WorldWideScience

Sample records for tetrazolium

  1. Orchid seeds viability identification by tetrazolium test

    Directory of Open Access Journals (Sweden)

    Jackeline Schultz Soares

    2014-10-01

    Full Text Available The knowledge of germinal potential optimizes the practice of in vitro germination of Orchidaceae. This work evaluated of the type and concentrations of tetrazolium solutions on reaction time and on the identification of viable orchid seeds. Were used seeds of the species Brassavola tuberculata Rchb f., Cattleya labiata Lindley var Amoema, Cattleya intermedia and Dendrobium antenatum and of two primary hybrids of Cattleya labiata autumnalis x Cattleya labiata Lindley var. Alba. The seeds (0.01g of each species or hybrid were soaked in 3 mL of aqueous and neutral solutions of 2, 3, 5 triphenyltetrazolium chloride at 0.0625%, 0.125%, 0.25%, 0.5% ou 1,0% concentrations and stored in a light-free environment at 25±2 °C, being evaluated every 30 minutes for identification of the time needed for embryos coloring and later counting of the number of viable seeds by milligram of seed. A completely randomized design was used, in a factorial scheme 6 x 2 x 5 (six genotypes of orchids, two types of tetrazolium solutions and five concentrations with 3 replications. The genotypes responded differently response both for viable seeds by milligram of seed and for the time required for embryos coloring and C. intermedia not respond to the test. Irrespective of solution type, concentrations only interfered in reaction time for the hybrids. It is recommended, therefore, the use of neutral tetrazolium solution at 0.5% for indentification of viable seeds of hybrids of Cattleya labiata autumnalis x Cattleya labiata var. Alba and use of aqueous solution at 0.125% for Brassavola tuberculata, Cattleya labiata var Amoema e Dendrobium antenatum.

  2. Neutrophil Ingestion Rate Of Nitroblue Tetrazolium In Subjects With ...

    African Journals Online (AJOL)

    Objectives: This study was designed to assess the WBC count, absolute neutrophil count, CD4 +T cell count and neutrophil ingestion rate of nitroblue tetrazolium in subjects with Malaria and HIV Co-morbidity. Method and materials: 134 participants were recruited and group as follow: Symptomatic HIV infected participants ...

  3. The nitroblue tetrazolium (NBT) test in renal transplantation

    Science.gov (United States)

    Gordon, A. M.; Briggs, J. D.; Bell, P. R. F.

    1974-01-01

    The nitroblue tetrazolium (NBT) test has been evaluated as a means of differentiating episodes of infection and rejection in 10 cadaver kidney transplant recipients. Normal NBT values were observed during all 11 episodes of acute rejection. A significant elevation of the NBT score was encountered in six of eight episodes of infection after transplantation. A positive NBT result is useful additional evidence in favour of infection in patients in whom the differentiation of infection and rejection is proving difficult. PMID:4610010

  4. Tetrazolium salt monomers for gel dosimetry I: Principles

    Science.gov (United States)

    Penev, Kalin I.; Wang, Meng; Mequanint, Kibret

    2017-05-01

    Tetrazolium salts (TS) have been previously used for radiochromic dosimetry in solutions, films and three dimensional (3D) gelatine-based gels. However, widespread application for 3D dosimetry has not been achieved due to the required high concentrations and associated high costs of the TS dimer used in prior research. Through careful selection of TS monomer, sensitivity-enhancing additives and inert gel forming material, we report the preparation of a non-diffusing, chemically stable, 3D dosimeter with linear sensitivity between 0 and 80 Gy with submillimolar requirements for the active TS.

  5. Tetrazolium test for assessment of seed viability of atemoya (Annona cherimola Mill. x A. squamosa L.)

    OpenAIRE

    Gimenez,Juliana Iassia; Ferreira,Gisela; Cavariani,Claudio

    2014-01-01

    The study aimed to adapt the tetrazolium test to assess the viability of atemoya (Annona cherimola Mill. x A. squamosa L.) seeds. The experimental design was completely randomized in 3 x 3 factorial arrangement (tetrazolium salt concentrations x exposure times), totaling 9 treatments with four replicates of 25 seeds. After immersion in water for 24 hours, the seeds were longitudinally sectioned and exposed to three tetrazolium salt concentrations (0.05%, 0.5% and 1%) for three periods of time...

  6. The nitroblue tetrazolium reduction test in canine leishmaniasis.

    Science.gov (United States)

    Gómez-Ochoa, Pablo; Lara, Ana; Couto, Guillermo; Marcen, Jose Miguel; Peris, Ana; Gascón, Manuel; Castillo, Juan Antonio

    2010-08-27

    The nitroblue tetrazolium reduction test (NBT) is a quick, easy and cheap assay based on the activation percentage of neutrophils in peripheral blood. The aim of this study was to evaluate the NBT on healthy dogs and in dogs affected by different degrees of leishmaniasis (Stages I and IV). Forty healthy dogs, 20 dogs in Stage I and 20 dogs in Stage IV were included in the study. Three millilitres of blood were extracted from all the dogs via jugular venipuncture in tubes with EDTA. Incubation with NBT was performed depositing 0.05 ml of the leukocyte suspension in the same quantity of 0.1% concentration NBT. The results of the test were reported as NBT reduction rate which represents the percentage of the total of neutrophils evaluated that presented cytoplasmatic accumulations of formazan, meaning a positive NBT reduction. The mean NBT reduction rate for the healthy dogs group was 4.57%, 34% for Stage I dogs (mild disease) and 3.7% for dogs in Stage IV (severe disease), showing that dogs affected with leishmaniasis but with no clinical development of disease have a significantly higher neutrophil reactivity (pNBT with other tests prior to and during treatment are needed, NBT could be a good assay in canine leishmaniasis evaluation. (c) 2010 Elsevier B.V. All rights reserved.

  7. Dose determination with nitro blue tetrazolium containing radiochromic dye films by measuring absorbed and reflected light

    DEFF Research Database (Denmark)

    Kovács, A.; Baranyai, M.; Wojnárovits, L.

    2000-01-01

    Tetrazolium salts as heterocyclic organic compounds are known to form highly coloured, water insoluble formazans by reduction, which can be utilized in radiation processing dosimetry. Radiochromic films containing nitro blue tetrazolium dissolved in a polymer matrix were found suitable for dose...... determination in a wide dose range both by absorbance and reflectance measurements. The concept of measuring reflected light from dose labels has been discussed earlier and emerged recently due to the requirement of introducing semiquantitative label dose indicators for quarantine control. The usefulness...

  8. CD4+ count and Nitro-Blue Tetrazolium reduction rate of neutrophil ...

    African Journals Online (AJOL)

    CD4+ cells were enumerated using flow Cytometric method and neutrophil phagocytic activity was determined using Nitro- Blue Tetrazolium (NBT) Reduction test. Data was analyzed using Microsoft Excel and independent sample t-test for comparison of means. P-value less than or equal to 0.05 (P ≤ 0.05) was regarded ...

  9. TETRAZOLIUM TEST FOR EVALUATING Peltophorum dubium (Sprengel Taubert LEGUMINOSAE CAESALPINIOIDEAE SEEDS QUALITY

    Directory of Open Access Journals (Sweden)

    Luciana Magda de Oliveira

    2005-06-01

    Full Text Available This research verified different methods of pre-conditioning and concentrations of tetrazolium solutions for evaluatingof canafistula seed lot quality. Seeds collected in Lavras-MG- were submitted to the following methods of pre-conditioning: ascarification and immersion in water of 25oC for 14 hours; and b immersion in water of 95oC and left in the same water to rest withoutheating for 24 hours at 25oC. The teguments of the seeds were removed and the embryos were immerged in 0.07, 0.1 and 0.3%tetrazolium solution during 150 minutes at 25oC. To verify the reliability of the results through the tetrazolium test, germination test,first counting, index of germination velocity and nursery emergency were done. Scarification and immersion in water of 25oC for 14hours were efficient in pre-conditioning canafistula seeds and 0,1% tetrazolium solution for 150 minutes at 25oC allowed evaluatingseed lots of this species.

  10. A rapid colorimetric assay for mold spore germination using XTT tetrazolium salt

    Science.gov (United States)

    Carol A. Clausen; Vina W. Yang

    2011-01-01

    Current laboratory test methods to measure efficacy of new mold inhibitors are time consuming, some require specialized test equipment and ratings are subjective. Rapid, simple quantitative assays to measure the efficacy of mold inhibitors are needed. A quantitative, colorimetric microassay was developed using XTT tetrazolium salt to metabolically assess mold spore...

  11. Nitroblue tetrazolium blocks BK channels in cerebrovascular smooth muscle cell membranes

    OpenAIRE

    Ye, D; Pospisilik, J A; Mathers, D A

    2000-01-01

    The effects of p-nitroblue tetrazolium (NBT) on large conductance, calcium-activated potassium channels (BK channels) in enzymatically dispersed rat cerebrovascular smooth muscle cells (CVSMCs) were examined.Patch clamp methods were employed to record single BK channel currents from inside-out patches of CVMC membrane maintained at 21–23°C.When applied to the cytoplasmic face of inside-out membrane patches (internally applied NBT), micromolar concentrations of NBT reversible reduced the mean ...

  12. Radiation-chemical reaction of 2,3,5-triphenyl-tetrazolium chloride in liquid and solid state

    DEFF Research Database (Denmark)

    Kovacs, A.; Wojnarovits, L.; McLaughlin, W.L.

    1996-01-01

    In pulse radiolysis of 2,3,5-triphenyl-tetrazolium chloride (TTC) at around 360 nm fast formation of intermediate tetrazolium radical was observed under both oxidizing and reducing conditions. In the latter case bimolecular formation of formazan, absorbing at around 480 nm, was observed. This rea......In pulse radiolysis of 2,3,5-triphenyl-tetrazolium chloride (TTC) at around 360 nm fast formation of intermediate tetrazolium radical was observed under both oxidizing and reducing conditions. In the latter case bimolecular formation of formazan, absorbing at around 480 nm, was observed....... This reaction is accompanied by combination to the diformazan dimer, absorbing over the spectral range 500-550 nm. A polyvinyl-alcohol-based TTC film was produced and tested for dosimetry purposes: it gave a measurable response in the 1-100 kGy dose range by evaluating the 50 mu m thick TTC films...

  13. A Simple Procedure for the Evaluation of Bone Vitality by Staining with a Tetrazolium Salt

    Directory of Open Access Journals (Sweden)

    René Schiffner

    2017-07-01

    Full Text Available Presently, no intra-operative method for a direct assessment of bone vitality exists. Therefore, we set out to test the applicability of tetrazolium-based staining on bone samples. The explanted femoral heads of 37 patients were used to obtain either cancellous bone fragments or bone slices. Samples were stained with 2,3,5-triphenyl-2H-tetrazolium chloride (TTC or 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (thiazolyl blue, MTT at different times (one to twelve hours after explantation. Staining was quantified either spectrophotometrically after extraction of the dyes or by densitometric image analysis. TTC-staining of cancellous bone fragments and bone slices, respectively, indicated the detectability of vital cells in both types of samples in a window of up to six hours after explantation. Staining intensity at later time-points was indistinguishable from the staining of untreated samples or sodium azide treated samples, which represent dead cells. In contrast, MTT-staining of bone slices revealed intense unspecific staining, which obscured the evaluation of the vitality of the samples. The lack of a detectable increase of colour intensity in TTC-stained bone samples, which were treated more than six hours after explantation, corresponds to reduced fracture healing. The described simple procedure could provide a basis for an intraoperative decision by the orthopaedic surgeon.

  14. Cell damage and dye reduction in the quantitative nitroblue tetrazolium (NBT) test.

    Science.gov (United States)

    Segal, A W; Levi, A J

    1975-01-01

    Nitroblue tetrazolium (NBT) is toxic to neutrophils; an effect which is greatly enhanced by endotoxin and latex particles. Cell damage, measured by the release of the cytoplasmic marker enzyme lactate dehydrogenase (LDH), was closely related to dye reduction. This suggests that, in this test, dye reduction occurs largely as a result of contact between intracellular reducing compounds and NBT following damage of the outer cell membrane. The expression of dye reduction as a function of LDH release should enhance the sensitivity of the quantitative NBT test by correcting for the observed intersubject variation in cell damage. The relationship between cell damage and dye reduction is a measure of the reducing capacity of the cell. This was normal in immature, bone marrow neutrophils, but diminished in neutrophils of patients with chronic granulomatous disease. Images Fig. 3 PMID:1212802

  15. The pyruvic acid analog 3-bromopyruvate interferes with the tetrazolium reagent MTS in the evaluation of cytotoxicity.

    Science.gov (United States)

    Ganapathy-Kanniappan, Shanmugasundaram; Geschwind, Jean-Francois H; Kunjithapatham, Rani; Buijs, Manon; Syed, Labiq H; Rao, Pramod P; Ota, Shinichi; Vali, Mustafa

    2010-04-01

    3-Bromopyruvate (3BrPA) is a pyruvate analog known for its alkylating property. Recently, several reports have documented the antiglycolytic and anticancer effects of 3BrPA and its potential for therapeutic applications. 3BrPA-mediated cytotoxicity has been evaluated in vitro by various methods including tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)-based assays such as MTT, MTS, and so on. However, growing body of evidences has shown that tetrazolium reagent may interfere with the test compounds. In this study, we investigated whether the tetrazolium reagent interferes with the assessment of 3BrPA cytotoxicity. The results of the tetrazolium-based MTS assay were compared with 3 distinct cell viability detection methods, that is, Trypan Blue staining, ATP depletion, and Annexin V staining in 2 different cell lines, Vx-2 and HepG2. The MTS assay data showed false positive results by indicating increased cell viability at 1 mM and 2 mM 3BrPA whereas the other cell viability assays demonstrated that both Vx-2 and HepG2 cells are not viable at the same treatment conditions. In order to validate the direct interaction of 3BrPA with MTS reagent, we tested cell-free media incubated with different concentrations of 3BrPA. The results of cell-free media showed an increase in absorbance in a dose-dependent manner confirming the interaction of MTS with 3BrPA. Thus, our data clearly demonstrate that 3BrPA interferes with the accuracy of MTS-based cytotoxicity evaluation. Hence, we suggest that employing multiple methods of biochemical as well as morphological cytotoxicity assays is critical to evaluate 3BrPA-mediated cell death.

  16. Ferrous iron mediated oxidation of arachidonic acid: studies employing nitroblue tetrazolium (NBT).

    Science.gov (United States)

    Peterson, D A; Gerrard, J M; Rao, G H; Krick, T P; White, J G

    1978-10-01

    The oxidation of arachidonic acid by ferrous sulfate provides a useful model to study the role of iron in lipid oxidation reactions. We have employed nitroblue tetrazolium (NBT) in the present investigation to evaluate the mechanism of this reaction. In the presence of arachidonic acid, Fe +++, and O2, the yellow dye NBT was rapidly reduced to the blue form, NBTH2. The molar ratio of arachidonic acid to Fe++ in this rapid reaction was 1:1, showing an interaction of one fatty acid molecule per iron molecule. Approximately one molecule of NBT was reduced per four molecules of arachidonic acid and Fe++. Reduction of NBT was accompanied by oxidation of Fe++ to Fe+++, suggesting the transfer of four electrons from the Fe++ to NBT to reduce the dye. Arachidonic acid was found to be unchanged when extracted at the end of the reaction, indicating formation of a complex that could dissociate leaving intact arachidonic acid. Evidence for the presence of such a complex which slowly dissociates during the reaction was obtained after longer incubations with small amounts of arachidonic acid. NBT reduction was not inhibited by agents which hydrolyze superoxide, by catalase or by agents which trap hydroxy radicals. We, therefore, propose a model in which NBT traps a radical generated on the arachidonic acid molecule. The proposed model suggests mechanisms for other fatty acid oxidation reactions such as prostaglandin and hydroperoxy fatty acid synthesis.

