-banding analyses. This new biological dosimeter should become useful to monitor both long term effects of single irradiation events and the cumulative effects of multiple or chronic irradiation exposure. In contrast to translocation scoring based on the analysis of banded chromosomes, this new approach has the particular advantage that a rapid, automated scoring of translocations can now be envisaged. (author)

  17. A Soluble Pyrophosphatase Is Essential to Oogenesis and Is Required for Polyphosphate Metabolism in the Red Flour Beetle (Tribolium castaneum

    Klébea Carvalho

    2015-03-01

    Full Text Available Polyphosphates have been found in all cell types examined to date and play diverse roles depending on the cell type. In eukaryotic organisms, polyphosphates have been mainly investigated in mammalian cells with few studies on insects. Some studies have demonstrated that a pyrophosphatase regulates polyphosphate metabolism, and most of them were performed on trypanosomatids. Here, we investigated the effects of sPPase gene knocked down in oogenesis and polyphosphate metabolism in the red flour beetle (Tribolium castaneum. A single sPPase gene was identified in insect genome and is maternally provided at the mRNA level and not restricted to any embryonic or extraembryonic region during embryogenesis. After injection of Tc-sPPase dsRNA, female survival was reduced to 15% of the control (dsNeo RNA, and egg laying was completely impaired. The morphological analysis by nuclear DAPI staining of the ovarioles in Tc-sPPase dsRNA-injected females showed that the ovariole number is diminished, degenerated oocytes can be observed, and germarium is reduced. The polyphosphate level was increased in cytoplasmic and nuclear fractions in Tc-sPPase RNAi; Concomitantly, the exopolyphosphatase activity decreased in both fractions. Altogether, these data suggest a role for sPPase in the regulation on polyphosphate metabolism in insects and provide evidence that Tc-sPPase is essential to oogenesis.

  18. The anti-cancer effect of Propranolol in K562 cell line: an in vitro study

    S Bastani

    2016-03-01

    Full Text Available Introduction: Β-AR receptors are one of the proteins involved in cancer and stress. The therapeutic activity of β-blockers such as propranolol is attributed to the blockade of β1-adrenergic receptors (ARs. In this study, the effect of propranolol on the viability of K562 cell line was examined. Material and methods: In order to assessment of anti-tumoral effects of propranolol, different concentrations of propranolol were prepared. K562 cells were treated with different concentrations of propranolol, then the percentage of inhibitory effect of propranolol on K562 cell viability at different times (24, 48 and 72 hours was estimated by MTT assay. Gel electrophoresis of DNA and DAPI staining were used for apoptosis investigation. Statistical comparisons were performed using two-sample t-test, Nominal significance level of each univariate test was 0.05. Results: Propranolol decreased viability of K562 cell line. The inhibitory effect of propranolol is time- and concentration-dependent, thus in higher concentrations and 72 hours after treatment, the maximum inhibitory effect was observed. (P<0.05. As the results showed, Propranolol induces apoptosis in K562 cell line. Conclusions: With respect to the inhibitory effect of propranolol on cell viability and its apoptotic effect on K562 cell line, this drug may be used for cancer therapy.

  19. Development of living cell microarrays using non-contact micropipette printing.

    Jonczyk, Rebecca; Timur, Suna; Scheper, Thomas; Stahl, Frank

    2016-01-10

    During the last 30 years cellular screening systems were unidirectional developed towards high throughput applications on single cell level. We developed living cell microarrays, which provide an in vivo-like microenvironment for an advanced method to measure cellular response to external stimuli. To print living cells on glass slides, the classic microarray equipment, which involves printer and scanner, was fully transferred to suspensions of living cells. The microarray production was optimized using a contact-free spotting procedure in order to enhanced cell adhesion and growth rates. The printed model cells, A-549 (lung cancer cell line), were analyzed with conventional cell staining assays like DAPI (cell nuclei staining), calcein acetoxymethyl ester (viable cell staining), and CellTiter-Blue(®) Cell Viability Assay. After optimization, a reproducible (spot-to-spot variation: ± 8.6 cells) printing method for small living cell amounts (1200 cells and fewer) was established that achieved cell viabilities of up to 88% for ≥ 0.6 μL and good proliferation characteristics. Hence, this method could be advantageous for use in biomedical and diagnostic applications. PMID:26603124

  20. An Immunomodulatory Protein (Ling Zhi-8 from a Ganoderma lucidum Induced Acceleration of Wound Healing in Rat Liver Tissues after Monopolar Electrosurgery

    Hao-Jan Lin

    2014-01-01

    Full Text Available The purpose of this study was to investigate the effect of an immunomodulatory protein (Ling Zhi-8, LZ-8 on wound healing in rat liver tissues after monopolar electrosurgery. Animals were sacrificed for evaluations at 0, 3, 7, and 28 days postoperatively. It was found that the wound with the LZ-8 treatment significantly increases wound healing. Western blot analysis clearly indicated that the expression of NF-κB was decreased at 3, 7, and 28 days when liver tissues were treated with LZ-8. Moreover, caspase-3 activity of the liver tissue also significantly decreases at 7 and 28 days, respectively. DAPI staining and TUNEL assays revealed that only a minimal dispersion of NF-κB was found on the liver tissue treated with LZ-8 at day 7 as compared with day 3 and tissues without LZ-8 treatment. Similarly, apoptosis was decreased on liver tissues treated with LZ-8 at 7 days when compared to the control (monopolar electrosurgery tissues. Therefore, the analytical results demonstrated that LZ-8 induced acceleration of wound healing in rat liver tissues after monopolar electrosurgery.