  17. Nitroblue tetrazolium (NBT) reduction by bacteria. Some properties of the reaction and its possible use.

    Science.gov (United States)

    Urban, T; Jarstrand, C

    1979-08-01

    All the S. albus, E. coli and P. aeruginosa strains examined reduced nitroblue tetrazolium (NBT) to dark blue formazan. The amount of formazan produced was proportional to the number of bacteria. Under the same growth conditions, an equal number of bacteria of various strains produced different amounts of formazan. However, there were statistically verified differences in the NBT-reduction between the three species examined. The NBT-reduction took place in all phases of growth but was most intense in the early logarithmic phase. NBT was found to be toxic for bacteria, and the different strains had varying sensitivity to that effect. The NBT-reaction was markedly enhanced by phenazine methosulphate (PMS). The blue colour of formazan produced from NBT has an advantage over the red colour from triphenyltetrazolium chloride (TTC) if the reaction occurs in the presence of haemoglobin often present in biological materials. With NBT and PMS, 10(6)--10(7) bacteria are needed to form detectable amounts of formazan. The NBT-reduction by bacteria may be useful for measuring the influence of bactericidal, bacteriostatic or growth-stimulating factors on bacteria.

  18. Subcellular distribution of nitroblue tetrazolium reductase (NBT-R) in human polymorphonuclear leukocytes (PMN).

    Science.gov (United States)

    Baehner, R L

    1975-11-01

    Subcellular distribution study of cytoplasmic organelles was performed on human polymorphonuclear leukocytes after homogenization in 0.34 molar sucrose by differential centrifugation and sucrose density gradient centrifugation of the homogenate. The whole homogenate and each fraction was assayed for nitroblue tetrazolium (NBT)-reductase with and without 1 mM potassium cyanide, and the distribution of this enzyme was compared to the distribution of lysozyme, peroxidase, beta-glucuronidase, and acid and alkaline phosphatase. Enzyme recovery was 97 per cent and ranged between 74 and 124 per cent. Latent activity of all enzymes except NBT-reductase, acid, and alkaline phosphatase was demonstrated by observing a four- to sixfold increase in activity after the addition of Triton-X 100. Maximal relative specific activity using either DPNH or without cyanide for NBT-reductase was found in the 100,000 x g differential centrifugation fraction and was concentrated in the less dense top fraction of the sucrose density gradient. The distribution pattern was similar to acid and alkaline phosphatase. In contrast, the maximal concentration of beta-glucuronidase and peroxidase was found in the heavier 7,200 x g granule fraction and in the more dense bottom fractions of the sucrose density gradient. Maximal lysozyme activity was concentrated in the 30,000 x g granule fraction and in the fractions located between the heaviest and lightest fractions of the sucrose density gradient. The lack of latent activity and the similarity of subcellular distribution of NBT-reductase to acid and alkaline phosphatase, two enzymes associated with microsomes and plasmalemal membranes in human polymorphonuclear leukocytes (PMN), indicates that NBT-reductase is also a nonlysosomal enzyme located in microsomes or in plasmalemal membranes. These findings support the previously described histochemical observations that initial reduction of NBT to formazan occurs on the PMN plasmalemal surface membrane at

  19. Succinate Dehydrogenase Activity Assay in situ with Blue Tetrazolium Salt in Crabtree-Positive Saccharomyces cerevisiae Strain

    Directory of Open Access Journals (Sweden)

    Joanna Berlowska

    2008-01-01

    Full Text Available A spectrophotometric method for determining succinate dehydrogenase (SDH activity assay in azide-sensitive yeast Saccharomyces cerevisiae has been developed. The permeabilization of yeast cells by 0.05 % digitonin permitted to study yeast enzymatic activity in situ. The reduction of blue tetrazolium salt (BT to blue tetrazolium formazan (BTf was conducted in the presence of phenazine methosulphate (PMS as an exogenous electron carrier, and sodium azide (SA as an inhibitor of cytochrome oxidase (Cyt pathway. Various factors such as type of substrate, BT concentration, cell number, temperature and time of incubation, and different Cyt pathway blockers were optimized. In earlier studies, dimethyl sulfoxide (DMSO had been selected as the best solvent for extraction of BTf from yeast cells. The linear correlation between permeabilized yeast cell density and amount of formed formazan was evidenced in the range from 9·10^7 to 5·10^8 cells per sample solution. Below the yeast cell concentration of 10^7 the absorbance values were too low to detect formazans with good precision. This standarized procedure allows the estimation of SDH activity in whole cells, depending on vitality level of yeast populations. Significant increases of succinate dehydrogenase activities were observed in sequential passages as the result of the increase of activity of the strain and adaptation to cultivation conditions.

  20. Quantitative aspects of the cytochemical demonstration of glucose-6-phosphate dehydrogenase with tetrazolium salts studied in a model system of polyacrylamide films

    NARCIS (Netherlands)

    van Noorden, C. J.; Tas, J.; Sanders, J. A.

    1981-01-01

    The enzyme cytochemical demonstration of glucose-6-phosphate dehydrogenase (G6PDH) with several tetrazolium salts has been studied with an artificial model of polyacrylamide films in corporated with the enzyme, which enabled teh correlation of cytochemical and biochemical data. In the model films no

  1. Improved localization of glucose-6-phosphate dehydrogenase activity in cells with 5-cyano-2,3-ditolyl-tetrazolium chloride as fluorescent redox dye reveals its cell cycle-dependent regulation

    NARCIS (Netherlands)

    Frederiks, Wilma M.; van Marle, Jan; van Oven, Carel; Comin-Anduix, Begonya; Cascante, Marta

    2006-01-01

    Since the introduction of cyano-ditolyl-tetrazolium chloride (CTC), a tetrazolium salt that gives rise to a fluorescent formazan after reduction, it has been applied to quantify activity of dehydrogenases in individual cells using flow cytometry. Confocal laser scanning microscopy (CLSM) showed that

  2. Differentiation of HL-60 promyelocytic leukemia cells monitored by flow cytometric measurement of nitro blue tetrazolium (NBT) reduction.

    Science.gov (United States)

    Blair, O C; Carbone, R; Sartorelli, A C

    1985-01-01

    Reduction of nitro blue tetrazolium (NBT) to insoluble blue formazan granules occurs during the stimulus-induced respiratory burst of mature granulocytes and is routinely used as an indicator of the extent of granulocytic differentiation of HL-60 acute promyelocytic leukemia cells. In the present study, the differentiation of HL-60 leukemia cells induced by dimethylsulfoxide (DMSO) or retinoic acid was monitored by flow cytometric (FCM) measurement of forward and 90 degree light scatter of NBT treated cells. Two-parameter correlated analysis permitted a distinction between cells with increased forward and decreased 90 degree light scatter (NBT-), and cells with decreased forward and increased 90 degree light scatter (NBT+). Fixation of NBT treated cells with 1% paraformaldehyde facilitated flow cytometric analysis, and allowed differences in NBT reduction to be quantitated. DMSO-induced cells expressed an all-or-none reduction of NBT to formazan, compared with retinoic acid treated cells that exhibited a graded response. Three parameter flow cytometric analysis of HL-60 leukemia cells stained with propidium iodide in combination with NBT allowed the determination of the cell cycle distribution of NBT-treated cells.

  3. An improved micromethod for evaluating the phagocytic potential of granulocytes by the nitroblue tetrazolium (NBT) slide test.

    Science.gov (United States)

    Murphy, K A; Kelleher, J J

    1982-04-01

    A comparison of granulocyte preparations obtained with the original endotoxin-stimulated nitroblue tetrazolium (NBT) slide test and a modified NBT slide test developed in our laboratory was performed as blind study on paired peripheral blood samples from 25 normal, adult humans and 25 normal, adult BALB/c mice. Distinct qualitative differences between granulocyte preparations obtained with the two NBT methods were observed. The original NBT procedure yielded cell preparations which were poorly stained and morphologically altered. In contrast the modified procedure consistently resulted in human and murine cell preparations which were intensely stained and morphologically intact. A blinded comparison of the number of NBT-positive cells showed quantitative differences between the original and modified NBT slide tests. The average percentage of NBT-positive cells of the 25 normal human subjects was 79.81 +/- 17.40 with the original method and 90.83 +/- 7.71 with the modified method (p less than 0.0002). The average percentage of NBT-positive cells obtained on 25 normal BALB/c mice was 29.52 +/- 17.26 and 69.22 +/- 9.01 for the original and modified procedures, respectively (p less than 0.0001). The increased numbers of NBT-positive cells observed with the modified NBT procedure may have resulted from variation in the original method due to the misinterpretation of weakly stained and morphologically altered granulocyte preparations.

  4. Investigations of riboflavin photolysis via coloured light in the nitro blue tetrazolium assay for superoxide dismutase activity.

    Science.gov (United States)

    Cheng, Chien-Wei; Chen, Liang-Yü; Chou, Chan-Wei; Liang, Ji-Yuan

    2015-07-01

    Determination of the superoxide dismutase activity is an important issue in the fields of biochemistry and the medical sciences. In the riboflavin/nitro blue tetrazolium (B2/NBT) method, the light sources used for generating superoxide anion radicals from light-excited riboflavin are normally fluorescent lamps. However, the conditions of B2/NBT experiments vary. This study investigated the effect of the light source on the light-excitation of riboflavin. The effectiveness of the photolysis was controlled by the wavelength of the light source. The spectra of fluorescent lamps are composed of multiple colour lights, and the emission spectra of fluorescent lamps made by different manufacturers may vary. Blue light was determined to be the most efficient for the photochemical reaction of riboflavin in visible region. The quality of the blue light in fluorescent lamps is critical to the photo-decomposition of riboflavin. A blue light is better than a fluorescent lamp for the photo-decomposition of riboflavin. The performance of the B2/NBT method is thereby optimized. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. [Study on the counting of Streptococcus mutans, Streptococcus sanguis, Haemophilus actinomycetemcomitans by methyl thiazolyl tetrazolium colorimetric method].

    Science.gov (United States)

    Wang, Zhong-Chao; Fan, Li-Yuan; Jiang, Jun-Qiang; Cai, Wei; Ding, Yi

    2010-06-01

    To explore the feasibility of methyl thiazolyl tetrazolium (MTT) colorimetric method and the applied condition for the normal bacteria in the mouth, as Streptococcus mutans (S. mutans), Streptococcus sanguis (S. sanguis), Haemophilus actinomycetemcomitans (H. actinomycetemcomitans). Colony forming units (CFU) which was the standard antitheses was used to count bacteria. This study would gain some parameters by changing wavelength, reactive time, dosage and so on. MTT colorimetric method was applied in the counting of S. mutans, S. sanguis and H. actinomycetemcomitans. When counting S. mutans, the best wavelength was 510 nm, the best range was 1.5 x 10(5) - 1.0 x 10(7) CFU x mL(-1). When counting S. sanguis, the best wavelength was 545 nm, the best range was 1.5 x 10(5) - 2.0 x 10(7) CFU x mL(-1). When counting H. actinomycetemcomitans, the best wavelength was 557 nm, the best range was 1.0 x 10(6) - 5.0 x 10(7) CFU x mL(-1). MTT colorimetric method can be used for different aged S. mutans, S. sanguis and H. actinomycetemcomitans. Oral bacteria could be counted by MTT colorimetric method, which is fast and convenient.

  6. Use of 5-Cyano-2,3-Ditolyl-Tetrazolium Chloride Staining as an Indicator of Biocidal Activity in a Rapid Assay for Anti-Acanthamoeba Agents

    OpenAIRE

    Kobayashi, Takeshi; Mito, Tsuyoshi; Watanabe, Narumi; Suzuki, Takashi; Shiraishi, Atsushi; Ohashi, Yuichi

    2012-01-01

    The usefulness of 5-cyano-2,3-ditolyl-tetrazolium chloride (CTC) staining to determine the respiratory activity of Acanthamoeba was evaluated in this study. Acanthamoeba trophozoites and cysts have a red fluorescence after staining with CTC. To determine the effectiveness of CTC staining as a CTC biocidal assay for Acanthamoeba, the trophozoites and cysts of Acanthamoeba castellanii (ATCC 5037) were treated with serial concentrations of disinfectant solutions, namely, polyhexamethylene biguan...

  7. Advantages and Limitations of Salmon-Gal/Tetrazolium Salt Histochemistry for the Detection of LacZ Reporter Gene Activity in Murine Epithelial Tissue.

    Science.gov (United States)

    Merkwitz, Claudia; Blaschuk, Orest; Winkler, Jana; Schulz, Angela; Prömel, Simone; Ricken, Albert Markus

    2017-04-01

    The Escherichia coli LacZ gene is a widely used reporter for gene regulation studies in transgenic mice. It encodes bacterial β-galactosidase (Bact β-Gal), which causes insoluble precipitates when exposed to chromogenic homologues of galactose. We and others have recently reported that Bact β-Gal detection with Salmon-Gal (S-Gal) in combination with nitro blue tetrazolium chloride (NBT) is very sensitive and not prone to interference by acidic endogenous β-galactosidases. Unfortunately, as we show here, the method appears to be inadequate for evaluation of Bact β-Gal expression in keratinized epithelial appendages but not in other keratinized epithelia. NBT in the reaction mixture, just as other tetrazolium salts, inevitably causes unwanted staining artifacts in lingual filiform papillae, penile spines, and hair fibers by interacting with keratin sulfhydryl-rich regions. The methodological limitation can be overcome in part by pretreating the tissues before the S-Gal/NBT staining with an iodine-potassium iodide solution. Alternatively, the use of iodonitrotetrazolium chloride instead of NBT in the S-Gal reaction mixture provides enough color resolution to distinguish the specific Bact β-Gal staining in orange from the artifact staining in dark red. In summary, we provide evidence that S-Gal/NBT histochemistry has limitations, when staining keratinized epithelial appendages.

  8. Conservação e teste de tetrazólio em sementes de jabuticabeira Conservation and tetrazolium test in jabuticaba seeds

    Directory of Open Access Journals (Sweden)

    Cristiano Hössel

    2013-03-01

    Full Text Available O objetivo deste trabalho foi avaliar embalagem a vácuo e biofilme sobre a viabilidade de sementes de jabuticabeira [Plinia trunciflora (O. Berg Kausel], bem como verificar a aplicabilidade do teste de tetrazólio em sementes dessa espécie. O experimento foi realizado no Laboratório de Fisiologia Vegetal e na Unidade de Ensino e Pesquisa Viveiro de Produção de Mudas Hortícolas, da Universidade Tecnológica Federal do Paraná - Câmpus Dois Vizinhos. O experimento foi em blocos ao acaso, em fatorial 2 (utilização de vácuo x 3 (tipo de revestimento x 5 (tempo de armazenamento, com quatro repetições de 50 sementes por unidade experimental. As sementes, após extraídas, foram separadas em dois lotes, sendo um para uso de embalagem a vácuo e outro não. A partir de cada lote, houve a divisão em três sublotes de acordo com o tipo de revestimento, quais sejam: fécula de mandioca (3% m v-1, quitosana (3% m v-1 e sem biofilme. Posteriormente, as sementes foram armazenadas em câmara fria, utilizando temperatura de 5± 1°C, por 7; 14;21; 28 e 35 dias. Foram analisadas as porcentagens de emergência, de viabilidade das sementes pelo teste de tetrazólio e o índice de velocidade de emergência. Recomenda-se armazenar as sementes de jabuticabeira em embalagem a vácuo. Na impossibilidade de utilizar embalagem a vácuo, as sementes desta fruteira devem ser revestidas com biofilme à base de quitosana ou fécula de mandioca. O teste de tetrazólio demonstrou-se viável e mais rápido para avaliar a viabilidade das sementes de jabuticabeira.The aim of this study was to evaluate the vacuum packing and biofilms in the viability seeds of jabuticaba tree [Plinia trunciflora (O. Berg Kausel], as well as, verify if the tetrazolium "test" can be applied in these seeds. The experiment was carried out at the Plant Physiology Laboratory and in the Nursery at the Universidade Tecnológica Federal do Paraná - Câmpus Dois Vizinhos, State of Paran

  9. A 2:2:2 Complex of Vanadium(V) with 4-(2-Thiazolylazo)orcinol and 2,3,5-Triphenyl-2H-Tetrazolium Chloride.

    Science.gov (United States)

    Gavazov, Kiril Blazhev; Delchev, Vassil Borisov; Mileva, Kremena Tomova; Stefanova, Teodora Stefcheva; Toncheva, Galya Kostadinova

    2016-01-01

    The complex formation in the vanadium(V)/4-(2-thiazolylazo)orcinol (TAO)/2,3,5-triphenyl-2H-tetrazolium chloride (TTC) liquid-liquid extraction-chromogenic system was studied. The chloroform-extracted complex has a composition of 2:2:2 under the optimum conditions (pH 4.8-5.2, extraction time 3 min, concentration of TAO 3.4 × 10(-4) mol dm(-3), and concentration of TTC 9.4 × 10(-4) mol dm(-3)) and could be regarded as a dimer (D) of two 1:1:1 species (S) presented by the formula (TT+)[VO2(TAO)]. The constant of extraction was calculated by two methods and some analytical characteristics were determined. The wavelength of maximum absorption (λmax), molar absorptivity (ελ) and fraction extracted (E) were found to be λ = 545 nm, ε545 = 1.97 × 10(4) dm(3) mol(-1) cm(-1), and E = 97.9%. The ground-state equilibrium geometries of the complexes S and D were optimized by quantum chemical Hartree-Fock calculations using 3-21G* basis functions. The bonding and interaction energies were calculated as well.

  10. Comparative study of nitro blue tetrazolium (NBT) reduction method for estimation of glycated haemoglobin with glycated HbA1c estimated on DCA2000+analyzer (immunoagglutination inhibition).

    Science.gov (United States)

    Sahu, Ashok; Gupta, Trapti; Sarkar, Purnima Dey

    2010-01-01

    Glycated haemoglobin is a diagnostic tool, used for the monitoring of the glycemic status among diabetic patients. The present study is designed to compare and correlate modified NBT reduction method for the estimation of Glycated protein (Glycated Haemoglobin) with HbA1C estimated on DCA+2000Analyzer. Glycated protein reduces Nitro Blue Tetrazolium (NBT) reagent in alkaline medium to tetrazinolyl radical NBT+ which is disproportional to yield a highly colored formazan dye (MF+) (monoformazen), absorbance of colored compound was measured which gives the concentration of glycated proteins present in the sample. Heme free globin (glycated hemoglobin) was extracted out and dissolved immediately in 1 ml normal saline. Dissolved globin was treated with modified NBT reagent, absorbance of color developed was recorded in milli delta A/min. The results of modified NBT were then compared with HbA1c estimated by immunoagglutination inhibition method. Correlation coefficient between Glycated hemoglobin and HbAlc was found to be r=0.926 using Schimadzu CL-750 spectrophotometer and r=0.902 using colorimeter. Results of this study were found to be statistically significant p NBT reduction method is as sensitive as HbAlc estimated by DCA2000+Analyzer (immunoagglutination inhibition). Hence it could be used for routine monitoring of blood glucose control level in diabetic subjects.

  11. [Effect of cysteine ethylester hydrochloride (Cystanin) on host defense mechanisms (III): Potentiating effects on phagocytosis and nitroblue tetrazolium (NBT) reduction by leukocytes of mice and guinea pigs].

    Science.gov (United States)

    Hisadome, M; Nakamura, Y; Okumoto, T; Ikegami, K

    1986-11-01

    ICR mice were treated orally with cysteine ethylester hydrochloride (ethylcysteine, 10 and 100 mg/kg) immediately before the intraperitoneal injection of yeast particles. This agent significantly potentiated phagocytosis of yeast particles by peritoneal polymorphonuclear leukocytes in mice obtained 2 hr after the yeast injection, and the treatment with this agent (3 and 30 mg/kg, p.o.) 4 hr before the injection of yeast potentiated phagocytosis of yeast particles by mouse peritoneal leukocytes. This agent (30 mg/kg, p.o.) restored the suppression of phagocytosis of mouse leukocytes by the intraperitoneal administration of cyclophosphamide (30 mg/kg, i.p.) 24 hr before the yeast injection. This agent (10-100 mg/kg, p.o.) had no effect on the decrease of peripheral leukocyte number in irradiated mice (560 rad), but restored the suppression of phagocytosis, nitroblue tetrazolium (NBT) reduction and stimulated NBT reduction by the addition of lipopolysaccharide. Furthermore, this agent (3-30 mg/kg, p.o.) potentiated phagocytosis, NBT reduction and stimulated NBT reduction of peripheral leukocytes obtained from guinea pigs 2 and 6 hr after ethylcysteine treatment. It is suggested that ethylcysteine potentiates phagocytosis and NBT reduction of leukocytes in animals, and it restores phagocytosis and NBT reduction inhibited by the treatment with cyclophosphamide or X-ray irradiation. It may be possible that this stimulating effect of ethylcysteine could be at least in part involved in the stimulation of nonspecific resistance to infection in the compromised host.

  12. Use of 5-cyano-2,3-ditolyl-tetrazolium chloride staining as an indicator of biocidal activity in a rapid assay for anti-Acanthamoeba agents.

    Science.gov (United States)

    Kobayashi, Takeshi; Mito, Tsuyoshi; Watanabe, Narumi; Suzuki, Takashi; Shiraishi, Atsushi; Ohashi, Yuichi

    2012-05-01

    The usefulness of 5-cyano-2,3-ditolyl-tetrazolium chloride (CTC) staining to determine the respiratory activity of Acanthamoeba was evaluated in this study. Acanthamoeba trophozoites and cysts have a red fluorescence after staining with CTC. To determine the effectiveness of CTC staining as a CTC biocidal assay for Acanthamoeba, the trophozoites and cysts of Acanthamoeba castellanii (ATCC 5037) were treated with serial concentrations of disinfectant solutions, namely, polyhexamethylene biguanide (PHMB) and commercial soft contact lens (SCL) disinfectant solutions. The treated Acanthamoeba organisms were stained with CTC, and their respiratory activity was determined by the intensity of fluorescence in a fluorescence microplate reader. The survival rates of the same samples were determined by a culture-dependent biocidal assay using the Spearman-Karber method. Our results showed that the respiratory activities determined by the CTC biocidal assay and the survival rates determined by the culture-dependent biocidal assay for Acanthamoeba trophozoites and cysts decreased in a dose-dependent way after PHMB treatments, and the results were significantly correlated (r = 0.83 and P activities in the trophozoites and cysts treated with SCL disinfectant solutions were significantly correlated with the survival rate (r = 0.70 and P biocidal assay can be used as an alternative method to a culture-dependent biocidal assay. The CTC biocidal assay is a rapid and simple method to test the effectiveness of disinfectant solutions against Acanthamoeba trophozoites and cysts.

  13. [Detection method for the ability of hemp (Cannabis sativa L.) seed germination by the use of 2,3,5-triphenyl-2H-tetrazolium chloride (TTC)].

    Science.gov (United States)

    Ogata, Jun; Kikura-Hanajiri, Ruri; Yoshimatsu, Kayo; Kiuchi, Fumiyuki; Goda, Yukihiro

    2008-11-01

    Cannabis plants show a high Delta9-tetrahydrocannabinol content and are used as a psychoactive drug. Therefore the cultivation of hemp and its possession are prohibited by law in Japan. Meanwhile, Cannabis seeds have been used as a component of shichimi-togarashi (a Japanese spice), bird feed, or a crude drug (mashinin). To exclude the possibility of germination, it is officially noticed that hemp seeds must be killed. However, the number of violators has increased in recent years. To judge the ability of seed germination, a germination test is performed. However, the test requires several days and thus has not been used for on-site inspection. In this study, we developed a rapid detection method to determine the ability of Cannabis seeds to germinate using 2,3,5-triphenyl-2H-tetrazolium chloride (TTC). The principle of the assay is as follows. The endogenous respiratory enzymes in hemp seeds convert added colorless TTC into red 1,3,5-triphenylformazan. Consequently, a living embryo is stained red, while red does not appear in the dead seeds. The reaction was active over a pH range of 8.0-9.0, and the optimum activity was found from 40 to 50 degrees C. Under the optimum conditions, we were able to determine the ability of seeds to germinate based on the presence of color within 20 min. Since this method is rapid and simple, it is applicable to on-site inspections. In addition, it could be used as an alternative technique to the germination test, because erroneous decisions is cannot occur under the assay principle.

  14. Limitations of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay when compared to three commonly used cell enumeration assays

    OpenAIRE

    van Tonder, Alet; Joubert, Annie M.; Cromarty, A Duncan

    2015-01-01

    Background The tetrazolium-based MTT assay has long been regarded as the gold standard of cytotoxicity assays as it is highly sensitive and has been miniaturised for use as a high-throughput screening assay. However, various reports refer to interference by different test compounds, including the glycolysis inhibitor 3-bromopyruvate, with the conversion of the dye to coloured formazan crystals. This study assessed the linear range and reproducibility of three commonly used cell enumeration as...

  15. Superoxide generated by pyrogallol reduces highly water-soluble tetrazolium salt to produce a soluble formazan: a simple assay for measuring superoxide anion radical scavenging activities of biological and abiological samples.

    Science.gov (United States)

    Xu, Chen; Liu, Shu; Liu, Zhiqiang; Song, Fengrui; Liu, Shuying

    2013-09-02

    Superoxide anion radical (O2(˙-)) plays an important role in several human diseases. The xanthine/xanthine oxidase system is frequently utilized to produce O2(˙-). However, false positive results are easily got by using this system. The common spectrophotometric probes for O2(˙-) are nitroblue tetrazolium (NBT) and cytochrome c. Nevertheless, the application of NBT method is limited because of the water-insolubility of NBT formazan and the assay using cytochrome c lacks sensitivity and is not suitable for microplate measurement. We overcome these problems by using 1,2,3-trihydroxybenzene (pyrogallol) as O2(˙-)-generating system and a highly water-soluble tetrazolium salt, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium sodium salt (WST-1) which can be reduced by superoxide anion radical to a stable water-soluble formazan with a high absorbance at 450 nm. The method is simple, rapid and sensitive. Moreover, it can be adapted to microplate format. In this study, the O2(˙-) scavenging activities of superoxide dismutase (SOD), L-ascorbic acid, N-acetyl-L-cysteine (NAC), albumin from human serum, flavonoids and herbal extracts were assessed by using this method. Meanwhile, the activities of tissue homogenates and serum were determined by using this validated method. This method, applicable to tissue homogenates, serum and herbal extracts, proved to be efficient for measuring O2(˙-) scavenging activities of biological and abiological samples. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Comparative study of ß-glucan induced respiratory burst measured by nitroblue tetrazolium assay and real-time luminol-enhanced chemiluminescence assay in common carp (Cyprinus carpio L.)

    DEFF Research Database (Denmark)

    Jiménez, Natalia Ivonne Vera; Pietretti, D.; Wiegertjes, G. F.

    2013-01-01

    -point measurement based on the intracellular reduction of nitroblue tetrazolium (NBT) and a real-time luminol-enhanced assay based on the detection of native chemiluminescence. Both assays allowed for detection of dose-dependent changes in magnitude of the respiratory burst response induced by β-glucans in head...... kidney cells of carp. However, whereas the NBT assay was shown to detect the production of only superoxide anions, the real-time luminol-enhanced assay could detect the production of both superoxide anions and hydrogen peroxide. Only the chemiluminescence assay could reliably record the production of ROS...

  17. Padronização do teste de tetrazólio em sementes de Parkia velutina Benoist (Leguminosae - Mimosoideae Tetrazolium test in Parkia velutina Benoist seeds (Leguminosae - Mimosoideae

    Directory of Open Access Journals (Sweden)

    Angela Maria da Silva Mendes

    2009-01-01

    Full Text Available O trabalho teve por objetivo determinar métodos de pré-condicionamento e concentrações da solução de tetrazólio na avaliação da qualidade de sementes de Parkia velutina Benoist. Os tratamentos pré-condicionantes das sementes foram avaliados com três métodos de escarificação: desponte na região oposta ao hilo, punção na região mediana e lixa nos dois lados da semente com posterior embebição em 200 ml de água para cada 25 sementes e permanência em câmara a 30 ºC por 16 horas. As concentrações da solução de tetrazólio testadas foram: 1,0%, 0,5% e 0,1% por duas horas na temperatura de 40 ºC. O tratamento de pré-condicionamento mais eficiente foi a combinação do desponte e lixamento nos dois lados da semente. A concentração de tetrazólio a 0,5% pode ser utilizada para avaliar a viabilidade das sementes de P. velutina como complemento ao teste de germinação. O teste de tetrazólio se mostrou eficiente na caracterização de lesões ocasionadas por insetos e danos mecânicos em sementes de P. velutina.This manuscript aimed to determine methods of daily pre-conditioning and concentrations of the tetrazolium solution for evaluating the quality of Parkia velutina Benoist seeds. The daily pre-condicionanting seed treatments were evaluated using three scarification methods: coating cutting on the opposite side of the hilum region, puncture in the medium region and scarification with sandpaper on both sides of the seed, followed by soaking each of the 25 seeds in 200 ml of wate and permanence in chamber 30 ºC for 16 hours. The concentrations of the tetrazolium solution tested were 1.0%, 0.5% and 0.1% for two hours at 40 ºC. The more efficient daily pre-conditioning treatlment was a combination of the coating cutting and the scarification n both sides of the seed. The 0.5% tetrazolium concentration can be used to evaluate the viability of P. velutina seeds as complement to the germination test. The tetrazolium test

  18. Assessment of cytotoxicity of carbon nanoparticles using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell viability assay.

    Science.gov (United States)

    Schrand, Amanda M; Lin, Jonathan B; Hussain, Saber M

    2012-01-01

    Ever since the discovery of carbon nanotubes, there has been an increasing interest in technologies that rely upon these incredibly small particles for their unique properties. However, assessment of their biological consequences has been riddled with assay limitations. Here, we describe application of a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium cell viability assay to study cytotoxicity of various carbon-based nanomaterials on cells and discuss some pitfalls of this method.

  19. An investigation on the physicochemical properties of the nanostructured [(4-X)PMAT][N(CN)2] ion pairs as energetic and tunable aryl alkyl amino tetrazolium based ionic liquids

    Science.gov (United States)

    Khalili, Behzad; Rimaz, Mehdi

    2017-06-01

    In this study the different class of tunable and high nitrogen content ionic liquids termed TAMATILs (Tunable Aryl Methyl Amino Tetrazolium based Ionic Liquids) were designed. The physicochemical properties of the nanostructured TAMATILs composed of para substituted phenyl methyl amino tetrazolium cations [(4-X)PMAT]+ (X = H, Me, OCH3, OH, NH2, NO2, F, CN, CHO, CF3, COMe and CO2Me) and dicyanimide anion [N(CN)2]- were fully investigated using M06-2X functional in conjunction with the 6-311++G(2d,2p) basis set. For all of the studied nanostructured ILs the structural parameters, interaction energy, cation's enthalpy of formation, natural charges, charge transfer values and topological properties were calculated and discussed. The substituent effect on the interaction energy and physicochemical properties also is taking into account. The results showed that the strength of interaction has a linear correlation with electron content of the phenyl ring in a way the substituents with electron withdrawing effects lead to make more stable ion pairs with higher interaction energies. Some of the main physical properties of ILs such as surface tension, melting point, critical-point temperature, electrochemical stability and conductivity are discussed and estimated for studying ion pairs using quantum chemical computationally obtained thermochemical data. Finally the enthalpy and Gibbs free energy of formation for twelve nanostructured individual cations with the general formula of [(4-X)PMAT]+ (X = 4-H, 4-Me, 4-OMe, 4-OH, 4-NH2, 4-NO2, 4-F, 4-CN, 4-CHO, 4-CF3, 4-COMe and 4-CO2Me) are calculated.

  20. Tetrazolium Compounds: Synthesis and Applications in Medicine

    Directory of Open Access Journals (Sweden)

    Cheng-Xi Wei

    2015-03-01

    Full Text Available Tetrazoles represent a class of five-membered heterocyclic compounds with polynitrogen electron-rich planar structural features. This special structure makes tetrazole derivatives useful drugs, explosives, and other functional materials with a wide range of applications in many fields of medicine, agriculture, material science, etc. Based on our research works on azoles and other references in recent years, this review covers reported work on the synthesis and biological activities of tetrazole derivatives.

  1. Comparison of NCCLS and 3-(4,5-Dimethyl-2-Thiazyl)-2,5-Diphenyl-2H-Tetrazolium Bromide (MTT) Methods of In Vitro Susceptibility Testing of Filamentous Fungi and Development of a New Simplified Method

    Science.gov (United States)

    Meletiadis, Joseph; Meis, Jacques F. G. M.; Mouton, Johan W.; Donnelly, J. Peter; Verweij, Paul E.

    2000-01-01

    The susceptibility of 30 clinical isolates belonging to six different species of filamentous fungi (Aspergillus fumigatus, Aspergillus flavus, Scedosporium prolificans, Scedosporium apiospermum, Fusarium solani, and Fusarium oxysporum) was tested against six antifungal drugs (miconazole, voriconazole, itraconazole, UR9825, terbinafine, and amphotericin B) with the microdilution method recommended by the National Committee for Clinical Laboratory Standards (NCCLS) (M38-P). The MICs were compared with the MICs obtained by a colorimetric method measuring the reduction of the dye 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to formazan by viable fungi. The levels of agreement between the two methods were 96 and 92% for MIC-0 (clear wells) and MIC-1 (75% growth reduction), respectively. The levels of agreement were always higher for Aspergillus spp. (97% ± 2.5%), followed by Scedosporium spp. (87% ± 10.3%) and Fusarium spp. (78% ± 7.8%). The NCCLS method was more reproducible than the MTT method: 98 versus 95% for MIC-0 and 97 versus 90% for MIC-1. However, the percentage of hyphal growth as determined visually by the NCCLS method showed several discrepancies when they were compared with the percentages of MTT reduction. A new simplified assay that incorporates the dye MTT with the initial inoculum and in which the fungi are incubated with the dye for 48 h or more was developed, showing comparable levels of agreement and reproducibility with the other two methods. Furthermore, the new assay was easier to perform and more sensitive than the MTT method. PMID:10921957

  2. Prediction of chemotherapeutic response in unresectable non-small-cell lung cancer (NSCLC) patients by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium (MTS) assay.

    Science.gov (United States)

    Chen, Juan; Cheng, Guo-Hua; Chen, Li-Pai; Pang, Ting-Yuan; Wang, Xiao-Le

    2013-01-01

    Selecting chemotherapy regimens guided by chemosensitivity tests can provide individualized therapies for cancer patients. The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium, inner salt (MTS) assay is one in vitro assay which has become widely used to evaluate the sensitivity to anticancer agents. The aim of this study was to evaluate the clinical applicability and accuracy of MTS assay for predicting chemotherapeutic response in unresectable NSCLC patients. Cancer cells were isolated from malignant pleural effusions of patients by density gradient centrifugation, and their sensitivity to eight chemotherapeutic agents was examined by MTS assay and compared with clinical response. A total of 37 patients participated in this study, and MTS assay produced results successfully in 34 patients (91.9%). The sensitivity rates ranged from 8.8% to 88.2%. Twenty-four of 34 patients who received chemotherapy were evaluated for in vitro-in vivo response analysis. The correlation between in vitro chemosensitivity result and in vivo response was highly significant (P=0.003), and the total predictive accuracy, sensitivity, specificity, positive predictive value, and negative predictive value for MTS assay were 87.5%, 94.1%, 71.4%, 88.9%, and 83.3%, respectively. The in vitro sensitivity for CDDP also showed a significant correlation with in vivo response (P=0.018, r=0.522). MTS assay is a preferable in vitro chemosensitivity assay that could be use to predict the response to chemotherapy and select the appropriate chemotherapy regimens for unresectable NSCLC patients, which could greatly improve therapeutic efficacy and reduce unnecessary adverse effects.

  3. Limitations of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay when compared to three commonly used cell enumeration assays.

    Science.gov (United States)

    van Tonder, Alet; Joubert, Annie M; Cromarty, A Duncan

    2015-02-20

    The tetrazolium-based MTT assay has long been regarded as the gold standard of cytotoxicity assays as it is highly sensitive and has been miniaturised for use as a high-throughput screening assay. However, various reports refer to interference by different test compounds, including the glycolysis inhibitor 3-bromopyruvate, with the conversion of the dye to coloured formazan crystals. This study assessed the linear range and reproducibility of three commonly used cell enumeration assays; the neutral red uptake (NRU), resazurin reduction (RES) and sulforhodamine B (SRB) assays, in comparison to the MTT assay. Interference between the MTT assay and three glycolysis inhibitors, 2-deoxyglucose, 3-bromopyruvate and lonidamine, was investigated. Data indicate that the NRU, RES and SRB assays showed the smallest variability across the linear range, while the largest variation was observed for the MTT assay. This implies that these assays would more accurately detect small changes in cell number than the MTT assay. The SRB assay provided the most reproducible results as indicated by the coefficient of determination after a limited number of experiments. The SRB assay also produced the lowest variance in the derived 50% inhibitory concentration (IC50), while IC50 concentrations of 3-bromopyruvate could not be detected using either the MTT or RES assays after 24 hours incubation. Interference in the MTT assay was observed for all three tested glycolysis inhibitors in a cell-free environment. No interferences were observed for the NRU, SRB or RES assays. This study demonstrated that the MTT assay was not the best assay in a number of parameters that must be considered when a cell enumeration assay is selected: the MTT assay was less accurate in detecting changes in cell number as indicated by the variation observed in the linear range, had the highest variation when the IC50 concentrations of the glycolysis inhibitors were determined, and interference between the MTT assay and

  4. Paradoxical proliferative potential of iron (II) sulphate on cancer cells after the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay.

    Science.gov (United States)

    Kok, Stanton Hon Lung; Gambari, Roberto; Chui, Chung Hin; Lau, Fung Yi; Cheng, Gregory Yin Ming; Lai, Paul Bo San; Lam, Wing Sze; Chan, Albert Sun Chi; Cheng, Chor Hing; Teo, Ivy Tuang Ngo; Yu, Michael Wing Yiu; Tang, Johnny Cheuk On; Cheung, Filly; Wong, Raymond Siu Ming

    2007-06-01

    There are several scientific approaches for the determination of cellular growth influences of known or novel substances under in vitro conditions, among which colourimetric absorption measurement is considered to be one of the convenient methods. [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (MTS) assay is one of the commonly used colourimetric absorption assays based on the ability of dehydrogenase from viable cells to produce the brown soluble formazan detectable at 490 nm. Here we have tested the possible growth influence of iron (II) sulphate on two human cancer cell lines, the K562 chronic myelogenous leukaemia and T47D breast carcinoma cells, based on the MTS assay. We found that iron (II) sulphate possessed an inhibitory effect when added at 16- to 125-microM concentrations, but iron (II) sulphate became growth stimulatory when its concentration was further increased to 1000 microM. In addition, a dose-dependent increase in absorbance at the same wavelength was observed when we repeated the experiments without the addition of MTS and phenazine methosulfate. When we further repeated the cell growth determinations using adenosine triphosphate content assay for K562 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for T47D, iron (II) sulphate showed a consistent dose-dependent growth inhibitory effect. Morphological investigation after methylene blue staining clearly demonstrated that iron (II) sulphate, at a concentration of 1000 microM, is cytotoxic to T47D cells. Interestingly, a consistent increment for the absorbance at 490 nm was further observed with increased iron (II) sulphate concentration either in the presence or absence of MTS even in a cell-free environment. Thus we conclude that iron (II) sulphate is actually growth inhibitory and even cytotoxic at high concentrations towards the K562 and T47D cancer cells and the paradoxical proliferative activity of iron (II) sulphate on

  5. 2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)

    African Journals Online (AJOL)

    abbreviara and Ziziphus abbyssinica were claimed to be used for the treatment of cancer. Table 2 shows the effect of the plant extracts on cell lines. At 100 pg/ml Albizia harveyi, Albizia anthelmintica, Dalbergia nitidula, Euphorbia grantii and Rauvolfia caffra exhibited up to 50% cytotoxicity on the three cell lines. Only one.

  6. Neutrophils ingestion rate of nitroblue tetrazolium in asymptomatic ...

    African Journals Online (AJOL)

    This study suggests that the presence of E. coli endotoxin could trigger aggressive antigen phagocytosis in pregnant women with asymptomatic malaria parasiteamia. This could have far reaching consequences in pregnancy. The possibility of co-morbidity of E. coli infection in malaria parasiteamia pregnant women in ...

  7. Tetrazolium chloride as an indicator of pine pollen germinability

    Science.gov (United States)

    Stanton A. Cook; Robert G. Stanley

    1960-01-01

    Controlled pollination in forest tree breeding requires pollen of known germination capacity. Methods of determining pollen viability include germination in a hanging drop, in a moist atmosphere, on agar gel, or in a sugar solution (DUFFIELD, 1954; DILLON et al., 1957). Errors commonly arise in the application of these techniques because maximum...

  8. Study on the triphenyl tetrazolium chloride– dehydrogenase activity ...

    African Journals Online (AJOL)

    The results indicate that dehydrogenase activity (DHA) can effectively facilitate the biochemical reaction of tomato paste wastewater treatment upon analysis of the influences of various DHA and kinetic factors. The biological activity of the activated sludge by TTC-DHA was changed to become applicable to aeration and ...

  9. Spontaneous nitroblue-tetrazolium (NBT) reduction related to granulocyte priming and activation.

    Science.gov (United States)

    Wikström, T; Braide, M; Bagge, U; Risberg, B

    1996-06-01

    The aim of the present study was to explore the relationship between the increasing level of spontaneous NBT-reduction and the tendency for PMNs to marginate during experimental hemorrhagic shock in rats. Rat PMNs, isolated on Percoll density gradients or suspended in blood, were examined by chemiluminescence (CL), NBT-test and by their CD-18 expression and F-actin formation. The NBT-test generally produced higher numbers of activated PMNs when the cells were suspended in buffer than in whole blood, probably due to the scavenging properties of blood. The level of spontaneous NBT-reduction of PMNs in blood correlated with the magnitude of the NBT-response to f-MLP stimulation in blood and buffer. On the contrary, there were no significant correlations between spontaneous NBT reduction, CD18 expression and F-actin content. Thus, high levels of spontaneous NBT reduction in blood were associated with priming of the separated PMNs rather than increased rigidity (F-actin) or adhesiveness (CD18).

  10. The spontaneous and stimulated nitroblue tetrazolium (NBT) tests in mononuclear cells of patients with tuberculosis.

    Science.gov (United States)

    Dubaniewicz, A; Hoppe, A

    2004-01-01

    The aim of this study was to evaluate the ability of the NBT reduction by non- and BCG-stimulated monocytes isolated from peripheral blood persons with pulmonary tuberculosis (TB) before treatment, after two-month antituberculosis therapy and in an inactive stage of this disease. The spontaneous and induced NBT tests were done in 24 healthy individuals and 59 patients with pulmonary tuberculosis: 33 before antituberculosis treatment and 26 with inactive stage of TB. Mononuclear cells were isolated from peripheral blood by the Bøyum method and identified by histochemical assay. The abilities of non- and BCG-stimulated monocytes of NBT reduction were estimated by the method according to Park with Szczylik modification. In an active state of TB and after 2 months treatment, the non- and BCG-stimulated monocytes capacity to reduce NBT was found to be significantly increased in comparison to controls. The NBT test parameters in the absence of cell stimulation and after administration of BCG were comparable in active TB and after two-month treatment. In an inactive TB, the ability of NBT reduction by non- and BCG-stimulated monocytes was comparable to the controls. The stimulation of mononuclear cells accompanied by the significantly higher capacity of monocytes to reduce NBT in controls and in TB patients with post-tuberculous changes in the lungs. These results of the spontaneous and induced NBT tests adequately reflect the status of the host's specific reactivity during tuberculosis and can be a simple, inexpensive and useful method for monitoring antituberculosis treatment.

  11. The nitroblue tetrazolium (NBT) test and white blood cell count in acute throat infections.

    Science.gov (United States)

    Björkstén, B; Ekstrand, T; Gothefors, L; Ostberg, Y

    1975-01-01

    The clinical value of the NBT test and of leucocyte counts in the aetiological differentiation of acute throat infections was investigated. In our hands a frequency of less than 13% NBT positive neutrophils is considered as normal and a test value above 19% as "positive", i.e. indicating a bacterial infection. More than 19% or more than 1 800 NBT positive neutrophils per mm-3 blood were found in 10 of 18 patients with an infection caused by beta-haemolytic streptococci, in 1 of 2 patients with a Mycoplasma pneumoniae infection and in 1 patient with both a streptococcal and mycoplasmal infection, but in none of 19 patients with a viral infection. Since 8 of 18 patients with streptococcal throat infection had normal NBT test results, the NBT test apparently is of limited value in the early recognition of these infections. A high NBT test value would however support the diagnosis. The white blood cell and neutrophil counts were of little value in the differentiation between streptococcal and viral throat infection.

  12. Tetrazolium/Formazan Test as an Efficient Method to Determine Fungal Chitosan Antimicrobial Activity

    Directory of Open Access Journals (Sweden)

    Shaaban H. Moussa

    2013-01-01

    Full Text Available Fungal chitosan was extracted from Aspergillus niger mycelia. The produced chitosan was characterized with deacetylation degree of 89.2%, a molecular weight of 2.4 × 104 Da, and 96.0% solubility in 1% acetic acid solution. The antibacterial activity of fungal chitosan was evaluated against two foodborne pathogens, that is, Salmonella typhimurium and Staphylococcus aureus, using the established antibacterial assays, for example, zone of growth inhibition and agar plat count tests, and using 2,3,5,-triphenyltetrazolium chloride (TTC as chromogenic marker for qualitative and quantitative determining of antibacterial potentiality. The TTC (0.5% w/v was added, at concentration of 10%, to cultured broth, containing chitosan with different concentrations then the formed formazan was separated. The formation of red formazan could be considered as a qualitative indication for antibacterial activity, whereas the measurement of color intensity for the resuspended red formazan, using spectrophotometer at 480 nm, provided a quantitative evidence for the strength of the used antibacterial agent. Regarding the rapidity, technical simplicity, and cost-effectiveness, TTC assay could be recommended as an efficient alternative method for qualitative and quantitative determination of chitosan antibacterial activity and could be suggested for general evaluation of antibacterial agents.

  13. CD4 count and Nitro-Blue Tetrazolium reduction rate of neutrophil in ...

    African Journals Online (AJOL)

    2Department of Medical Laboratory Science, College of Health Sciences,. Nnamdi Azikiwe University, Nnewi Campus, Anambra State, Nigeria. 3Department of Internal Medicine, Usmanu Danfodiyo University/Teaching Hospital, Sokoto, Nigeria. 4Department of Veterinary Microbiology, Faculty of Veterinary Medicine,.

  14. Inhibition of prostaglandin (PG) synthesis in sheep vesicular gland microsomes (SVGM) by nitroblue tetrazolium (NBT) and vitamin E (VE).

    Science.gov (United States)

    Rao, G H; Burris, S M; Gerrard, J M; White, J G

    1979-02-01

    Previous studies have shown that NBT and VE together are potent inhibitors of platelet aggregation, secretion and PG synthesis. In this study, we evaluated the capacity of NBT to detect PG synthesis by SVGM. Aspirin pretreatment of SVGM decreased the amount of NBT reduced after addition of arachidonic acid, demonstrating that products generated by the cyclo-oxygenase were involved in NBT reduction. The influence of NBT and VE on PG synthesis by SVGM was then evaluated by measuring malondialdehyde (MDA) production. NBT or VE alone had no significant effect, but together these agents were as effective as aspirin in preventing MDA formation. The effect of NBT and VE on 14C-arachidonic acid conversion was followed by thin layer chromatography and radioscanning. Again, NBT or VE alone were ineffective, whereas the combination was as effective as aspirin in preventing conversion of arachidonic acid. We speculate NBT and VE together inhibit pg synthesis by scavenging a free radical species of arachidonic acid generated in the initial step of fatty acid peroxidation.

  15. Use of tetrazolium (TTC, Germ's and greenhouse plant emergences methods for testing seed vigour of selected ornamental plant species

    Directory of Open Access Journals (Sweden)

    Roman Hołubowicz

    2013-12-01

    Full Text Available In the years 1996-1997 the experiments were carried out on methods to investigate seed vigour of tassel flower (Amaranthus caudatus L., sand pink (Dianthus chinensis L., babies' breath (Gypsophila elegans M.B., sweet pea (Lathyrus odorathus L., African marigold (Tagetes erecta L. and zinnia (Zinnia elegans Jasq.. The main goals of this research were to specify conditions for accelerated ageing (AA of the seeds of a few selected ornamental plant species and to choose the most appropriate methods for their seed vigour evaluation in the laboratory and greenhouse conditions. All used in the experiments seeds came from the commercial seed lots from Polish seed company. Evaluation was carried out on the seed samples with high and low vigour. The latter ones were received through subjecting the seed samples to AA, i.e. by placing them in 100% relative humidity (RH at 44°C, except African marigold-at 42°C, in the darkness and keeping them for 144, 88, 100, 48, 72 and 72 hours, respectively. The tested seed vigour estimated methods included the Germ's method, the 2,3,5-triphenyl tetrazoilum chloride (TTC method and the test of plant emergences in the greenhouse. The high vigour seeds samples were used as a check. The Germ's method was found to be useful to evaluate sand pink, babies' breath and African marigold seed vigour, whereas the TTC method was found to be suitable for vigour evaluation of sand pink, babies' breath and zinnia. At present stage of our knowledge about seed vigour, the plant emergences in the greenhouse method was found to be the best for evaluation of seed vigour of tassel flower, sand pink, babies' breath, sweet pea and zinnia. It is reasonable to combine a few methods of seed vigour evaluation for ornamental plant species.

  16. Extraction spectrophotometric determination of vanadium in natural waters and aluminium alloys using pyridyl azo resorcinol (PAR) and iodo-nitro-tetrazolium chloride (INT).

    Science.gov (United States)

    Gavazov, K; Simeonova, Z; Alexandrov, A

    2000-06-30

    Extraction-spectrophotometric methods are developed for the determination of vanadium content in natural waters and aluminium alloys. They are based on the formation and subsequent extraction into chloroform of the ternary ion association complex of V(V) with PAR and INT in the presence of CDTA and NH(4)F as masking agents. Optimum pH range of the reaction is 5.5-7.5. Maximum absorbance of the extracted complex is at 560 nm. The method for determination of V(V) in drinking waters can be successfully applied at a concentration level of 3 ppb and higher without additional pre-concentration. Among studied more than 30 foreign ions potentially present in natural waters only Ca(II) can interfere. It is removed by precipitation as CaF(2) and filtration. A 40-fold excess of V(IV) does not interfere with determination of V(V) and can also be determined indirectly (after oxidation to V(V)). The proposed method is applied to analysis of model mixtures as well as to the analysis of tap and mineral waters. Beer's law is obeyed for up to 15 mug of V(V) in 40 ml aqueous phase. The accuracy and precision are reasonable. The RSD is in the range 6.5-23.2% for determination of 6.3 ppb V(V). The procedure for analysis of aluminium alloys differs from the procedure for analysis of waters by the order of introduction of the reagents. The macrocomponent does not interfere and is not separated. Mg, Mn, Cu, Zn, Fe, Cr, Ti and Zr do not interfere. A 25-fold excess of Ni interferes. The method is tested in the analysis of reference standards containing 0.005 and 0.007% V, respectively. The RSD is 1.4%.

  17. A Spectrophotometric Assay for Robust Viability Testing of Seed Batches Using 2,3,5-Triphenyl Tetrazolium Chloride: Using Hordeum vulgare L. as a Model

    Directory of Open Access Journals (Sweden)

    Laura Lopez Del Egido

    2017-05-01

    Full Text Available A comparative analysis was carried out of published methods to assess seed viability using 2,3,5-triphenyltetrazolium chloride (TTC based assays of seed batches. The tests were carried out on seeds of barley (Hordeum vulgare cv. Optic as a model. We established that 10% [w/v] trichloroacetic acid (TCA/methanol is superior to the acetone and methanol-only based methods: allowing the highest recovery of formazan and the lowest background optical density (OD readings, across seed lots comprising different ratios of viable and dead seeds. The method allowed a linear-model to accurately capture the statistically significant relationship between the quantity of formazan that could be extracted using the method we developed and the seed temperature-response, and seed viability as a function of artificially aged seed lots. Other quality control steps are defined to help ensure the assay is robust and these are reported in a Standard Operating Procedure.

  18. Comparative study of B-glucan induced respiratory burst measured by nitroblue tetrazolium assay and real-time luminol-enhanced chemiluminescence assay in common carp (Cyrpinus carpio L.)

    NARCIS (Netherlands)

    Vera-Jimenez, N.I.; Pietretti, D.; Wiegertjes, G.F.; Nielsen, M.E.

    2013-01-01

    The respiratory burst is an important feature of the immune system. The increase in cellular oxygen uptake that marks the initiation of the respiratory burst is followed by the production of reactive oxygen species (ROS) such as superoxide anion and hydrogen peroxide which plays a role in the

  19. Accurate Thermochemical Properties for Energetic Materials Applications. II. Heats of Formation of Imidazolium-, 1,2,4-Triazolium-, and Tetrazolium-Based Energetic Salts fromIsodesmic and Lattice Energy Calculations.

    Energy Technology Data Exchange (ETDEWEB)

    Gutowski, Keith E; Rogers, Robin D; Dixon, David A

    2007-03-01

    The research described in this product was performed in part in the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the Department of Energy's Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory. A computational approach to the prediction of the heats of formation (ΔHf°’s) of solid-state energetic salts from electronic structure and volume-based thermodynamics (VBT) calculations is described. The method uses as its starting point reliable ΔHf°’s for energetic precursor molecules and ions. The ΔHf°’s of more complex energetics species such as substituted imidazole, 1,2,4-triazole, and tetrazole molecules and ions containing amino, azido, and nitro (including methyl) substituents are calculated using an isodesmic approach at the MP2/complete basis set level. On the basis of comparisons to experimental data for neutral analogues, this isodesmic approach is accurate to <3 kcal/mol for the predicted cation and anion ΔHf°’s. The ΔHf°’s of the energetic salts in the solid state are derived from lattice energy (UL) calculations using a VBT approach. Improved values for the ∝ and β parameters of 19.9 (kcal nm)/mol and 37.6 kcal/mol for the UL equation were obtained on the basis of comparisons to experimental UL’s for a series of 23 salts containing ammonium, alkylammonium, and hydrazinium cations. The total volumes are adjusted to account for differences between predicted and experimental total volumes due to different shapes of the ions (flat vs spherical). The predicted ΔHf°’s of the energetic salts are estimated to have error bars of 6-7 kcal/mol, on the basis of comparisons to established experimental ΔHf°’s of a subset of the salts studied. Energetic salts with the highest positive ΔHf°’s are predicted for azido-containing cations, coupled with heterocyclic anions containing nitro substituents. The substitution of functional groups on carbon versus nitrogen atoms of the heterocyclic cations has interesting stabilization and destabilization effects, respectively.

  20. Lesão intestinal após isquemia-reperfusão: estudo comparativo usando sal tetrazólico (MTT e histologia Ischemia-reperfusion injury of intestine: comparative study using tetrazolium salt (MTT and histology

    Directory of Open Access Journals (Sweden)

    Marcus Vinicius Henriques Brito

    2001-03-01

    Full Text Available Vários métodos são utilizados para avaliar e estimar as lesões intestinais de isquemia e reperfusão (IR. Assim, o objetivo do presente trabalho é realizar o estudo comparativo dos aspectos colorimétrico e histológico da lesão intestinal após IR. Para tal, foram utilizados 30 ratos Wistar, machos, pesando entre 310 a 410g, distribuídos em 3 grupos: Grupo Controle (GC, Grupo Isquemia e Reperfusão-1 (GIR-1 e Grupo Isquemia e Reperfusão-3 (GIR-3, com 10 animais cada. Nos grupos GIR-1 e GIR-3 foi realizada isquemia intestinal, por meio de falsa ligadura da artéria mesentérica anterior, durante 30 minutos e após esta a perfusão sangüínea foi restaurada. Estes animais foram submetidos a eutanásia após 1 e 3 dias de reperfusão, respectivamente, sendo colhido material para realização dos estudos colorimétrico, usando o Methyl Thiazolyl Blue (MTT e histológico pela hematoxilina e eosina. Os resultados obtidos demonstraram uma menor proporção de células viáveis e um maior grau de lesão da túnica mucosa nos animais do grupo GIR-3 em relação ao controle (pMany methods are used to evaluate ischemia reperfusion injury, but the most employed one is the histological study. However, it only demonstrates on mucosal tunic, the index of lesion and morphologic preserved cells, but not the index of viable functional cells, present in the sample. With this purpose, a colorimetric method was used, employing Methyl Thiazolyl Blue (MTT. The experiment was conducted in 30 Wistar male rats, distributed in 3 groups: Group Control (GC, Group ischemia and reperfusion-1 (GIR-1 and Group ischemia and reperfusion-3 (GIR-3, with 10 animals each. The Groups GIR-1 and GIR-3 were submitted to intestinal ischemia for 30 minutes by a false ligature of superior mesenteric artery, and submitted to euthanasia after 1 and 3 days of reperfusion, when material was picked for absorbency and histological procedures. It was observed a smaller proportion of viable cells and a larger degree of mucosal lesion in GIR-3 group (p<0.05, while GC group was the one with the larger proportion of viable cells and smaller degree of the mucosal injury (p<0.05. This way we concluded that MTT is as effective and reliable as the histological study at evaluating alterations produced by intestinal ischemia-reperfusion.

  1. INT (2-(4-Iodophenyl)-3-(4-Nitrophenyl)-5-(Phenyl) Tetrazolium Chloride) Is Toxic to Prokaryote Cells Precluding Its Use with Whole Cells as a Proxy for In Vivo Respiration.

    Science.gov (United States)

    Villegas-Mendoza, Josué; Cajal-Medrano, Ramón; Maske, Helmut

    2015-11-01

    Prokaryote respiration is expected to be responsible for more than half of the community respiration in the ocean, but the lack of a practical method to measure the rate of prokaryote respiration in the open ocean resulted in very few published data leaving the role of organotrophic prokaryotes open to debate. Oxygen consumption rates of oceanic prokaryotes measured with current methods may be biased due to pre-incubation size filtration and long incubation times both of which can change the physiological and taxonomic profile of the sample during the incubation period. In vivo INT reduction has been used in terrestrial samples to estimate respiration rates, and recently, the method was introduced and applied in aquatic ecology. We measured oxygen consumption rates and in vivo INT reduction to formazan in cultures of marine bacterioplankton communities, Vibrio harveyi and the eukaryote Isochrysis galbana. For prokaryotes, we observed a decrease in oxygen consumption rates with increasing INT concentrations between 0.05 and 1 mM. Time series after 0.5 mM INT addition to prokaryote samples showed a burst of in vivo INT reduction to formazan and a rapid decline of oxygen consumption rates to zero within less than an hour. Our data for non-axenic eukaryote cultures suggest poisoning of the eukaryote. Prokaryotes are clearly poisoned by INT on time scales of less than 1 h, invalidating the interpretation of in vivo INT reduction to formazan as a proxy for oxygen consumption rates.

  2. Testing of the in vitro susceptibilities of Madurella mycetomatis to six antifungal agents by using the Sensititre system in comparison with a viability-based 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5- [(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) assay and a modified NCCLS method.

    NARCIS (Netherlands)

    W.W.J. van de Sande (Wendy); A. Luijendijk (Ad); A. Ahmed (Asif); I.A.J.M. Bakker-Woudenberg (Irma); A.F. van Belkum (Alex)

    2005-01-01

    textabstractThe in vitro susceptibilities of 36 clinical isolates of Madurella mycetomatis, the prime agent of eumycetoma in Africa, to ketoconazole, itraconazole, fluconazole, voriconazole, amphotericin B, and flucytosine were determined by the Sensititre YeastOne system. This system appeared to be

  3. Teste de tetrazólio para a avaliação da viabilidade de sementes de atemoia (Annona cherimola Mill. x A. squamosa L.)

    OpenAIRE

    Gimenez, Juliana Iassia; Ferreira, Gisela; Cavariani, Claudio

    2014-01-01

    The study aimed to adapt the tetrazolium test to assess the viability of atemoya (Annona cherimola Mill. x A. squamosa L.) seeds. The experimental design was completely randomized in 3 x 3 factorial arrangement (tetrazolium salt concentrations x exposure times), totaling 9 treatments with four replicates of 25 seeds. After immersion in water for 24 hours, the seeds were longitudinally sectioned and exposed to three tetrazolium salt concentrations (0.05%, 0.5% and 1%) for three periods of time...

  4. Page 1 Agro-Science ISBN 1119-7455 Journal of Tropical ...

    African Journals Online (AJOL)

    oxidizes the carbon source in the well, NADH (Nicotina- mide Adenine Dinucleotide Hydrogenase) is formed and it donates electrons to the electron transport chain. The redox dye taps the electrons from the flow, converting the tetrazolium to a highly coloured formazan (Bochner, l989). Thus the redox dye tetrazolium violet ...

  5. Phytochemical investigation of Rhus tripartita and its activity against ...

    African Journals Online (AJOL)

    azino-bis(3-ethylbenzthiazoline-. 6-sulfonic) acid diammonium salt, phenazine methosulphate (PMS)-nicotinamide adenine dinucleotide (NADH), nitroblue tetrazolium. (NBT), Tris-HCl buffer, Greiss reagent, sodium nitroprusside ascorbic acid (Vc), ...

  6. In vitro and in vivo anticancer activities of a novel antibiotic | Xiaoxi ...

    African Journals Online (AJOL)

    -diphenyl tetrazolium bromide (MTT) assays. In KM mice bearing sarcoma S180 that was treated with subcutaneous (s.c.) injection of antibiotic BS, marked cures were obtained. In terms of tumor weight, the treated/control = 0.43.

  7. Inhibition of lipopolysaccharide-induced neuroinflammatory events ...

    African Journals Online (AJOL)

    tetrazolium bromide (MTT) assay. Lipopolysaccharide (LPS) is used to activate BV-2 microglia. Nitric oxide (NO) levels were measured using Griess assay. Inducible NO synthase (iNOS) expressional levels were measured by Western blot analysis.

  8. Discrepancies between metabolic activity and DNA content as tool to assess cell proliferation in cancer research

    OpenAIRE

    Quent, Verena MC; Loessner, Daniela; Friis, Thor; Reichert, Johannes C.; Hutmacher, Dietmar W.

    2010-01-01

    Abstract Cell proliferation is a critical and frequently studied feature of molecular biology in cancer research. Therefore, various assays are available using different strategies to measure cell proliferation. Metabolic assays such as AlamarBlue, water-soluble tetrazolium salt and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, which were originally developed to determine cell toxicity, are used to assess cell numbers. Additionally, proliferative activity can be determined by...

  9. Identification of Small Molecules against Botulinum Neurotoxin B Binding to Neuronal Cells at Ganglioside GT1b Binding Site with Low to Moderate Affinity

    Science.gov (United States)

    2014-10-01

    incubated with BCIP and NBT for chromogenic development. BSA (50 mg/mL) was used as negative control. ELISA: The binding of HCD-BoNT/B to...AP enzyme detection was completed by adding 150 µL of BCIP (5-bromo-4- chloro-3’-indolyl phosphate)/ NBT (nitro-blue tetrazolium) each diluted 1:250...HCC, heavy chain C- terminal sub-domain region of the BD LC, light chain domain NBT , nitro-blue tetrazolium chloride NCI/DTP (National Cancer

  10. Development of Pantothenate Analogs That Can Treat Combat-Related Infections

    Science.gov (United States)

    2012-01-01

    P3350, 0.2 M LiSO4, 0.1 M HEPES and pH 7.5. For KpPanK + N5, a reservoir solution contained 20% PEG3350, 0.2M tri- lithium Citrate and 0.2 ul...MTT dye (Thiazolyl Blue Tetrazolium Bromide ) in two time intervals: 24 and 48 hrs. MTT is reduced to purple formazan in living cells; the absorbance...incubated at 37°C in 5% CO2 for 72 hrs. Following drugs exposure, cell viability was determined with the thiazolyl blue tetrazolium bromide dye (M2128

  11. Protective roles of nitric oxide on antioxidant systems in tall fescue ...

    African Journals Online (AJOL)

    ONOS

    2010-01-18

    Jan 18, 2010 ... The activity of SOD was measured by nitroblue tetrazolium (NBT) method of Beauchamp and Fridovich (1971). One unit of SOD was defined as the amount of enzyme required to cause 50% inhibition of the reduction of NBT as monitored at 560 nm. The activity of CAT was determined by following the ...

  12. Beneficial effects of hesperidin following cis ...

    African Journals Online (AJOL)

    inhibition of nitro blue tetrazolium (NBT) reduction due to. O2. − generated by the xanthine/xanthine oxidase system.[12]. One unit of SOD activity was defined as the amount of protein causing 50% inhibition in the NBT reduction rate. The product was evaluated spectrophotometrically at 560 nm. Results were expressed as ...

  13. The antioxidant properties, cytotoxicity and monoamine oxidase ...

    African Journals Online (AJOL)

    The antioxidant activities were assessed using the thiobarbituric acid-reactive substances (TBARS) assay and the nitroblue tetrazolium (NBT) assay. The cytotoxicity assays were performed according to the microculture MTT method. From the MTT assay, it was determined that at a concentration of 10 mg/ml of crude extract, ...

  14. parasite lactate dehydrogenase assay for the determination

    African Journals Online (AJOL)

    hi-tech

    2005-03-01

    Mar 1, 2005 ... tetrazolium (NBT), phenazine ethosulphate (PES), 3- acetyl pyridine dinucleotide (APAD) and nucleic acid(NAD). The malstat reagent, which is a formulation for parasite LDH detection, was obtained from flow, Inc. (Portland, OR.) and used as outlined earlier (1). The RPMI 1640 medium was obtained from ...

  15. Spo0A positively regulates epr expression by negating the ...

    Indian Academy of Sciences (India)

    2013-03-07

    Mar 7, 2013 ... Migration was monitored by Bromophenol blue and. Xylene cyanol mobility on the gel. Gel was electroblotted onto a Nylon membrane and DNA was detected with nitroblue tetrazolium/5-bromo-4-chloro-3-indolylphosphate (NBT/. BCIP) in ALP buffer (100 mM Tris-Cl, pH 9.5, 100 mM. Table 2. List of primers.

  16. Overexpression of an abiotic-stress inducible plant protein in the ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-09-17

    Sep 17, 2008 ... nitroblue-tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl- phosphate (BCIP) substrates. RESULTS AND DISCUSSION. Because of the vast fund of knowledge about its genetics, biochemistry and molecular biology, E. coli is the system of first choice for expression of many heterologous proteins. Genetic ...

  17. Effect of Controlled Atmosphere Storage (CAS) on Antioxidant ...

    African Journals Online (AJOL)

    photochemical reduction of nitroblue tetrazolium (NBT) [9]. Enzyme activity was measured spectrophotometrically at 560 nm. One unit of SOD was considered to be the amount of enzyme that inhibited NBT reduction by 50%. The decomposition of H2O2 by CAT was measured spectrophotometrically at 240 nm. One unit of ...

  18. Detection of carriers of the autosomal form of chronic granulomatous disease

    NARCIS (Netherlands)

    Verhoeven, A. J.; van Schaik, M. L.; Roos, D.; Weening, R. S.

    1988-01-01

    The NADPH:O2 oxidoreductase catalyzing the respiratory burst in activated phagocytes from healthy individuals is not operative in phagocytes from patients with chronic granulomatous disease (CGD). In a microscopic slide test using the dye nitroblue tetrazolium (NBT), carriers of X-linked CGD can be

  19. Leucocyte Phagocytosis In Children With Urinary Schistosomiasis ...

    African Journals Online (AJOL)

    In the participants considered for this study, leucocyte migration, neutrophil candidacidal activity and ability to generate reactive oxygen were determined as percentage migration index (%M. I), candidacidal phagocytic index (%C.I) and bacterial stimulated nitroblue tetrazolium (NBT) dye reduction index (%NBT) respectively ...

  20. International Journal of Health Research

    African Journals Online (AJOL)

    Erah

    nitroblue tetrazolium (NBT), phenazine methosulphate (PMS), nicotinamide adenine dinucleotide(NADH), trichloroacetic acid. (TCA) and ferric chloride were purchased from Sigma Chemical Co. (St Louis, MO,. USA). All other chemicals and reagents used were of analytical grade. Plant Material. The plant materials were ...

  1. Biokemistri

    African Journals Online (AJOL)

    Chibuike

    the photochemical reduction of nitro blue tetrazolium (NBT). (Beauchamp and Fridovich, 1971) and the method of Martinez et al.,. (2001) to determine superoxide dismutase as modified by Awah et al. (2010). The percentage inhibition of superoxide generation was also estimated by comparing the absorbance of the control ...

  2. pLIVE-EGFP: A liver specific vector carrying the EGFP reporter for ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-10-19

    Oct 19, 2009 ... Subsequently, the slides were tested for AP expression, using a colorimetric assay,. Nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (BCIP/. NBT, Ameresco) according to the manufacturer's instructions. RESULTS AND DISCUSSION. The directional IRES-EGFP insertion was confirmed by.

  3. Cytotoxic and Antioxidant Activities of Antidesma thwaitesianum Müll ...

    African Journals Online (AJOL)

    methods: nitroblue tetrazolium (NBT) dye reduction assay and nitric oxide (NO) scavenging assay. The phenolic and flavonoid contents were assessed colorimetrically at 765 nm and 415 nm respectively. Results: Among the test extracts, the ethanol extracts of fresh fruits (FME) and marc left after squeezing fresh fruits ...

  4. Free radical-scavenging and antimutagenic potential of acetone ...

    African Journals Online (AJOL)

    The antioxidant potency of acetone, chloroform and methanol extracts of Argemone mexicana was investigated by employing in vitro systems like nitroblue tetrazolium (NBT) and 1,1-Diphenyl-2- picrylhydrazyl (DPPH) free radical scavenging assay whereas antimutagenic activity was determined by Maron and Ames assay ...

  5. The high performance liquid chromatography (HPLC) analysis of ...

    African Journals Online (AJOL)

    use

    2011-11-23

    Nov 23, 2011 ... malondialdehyde; NBT, nitro-blue tetrazolium; IR, infrared;. DNA, deoxyribonucleic acid; UV-Vis, ultraviolet/visible. internal enzymatic mechanisms fail or become inefficient against oxidative damage (Ślusarczyk et al., 2009). These natural antioxidants could be replacement for the synthetic antioxidants ...

  6. In vitro cytotoxic and antioxidant properties of the aqueous ...

    African Journals Online (AJOL)

    user

    2010-09-23

    Sep 23, 2010 ... The superoxide anion radical scavenging activity was performed using the method of Okamura et al. (1993) with some modification. This assay is based on the removal rate of xanthine/xanthine oxidase-generated superoxide by measuring the reduction of nitro blue tetrazolium (NBT). The sample solution ...

  7. Effects of Zinc Exposure on Accumulation, Haematology and ...

    African Journals Online (AJOL)

    BILGISAYAR1

    2013-02-13

    Feb 13, 2013 ... A decrease was found in nitroblue tetrazolium (NBT) activity with medium and high concentrations; in the lysozyme and myeloperoxidase activities was found with high concentrations (P<0.05). In this study, the highest zinc accumulation rate was found in the liver tissue, and the lowest rate was found in the ...

  8. Extremophilic Enzymatic Response for Protection against UV-Radiation Damage

    Science.gov (United States)

    2012-09-17

    assayed by the method of Winterbourn et al. based on the ability of SOD to inhibit the reduction of nitroblue tetrazolium ( NBT ) by superoxide. One...unit was defined as the amount of enzyme causing one half of the maximum inhibition of NBT reduction. The graph shows that the enzyme is active at a

  9. Effect of 1-methylcyclopropene (1- MCP) treatment on antioxidant ...

    African Journals Online (AJOL)

    ajl2

    2013-02-13

    Feb 13, 2013 ... *Corresponding author. E-mail: gaozhihong@njau.edu.cn. Abbreviations: PODs, Peroxidases; SODs, superoxide dismutases; CAT, catalase; SSC, soluble solids content; TA, titratable acidity; NBT, nitroblue tetrazolium. effective than the control group at retarding the decrease in hardness and slowing down ...

  10. Effect of garlic, black seed and Biogen as immunostimulants on the ...

    African Journals Online (AJOL)

    Significant changes were seen in nitroblue tetrazolium (NBT) values in groups treated with 1.0 and 3.0% garlic. Mortalities following challenge with Pseudomonas fluorescens were lower in the groups that received garlic compared to the other two treatments. At the end of winter a significant increase in body weight gain ...

  11. Evaluation of the membrane attack complex of complement for the detection of a recent myocardial infarction in man

    NARCIS (Netherlands)

    Robert-Offerman, S. R.; Leers, M. P.; van Suylen, R. J.; Nap, M.; Daemen, M. J.; Theunissen, P. H.

    2000-01-01

    The diagnosis of an acute myocardial infarction (MI) can be cumbersome for pathologists. Even with a positive nitroblue tetrazolium (NBT) reaction, haematoxylin and eosin (H&E) evaluation of the myocardial tissue can remain inconclusive. Early signs presumed diagnostic for myocardial infarction,

  12. Volume 9 No. 5 2009 August 2009 1210 EFFECT OF PRE ...

    African Journals Online (AJOL)

    The supernatant collected was used for estimation of POX activity. Superoxide dismutase Assay. The activity of SOD was assayed by measuring the ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT) using a published procedure. [16]. Enzyme activity was measured spectrophotometrically at 560 nm.

  13. PAX6 can substitute for LHX2 and override NFIA-induced ...

    Indian Academy of Sciences (India)

    Veena Kinare

    2018-01-24

    Jan 24, 2018 ... colour reaction was performed using NBT/BCIP (Roche,. 4-nitroblue tetrazolium chloride, cat no. 70210625;. 5-Bromo-4-chloro-3-idolyl phosphate, cat no. 70251721) as per manufacturer's instructions. Slides were counterstained with Fast Red (Sigma N3020), coverslipped using DPX mountant and imaged ...

  14. Author Details

    African Journals Online (AJOL)

    Use of absolute lymphocyte count or neutrophil ingestion rate of nitroblue tetrazolium (NBT) as alternative index to CD4+T-cell count to initiate ART in the management of HIV/AIDS disease. Abstract PDF · Vol 6, No 4 (2012) - Articles Serum hormonal levels in HIV/AIDS infected male subjects on antiretroviral therapy (ART) ...

  15. [Peripheral blood neutrophil subpopulations and capacities of NBT test in the diagnosis of neonatal diseases].

    Science.gov (United States)

    Gerasimov, I G

    2011-04-01

    By analyzing the data available in the literature, the author shows new capacities of the nitroblue tetrazolium (NBT) test in the diagnosis of neonatal diseases and conditions. The findings versus the, data obtained in adult patients are characterized. The NBT test has been used to determine changes in neutrophil subpopulations. The kinetic parameters of the process are analyzed.

  16. Ascorbic acid in bronchial asthma

    African Journals Online (AJOL)

    1983-04-23

    Apr 23, 1983 ... Polymorphonuclear leucocyte (PMNL) functions. PMNLs were obtained from heparinized venous blood and resuspended in Hanks's balanced salt solution as previously describ.ed. 1 Investigations of PMNL motility, phagocytosis and nitroblue tetrazolium (NBT) reduction were performed using ...

  17. Mechanism of bacterial adaptation to low temperature

    Indian Academy of Sciences (India)

    Madhu

    cyano-2,3-ditoyl tetrazolium chloride ... –12°C, in Arctic sea ice has also been reported. This strain named Psychromonas ingrahamii, was .... proteins, called cold acclimation proteins (Caps), is found to be produced exclusively in psychrophilic ...

  18. Linking surface-fire behavior, stem heating, and tissue necrosis

    Science.gov (United States)

    A.S. Bova; M.B. Dickinson; M.B. Dickinson

    2005-01-01

    Data from 69 experimental, small-plot fires are used to describe relationships among fire intensity, barksurface heat flux, and depth of necrosis in stem tissue for red maple (Acer rubrum L.) and chestnut oak (Quercus prinus L.j. A tetrazolium staining technique was used to determine the depth of necrosis in tree boles subjected to fires with intensities of 20 to 2000...

  19. In vitro and in vivo anticancer activities of a novel antibiotic

    African Journals Online (AJOL)

    Jane

    2011-08-03

    Aug 3, 2011 ... In vitro and in vivo anticancer activities of a novel antibiotic. Chen Xiaoxi. Basic Medicine College, Zhejiang ... paper is to assay the BS's in vitro anticancer activity via. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium .... practice of oncology. Philadelphia, Lippincott Raven, pp. 328-339. Karikas GA (2010) ...

  20. Anti-tumor effect of polysaccharides isolated from Taraxacum ...

    African Journals Online (AJOL)

    The effects of extraction temperature, liquid-solid ratio and extraction time on the yield of PTM were investigated using a Box-Behnken design (BBD). The in vitro anti-tumor effect of PTM on MCF-7 cells was investigated by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, while the mechanism of PTM-induced ...

  1. Anticancer and antibacterial secondary metabolites from the ...

    African Journals Online (AJOL)

    according to morphologic traits and a molecular biologi- cal protocol by DNA amplification and sequencing as de- scribed by Douanla et al (2013).18. Fermentation .... This cell viability assay is based on living cells' property to transform the MTT dye tetrazolium ring into a purple-colored formazan structure due to the action ...

  2. Tropical Journal of Medical Research - Vol 12, No 1 (2008)

    African Journals Online (AJOL)

    Neutrophil Ingestion Rate Of Nitroblue Tetrazolium In Subjects With Malaria-Hiv Co-Morbidity · EMAIL FULL TEXT EMAIL FULL TEXT · DOWNLOAD FULL TEXT DOWNLOAD FULL TEXT. CC Onyenekwe, NR Ukibe, SC Meludu, CM Ifeanyi, AO Igwegbe, M Ezeani, I Ezeugwunne, N Ofiaeli, A Onochie, A Ilika, N Abor, 1-5.

  3. Soil-seed bank survival in forests of the southern United States

    Science.gov (United States)

    James S. Meadows; Frank T. Bonner; James D. Haywood

    2006-01-01

    We evaluated the longevity of seeds of 12 common woody species buried in fresh condition in the forest floor at three forest locations in Mississippi and Louisiana. Seed samples of each species were retrieved annually for 5 years from each location. Germination and tetrazolium chloride staining tests were conducted on the samples to determine germinative capacity. When...

  4. Temperature-controlled radiofrequency ablation of cardiac tissue

    DEFF Research Database (Denmark)

    Petersen, H H; Chen, X; Pietersen, Adrian

    1999-01-01

    was induced by circulating the saline in the tissue bath at a flow-velocity of 0.1 m/s. Lesion volume was determined by cutting lesions in 1 mm thick slices, staining with nitroblue tetrazolium and planimetering. A total of 107 lesions was created. Lesion size was significantly larger for perpendicular...

  5. Effects of RNAi-mediated cathepsin L gene silencing on bionomics ...

    African Journals Online (AJOL)

    The expression of cathepsin L in hepatoma carcinoma cells was detected by immunofluorescence, reverse transcription polymerase chain reaction (RT-PCR) and western blot. Cell viability was detected by methyl thiazolyl tetrazolium (MTT) assay. The changes of cell cycle and apoptosis were observed by flow cytometry.

  6. Disease: H00657 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available stinct human myopathies: a comprehensive review of the clinical, histological and pathological features. Neu...es fatal, X-linked disorder characterized by progressive muscle weakness and the presence of intracytoplasmic aggregates in histologi...cal muscle sections which exert a reducing activity on nitro-blue tetrazolium (NBT)

  7. Physiological and antioxidant responses of three leguminous ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-11-02

    Nov 2, 2009 ... play a key role by reducing H2O2 to water through the. Halliwell–Asada pathway (Noctor and Foyer, 1998). .... as the reduction of nitro-blue tetrazolium (NBT) at 560 nm. (Beauchamp and Fridovich, 1971). ..... assays and an assay applicable to acrylamide gels. Anal. Biochem. 44: 276-287. Bergmeyer N ...

  8. Anti-Cancer Properties of Diethylether Extract of Wood from Sukun ...

    African Journals Online (AJOL)

    Purpose: To evaluate the anti-cancer properties of the diethylether extract of Sukun (Artocarpus altilis) wood. Methods: The extract was tested in human T47D breast cancer cells and examined for its effect on cell viability, nuclear morphology and sub-G1 formation. Cell viability was determined by microculture tetrazolium ...

  9. Modulatory effects of Thai medicinal plant extract on ...

    African Journals Online (AJOL)

    STORAGESEVER

    2010-08-02

    Aug 2, 2010 ... diphenyl-tetrazolium bromide; DMEM, Dulbecco's modified eagle's medium. interface between the body and the environment and is vulnerable to pathological conditions caused by various factors including chemical and microbial agents, thermal and electromagnetic radiation, and mechanical trauma.

  10. (MTT) dye reduction assay.

    African Journals Online (AJOL)

    to inhibit proliferation of HeLa cells was determined using the 3443- dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) dye reduction assay. Extracts from roots of Agathisanthemum bojeri, Synaptolepis kirkii and Zanha africana and the leaf extract of Physalis peruviana at a concentration of 10 pg/ml inhibited cell ...

  11. Colorimetric micro-assay for accelerated screening of mould inhibitors

    Science.gov (United States)

    Carol A. Clausen; Vina W. Yang

    2013-01-01

    Since current standard laboratory methods are time-consuming macro-assays that rely on subjective visual ratings of mould growth, rapid and quantitative laboratory methods are needed to screen potential mould inhibitors for use in and on cellulose-based products. A colorimetric micro-assay has been developed that uses XTT tetrazolium salt to enzymatically assess...

  12. Accelerated Colorimetric Micro-assay for Screening Mold Inhibitors

    Science.gov (United States)

    Carol A. Clausen; Vina W. Yang

    2014-01-01

    Rapid quantitative laboratory test methods are needed to screen potential antifungal agents. Existing laboratory test methods are relatively time consuming, may require specialized test equipment and rely on subjective visual ratings. A quantitative, colorimetric micro-assay has been developed that uses XTT tetrazolium salt to metabolically assess mold spore...

  13. CYTOTOXICITY OF FLAVONOIDS AND SESQUITERPENE LACTONES FROM ARNICA SPECIES AGAINST THE GLC(4) AND THE COLO-320 CELL-LINES

    NARCIS (Netherlands)

    WOERDENBAG, HJ; MERFORT, [No Value; PASSREITER, CM; SCHMIDT, TJ; WILLUHN, G; VANUDEN, W; PRAS, N; KAMPINGA, HH; KONINGS, AWT

    1994-01-01

    The cytotoxicity of 21 flavonoids and 5 sesquiterpene lactones, as present in Arnica species, was studied in GLC(4), a human small cell lung carcinoma cell line, and in COLO 320, a human colorectal cancer cell line, using the microculture tetrazolium (MTT) assay. Following continuous incubation,

  14. Effects of RNAi-mediated cathepsin L gene silencing on bionomics ...

    African Journals Online (AJOL)

    USER

    2010-08-02

    Aug 2, 2010 ... Cell viability was detected by methyl thiazolyl tetrazolium (MTT) assay. The changes of cell cycle and apoptosis were observed by flow cytometry. The changes of invasiveness of hepatoma carcinoma cells were detected by Boyden chamber. Compared with the blank group and fluorescence control group ...

  15. Effect of Triptolide on Functions of Monocytes/ Macrophages in ...

    African Journals Online (AJOL)

    The number of monocytes/macrophages under the varying conditions was subsequently determined by methyl thiazolyl tetrazolium (MTT) assay. The supernatants were collected after 24-h culture, and the content of VEGF and VEGF-C in each supernatant measured by enzyme-linked immunosorbent assay (ELISA).

  16. Heating ability and biocompatibility study of silica-coated magnetic ...

    Indian Academy of Sciences (India)

    The core–shell structure of nanoparticles was confirmed by X-ray diffraction, fourier transform infrared spectroscopy and transmission electron microscopy analyses. Cytotoxicity of bare and silica-coated nanoparticles was evaluated by methyl thiazol tetrazolium bromide assay with MCF-7 cell line. The results revealed that ...

  17. In vitro activity of Piper sarmentosum ethanol leaf extract against ...

    African Journals Online (AJOL)

    Non-toxic concentrations of the ethanol extract for Vero cells were determined by methyl thiazolyl tetrazolium (MTT) cell proliferation. The presence of Toxoplasma gondii was observed by Giemsa staining. Results: The results showed that significant (p < 0.05) anti-toxoplasma activity of the ethanol extract, though lower than ...

  18. Anticancer Activity of Tetrahydrocorysamine against Pancreatic ...

    African Journals Online (AJOL)

    The effects of TCSM on the proliferation and apoptosis of PANC-1 cells were determined by methyl thiazolyl tetrazolium (MTT) and flow cytometry assays. The effect of TCSM on the expressions of mitochondria-mediated apoptotic proteins were investigated by Western blot assay. Xenograft assay was used to evaluate the ...

  19. Apigenin inhibits proliferation and migratory properties of Barrett's ...

    African Journals Online (AJOL)

    Methods: Proliferation index of OE33 in the absence and presence of apigenin was determined by methyl-thiazolyl-tetrazolium (MTT) assay and apoptosis was determined by enzyme-linked immunosorbent assay (ELISA) method. Boyden Chamber's assay was applied to determine the migration and invasion of control and ...

  20. In vitro antitumor effects of a new cultivar (Gürarslan) of Trigonella ...

    African Journals Online (AJOL)

    Materials and Methods: The effect on cell proliferation of these extracts was detected by using methyl thiazolyl diphenyl tetrazolium (MTT) assay. Mitotic index and labelling index was determined using the Feulgen staining and autoradiography methods, respectively. Results: Our findings show that the methanolic extract of ...

  1. Chloroquinone Inhibits Cell Proliferation and Induces Apoptosis in ...

    African Journals Online (AJOL)

    Purpose: To demonstrate the role of chloroquinone (CQ) in inducing apoptosis in HONE-1 and HNE-1, nasopharyngeal carcinoma (NPC) cell lines. Methods: Water-soluble tetrazolium salt (WST)-1 assay was used for the determination of cell proliferation while an inverted microscope was employed for the analysis of ...

  2. Studies on cytotoxicity and antitumor screening of red and white ...

    African Journals Online (AJOL)

    The cytotoxicity study with cancer cell line A-549 indicated that the methanol insoluble fraction of crude red forms of Abrus precatorius is toxic (CTC50 175,100 mg/ml) in microculture tetrazolium assay and sulphorhodamine B assays, whereas crude and fractions of the red and white forms exhibited toxicity at still higher ...

  3. Homogeneous distribution of phosphofructokinase in the rat liver acinus: a quantitative histochemical study

    NARCIS (Netherlands)

    Frederiks, W. M.; Marx, F.; van Noorden, C. J.

    1991-01-01

    A quantitative histochemical method was developed for the demonstration in rat liver of the activity of phosphofructokinase, one of the enzymes assumed to be rate-limiting for glycolysis. The procedure was based on the reduction of a tetrazolium salt as final electron acceptor and a multistep

  4. Development of an Isoelectric Focusing Technique for ...

    African Journals Online (AJOL)

    ... dehydrogenase ( -GPD); hexokinase (HK); leucine-alanine peptidase (LeAP); malate dehydrogenase (MTDH); malic enzyme (ME); malic enzyme dehydrogenase (MDH); peptidase P (PEP-P); peroxidase (POD); phosphoglucomutase (PGM); sorbitol dehydrogenase (SOD) and tetrazolium oxidase (TOD) were screened in ...

  5. Structure-cytotoxicity relationships of a series of natural and semi-synthetic simple coumarins as assessed in two human tumour cell lines

    NARCIS (Netherlands)

    Kolodziej, H; Kayser, O; Woerdenbag, HJ; vanUden, W; Pras, N

    1997-01-01

    The cytotoxicity of 22 natural and semi-synthetic simple coumarins was evaluated in GLC(4), a human small cell lung carcinoma cell line, and in COLO 320, a human colorectal cancer cell line, using the microculture tetrazolium (MTT) assay. With IC50 values > 100 mu M, following a continuous (96h)

  6. Liquid-liquid extraction of ion-association complexes of cobalt(II-4-(2-pyridylazoresorcinol with ditetrazolium salts

    Directory of Open Access Journals (Sweden)

    Divarova Vidka V.

    2015-01-01

    Full Text Available The formation and liquid-liquid extraction of ion-association complexes between Co(II-4-(2-Pyridylazoresorcinol (PAR anionic chelates and cations of three ditetrazolium chlorides were studied: Blue Tetrazolium chloride (BTC, Neotetrazolium chloride (NTC and Nitro Blue Tetrazolium chloride (NBT. The optimum conditions for the formation and solvent extraction of the ion-association comlpex chelates were determined. It has been found that in the systems of Co(II-PAR-DTS, the reactants are reacted in molar ratios 1:2:1 and the general formula of complexes was suggested. The extraction equilibria were investigated and quantitatively characterized by the equilibrium constants and the recovery factors. The analytical characteristics of the complexes were calculated.

  7. Interactions of selected bacterial isolates with DBT and solubilized coal

    Energy Technology Data Exchange (ETDEWEB)

    Key, D.H.; Fox, R.V.; Kase, R.S.; Willey, M.S.; Stoner, D.L.; Ward, T.E.

    1990-01-01

    We are studying the interactions of isolated bacteria with dibenzothiophene (DBT), a sulfur-containing model compound, and with a solubilized coal product derived from a high-organic-sulfur lignite. The sensitivity of the tetrazolium assay used to identify and study these strains was improved by substituting tetrazolium violet for triphenyltetrazolium. DBT metabolism by thirteen strains was investigated using qualitative and quantitative GC and GC-MS analyses. Growth medium and incubation time affect the extent of DBT degradation and the production of DBT metabolites. Under specific conditions, seven of the strains produce metabolites which elute close to the position of one or another of the biphenyl standards. However, when these samples are spiked with the standard compounds, the bacterial metabolites do not co-elute with the standards. The modification of solubilized high-organic-sulfur coal by six of these strains was also studied. No selective removal of sulfur relative to carbon was observed. 13 refs., 1 fig., 2 tabs.

  8. Influence of silicon doping of titanium nickelide near-surface layers on alloy cytocompatibility

    Science.gov (United States)

    Lotkov, A. I.; Matveev, A. L.; Artemyeva, L. V.; Meysner, S. N.; Matveeva, V. A.; Kudryashov, A. N.

    2017-12-01

    The cytocompatibility of titanium nickelide (TiNi) with near-surface layers doped with silicon ions was studied on mesenchymal stem cells of rat bone marrow cultivated in vitro. The cytotoxic effect of eluted components of material on the mesenchymal stem cells was determined using a RTCA iCELLigence cellular analyzer. The proliferative activity of mesenchymal stem cells cultivated in the presence or on the surfaces of titanium nickelide samples was estimated from the cell mitochondrial respiration rate in MTT tests using [2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium] tetrazolium salt. It is shown that ion plasma modification of near-surface layers of titanium nickelide with silicon improves the cytocompatibility of the alloy.

  9. Sealing of Corneal Lacerations Using Photo Activated Rose Bengal Dye and Amniotic Membrane

    Science.gov (United States)

    2017-04-22

    compared to the mean temperature between 60 and 360 176 minutes irradiation. 177 The lactate dehydrogenase-nitro blue tetrazolium (LDH- NBT ) staining...temperature increased 8 degrees. 223 In order to assess damage to iris cells during the procedure. the LDH- NBT assay was used which 224 shows deposition...Using the LDH- NBT staining method, an accepted method for detecting 292 thermal damage in tissues (Sherwood and Flotte, 2007), our results indicated

  10. 749-IJBCS-Article-Awah Francis M

    African Journals Online (AJOL)

    DR GATSING

    reduction of nitro blue tetrazolium (NBT) as described by Martinez et al. (2001) with slight modifications (Awah et al., 2010). Briefly, each 3.0 ml reaction mixture contained 0.05. M PBS (pH 7.8), 13 mM methionine, 2 μM riboflavin, 100 μM EDTA, NBT (75 μM) and. 1.0 ml of test sample solutions. The tubes were kept in front of ...

  11. A comparative study of the curative effect of two different species of

    African Journals Online (AJOL)

    sgeorge

    inhibition of NBT (Nitroblue tetrazolium) reduction /min/mg protein. The reaction mixture contained 150μl EDTA, 600μl L-methionine, 300μl NBT and make the volume up to 2.8ml by the addition of SOD buffer. To the reaction mixture, was added 200μl of the lysate except in the control. Finally to this was added 200μl of.

  12. A modified Ce/Mg-BCIP-NBT formazan/indigoblue technique for demonstration of non-specific alkaline phosphatase activity.

    Science.gov (United States)

    Halbhuber, K J; Krieg, R; Geidel, O; Dietz, W

    2004-01-01

    The wide ranged structurally variability of formazans and their accessibility for auxiliary additives as redoxmediators or metals provide an easy tunable chromogenic visualization technique. We present here an improved nitro blue tetrazolium (NBT) 5-bromo-4-chloro-3-indolyl phosphate (BCIP) method which is superior to the classical McGadey's procedure regarding proper precipitation and localization as well as sensitivity. Different metal additives as well as the overall reaction course modifying additives (redox mediators, chelating additives, buffer) were optimized.

  13. Physiological and antioxidant responses of three leguminous ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-11-02

    Nov 2, 2009 ... methionine, 75 µM nitroblue tetrazolium (NBT), 10 µM EDTA-Na2,. 2.0 µM riboflavin and 0.3 ml enzyme extract. The reaction mixture was kept at 35°C for 10 min under 4,000 lx. One unit SOD activity was defined as the amount of enzyme required to result in a 50% inhibition of the rate of NBT reduction ...

  14. Fluorescent in situ hybridization employing the conventional NBT/BCIP chromogenic stain.

    Science.gov (United States)

    Trinh, Le A; McCutchen, Marshall D; Bonner-Fraser, Marianne; Fraser, Scott E; Bumm, Lloyd A; McCauley, David W

    2007-06-01

    In situ hybridization techniques typically employ chromogenic staining by enzymatic amplification to detect domains of gene expression. We demonstrate the previously unreported near infrared (NIR) fluorescence of the dark purple stain formed from the commonly used chromogens, nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP). The solid reaction product has significant fluorescence that enables the use of confocal microscopy to generate high-resolution three-dimensional (3-D) imaging of gene expression.

  15. ORIGINAL ARTICLE

    African Journals Online (AJOL)

    tetrazolium (NBT) according to the method of. Gianopolids and Ries (11). A 3ml of reaction mixture containing 50mM potassium phosphate buffer (pH 8.0), l33mM methionine, 75um NBT,. Zurn riboflavin, 0.1 mM EDTA and 100pl of enzyme extract, the reaction mixture illuminated for 20 minutes at a light intensity of.

  16. Effects of oxyresveratrol and its derivatives on cultured P19-derived ...

    African Journals Online (AJOL)

    established protocols. The assay was based on the capacity of the test sample to inhibit the reduction of nitroblue tetrazolium (NBT) in the riboflavin-light-NBT system. A 96-well microplate was used. The reaction mixture (200 μL) in each well contained 20 μL of 50 mM potassium phosphate buffer,100 μL of 266 μM riboflavin, ...

  17. Telomerase Inhibition Decreases Alpha-Fetoprotein Expression and Secretion by Hepatocellular Carcinoma Cell Lines: In Vitro and In Vivo Study

    OpenAIRE

    Tahtouh, Roula; Azzi, Anne-Sophie; Alaaeddine, Nada; Chamat, Soulaima; Bouharoun-Tayoun, Hasnaa; Wardi, Layal; Raad, Issam; Sarkis, Riad; Antoun, Najibe Abou; Hilal, George

    2015-01-01

    Alpha-fetoprotein (AFP) is a diagnostic marker for hepatocellular carcinoma (HCC). A direct relationship between poor prognosis and the concentration of serum AFP has been observed. Telomerase, an enzyme that stabilizes the telomere length, is expressed by 90% of HCC. The aim of this study was to investigate the effect of telomerase inhibition on AFP secretion and the involvement of the PI3K/Akt/mTOR signaling pathway. Proliferation and viability tests were performed using tetrazolium salt. A...

  18. Antitumor evaluation of epigallocatechin gallate by colorimetric methods

    Energy Technology Data Exchange (ETDEWEB)

    Baek, Soon Ok [Korean Ginseng and Tobacco Research institute, Daejon (Korea, Republic of); Kim, Il Kwang; Baek, Seung Hwa; Han, Du Seok [Wonkwang Unvi., Iksan (Korea, Republic of)

    1998-08-01

    In the present study, we were evaluated cytotoxic effects of epigallocatechin gallate in human skin melanoma cells such as HTB-69. The light microscopic study showed morphological changes of the treated cells. Disruptions in cell organelles were determined by colorimetric methods; 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, neutral red (NR) assay and sulforhodamine B protein (SRB) as-say. These results suggest that epigallocatechin gallate retains a potential antitumor activity.

  19. Chemiluminescence by polymorphonuclear leukocytes adhering to surfaces.

    OpenAIRE

    Yanai, M.; Quie, P. G.

    1981-01-01

    Stimulation of the plasma membranes of granulocytes results in an oxidative metabolic response. This response can be measured by measuring the reduction of oxidizable substrates, such as Nitro Blue Tetrazolium, as well as by measuring the energy released as light (chemiluminescence). While investigating the oxidative response of human granulocytes, we observed a marked variation in the chemiluminescence response when leukocytes were suspended in a balanced salt solution without gelatin or any...

  20. Chemical composition of the essential oil of whole plant of ...

    African Journals Online (AJOL)

    The effect of the essential oil on proliferation of SMMC-7721 cells was studied by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, with L02 and HeLa cells serving as control groups. Results: GC-MS results show that the essential oil of E. dense contains 40 components. Thirty seven ...

  1. Effects of moisture stress on germination and protein synthesis in ...

    African Journals Online (AJOL)

    ... 3, 5 triphenyl tetrazolium chloride (TTC), and their abilities to synthesize protein after stress by incorporating L- 4,5-3H leucine into their root tips. Les graines de dolique non pigmentées, TVX 3236 (crème et brune) et IT81S-818 (blanche), étaient exposées aux conditions d'humidité constantes plus stressantes (-0.1 et ...

  2. Screening for antibacterial and antibiofilm activity in Thai medicinal plant extracts against oral microorganisms

    OpenAIRE

    Teanpaisan, Rawee; Kawsud, Pajaree; Pahumunto, Nuntiya; Puripattanavong, Jindaporn

    2016-01-01

    To evaluate the antibacterial activity of 12 ethanol extracts of Thai traditional herb against oral pathogens. The antibacterial activities were assessed by agar well diffusion, broth microdilution, and time-kill methods. Antibiofilm activity was investigated using a 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium-bromide (MTT) assay. High performance liquid chromatography (HPLC), thin layer chromatography (TLC) fingerprinting, and TLC-bioautography were used to determine the active ...

  3. Cell Enumeration Assays: Application of the MTT and Sulforhodamine B Assays to Lipopolysaccharide-Stimulated Neonatal Rodent Microglia.

    Science.gov (United States)

    Facci, Laura; Skaper, Stephen D

    2018-01-01

    Glial cell activation, in particular microglia, plays an important role in the pathogenesis of various neurodegenerative disorders as well as in chronic and neuropathic pain. This chapter compares two established cell enumeration assays, namely, the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay and the protein-binding sulforhodamine B assay for microglia as a function of culture condition and activation state. The pros and cons of each are then described.

  4. Identificação da viabilidade de sementes de orquídeas pelo teste de tetrazólio

    OpenAIRE

    Soares, Jackeline Schultz; Universidade Federal da Grande Dourados; Rosa, Yara Brito Chaim Jardim; Universidade Federal da Grande Dourados; Tatara, Mariana Bento; Faculdade de Ciências da Saúde; Sorgato, José Carlos; Universidade Federal da Grande Dourados; Lemes, Camila Soares Rosa; Universidade Federal da Grande Dourados

    2014-01-01

    The knowledge of germinal potential optimizes the practice of in vitro germination of Orchidaceae. This work evaluated of the type and concentrations of tetrazolium solutions on reaction time and on the identification of viable orchid seeds. Were used seeds of the species Brassavola tuberculata Rchb f., Cattleya labiata Lindley var Amoema, Cattleya intermedia and Dendrobium antenatum and of two primary hybrids of Cattleya labiata autumnalis x Cattleya labiata Lindley var. Alba. The seeds (0.0...

  5. Petiveria alliacea extracts uses multiple mechanisms to inhibit growth of human and mouse tumoral cells

    OpenAIRE

    Urue?a, Claudia; Cifuentes, Claudia; Casta?eda, Diana; Arango, Amparo; Kaur, Punit; Asea, Alexzander; Fiorentino, Susana

    2008-01-01

    Abstract Background There is ethnopharmacological evidence that Petiveria alliacea can have antitumor activity; however, the mechanism of its cytotoxic activity is not well understood. We assessed multiple in vitro biological activities of an ethyl acetate soluble plant fraction over several tumor cell lines. Methods Tumor cell lines were evaluated using the following tests: trypan blue exclusion test, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], flow cytometry, cytosk...

  6. Comparative analysis of quantitative methodologies for Vibrionaceae biofilms

    OpenAIRE

    Chavez-Dozal, Alba A.; Nourabadi, Neda; Erken, Martina; McDougald, Diane; Nishiguchi, Michele K.

    2016-01-01

    Multiple symbiotic and free living Vibrio sp. grow as a form of microbial community known as biofilm. In the laboratory, methods to quantify Vibrio biofilm mass include crystal violet staining, direct Colony Forming Unit (CFU) counting, dry biofilm cell mass measurement, and observation of development of wrinkled colonies. Another approach for bacterial biofilms also involves the use of tetrazolium (XTT) assays (used widely in studies of fungi) that are an appropriate measure of metabolic act...

  7. Ultraviolet Photoionization Efficiency of the Vaporized Ionic Liquid 1-Butyl-3-Methylimidazolium Tricyanomethanide: Direct Detection of the Intact Ion Pair (Post Print)

    Science.gov (United States)

    2012-09-21

    papers were published on the subject, whereas in 2010, over 6000 papers were published [SciFinder results searching for the concept “ionic liquid” (August...not limited to, ammonium, imidazolium, triazolium, tetrazolium, and phosphonium ions. Common anions include the halide , nitrate, dicyanamide...are more acidic, and those ILs have been termed “protic” ILs.9 In general, protic ILs have lower thermal stability than ILs with alkyl substituents on

  8. Expression of mineralization markers during pulp response to biodentine and mineral trioxide aggregate.

    OpenAIRE

    Dalto é, Mariana O.; Paula-Silva, Francisco Wanderley G.; Faccioli, Lucia H.; Gatón Hernández, Patrícia; Rossi, Andiara de; Silva, Léa Assed Bezerra da

    2016-01-01

    INTRODUCTION: The purpose of this study was to compare the cell viability of dental pulp cells treated with Biodentine (Septodont, Saint-Maur, France) and mineral trioxide aggregate (MTA) and the in vitro and in vivo expression of mineralization markers induced by the 2 materials. METHODS: Human dental pulp cells isolated from 6 permanent teeth were stimulated with Biodentine and MTA extracts. Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromid...

  9. Reference: 526 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available tic suberin in young roots and produced seed coats with a severalfold reduction in very long chain dicarboxy...mposition or content of membrane or storage glycerolipids or surface waxes. Consistent with their altered suberin, seed coats... of gpat5 mutants had a steep increase in permeability to tetrazolium salts compared with wild-type seed coats... a critical role of GPAT5 in polyester biogenesis in seed coats and roots and for the importance of lipid po

  10. Biological activities and phenolic contents of Argania spinosa L ...

    African Journals Online (AJOL)

    Cytotoxic activity was evaluated by methyl-thiazolyldiphenyl-tetrazolium bromide (MTT) assay. Results: The results revealed abundant polyphenols and flavonoids (221.39 ± 5.70 μg GAEq/1 g and 66.86 ± 3.36 μg CAEq/1 g, respectively) in the leaf extract. UPLC-DAD-ESI-QTOF-MS profiling showed the presence of ...

  11. Effect of pre-germination treatments on seed physiology and germination of central Himalayan oaks?

    OpenAIRE

    Purohit, Vijay K.; Palni, L. M. S.; Nandi, Shyamal K.

    2009-01-01

    The continuous decline in regeneration of two important species of central Himalayan oak, namely Quercus glauca and Q. leucotrichophora, is of great concern. A study was therefore, carried out to improve germination ability of these species using various presoaking treatments. Seeds of both the species lost viability following storage; tetrazolium staining pattern and germination capacity of seeds following different period of storage at 4 °C and 20 °C indicated retainment of viability for a ...

  12. [Cytotoxicity of the strontium-substituted hydroxyapatite evaluated by MTT colorimetry].

    Science.gov (United States)

    Fu, Y; Chen, D; Zhang, J

    2001-09-01

    This study was conducted to evaluate the cytotoxicity of strontium-substituted hydroxyapatite by methyl thiazolyl tetrazolium (MTT) colorimetry. We used the MTT method to assay the cytotoxicity of the strontium-substituted hydroxyapatite containing different strontium concentrations (1%, 5%, 10%, 100% Sr2+) and the pure hydroxyapatite. The results showed that the cytotoxicity scores of the different materials were grade 0 or grade 1. These led us to the conclusion that strontium-substituted hydroxyapatite has good biocompatibility.

  13. Preliminary study of cell metabolism, by use of NBT test, determination the intensity of lipid peroxidation and antioxidant activity

    Directory of Open Access Journals (Sweden)

    Diana BEI

    2009-05-01

    Full Text Available Otto Warburg, in the early part of the 20th century, originated a hypothesis, that the cause of cancer is primarily a defect in energy metabolism.A decrease in the capacity of mitochondria to reduce NAD(P, together with a decline in the NAD(PH/NAD(P redox couple, uncouples oxidative phosphorylation, lead to depletion of ATP and decrease the cell viability.Nitro-bleu tetrazolium have been used to assay cell proliferation and viability. The method to measure cell proliferation is based on enzymatic cleavage of the tetrazolium salts to a water-soluble formazan dye.Succinate-tetrazolium reductase, is an enzymatic sistem, which belongs to the respiratory chain of the mitochondria and it is active only in viable cells. The reagent diffuses into the cells and it is cleaved to formazan. The absorption change is measured and analysed.Free radicals such as superoxide, can cause a damage in cellular components, but several antioxidants inhibiting the lipid peroxidation and limiting the level of free radicals in cells.In the present study we had in view the proliferation and viability of leukemia cells during antineoplastic treatment along with the alteration of the serum level of malondialdehyde (MDA and ceruloplasmin (CP. With serum level of malondialdehyde we monitored the presence of the lipid peroxidation by the reactive oxygen species, and with the oxidized ceruloplasmin level in blood serum we evidenced the activity of antioxidant system in blood.

  14. Critical Appraisal of the MTT Assay in the Presence of Rottlerin and Uncouplers

    Directory of Open Access Journals (Sweden)

    Maioli Emanuela

    2009-12-01

    Full Text Available Abstract Rottlerin is a natural product isolated from Mallotus philippinensis. This polyphenolic compound, originally described as a selective inhibitor of PKCδ, can inhibit many other PKC-unrelated kinases and has a number of biological actions, including mitochondrial uncoupling effects. We recently found that Rottlerin inhibits the transcription factor nuclear factor κB in different cell types, causing downregulation of cyclin D1 and growth arrest. The present study was carried out to clarify the surprising lack of effect of Rottlerin on MCF-7 cell viability, assessed by the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT test. We found that Rottlerin causes overestimation of the MTT test, leading to inconsistent results between cell number and cell viability. Rottlerin, however, strongly differs from other antioxidant polyphenols, which directly reduce tetrazolium salts, since it does not exhibit any reactivity toward the tetrazolium salts in vitro nor does it modulate lactate dehydrogenase activity. The interference in the MTT assay occurred only in cultured cells, concomitantly with a decrease in the energy charge. Because the same MTT overestimation was observed in the presence of uncoupling agents, we conclude that the Rottlerin artifact is linked to its uncoupling action that, by accelerating oxidative chain, accidentally results in enhanced MTT reduction. These results suggest caution in the use of the MTT assay in the presence of Rottlerin and uncouplers in general.

  15. Critical Appraisal of the MTT Assay in the Presence of Rottlerin and Uncouplers

    Directory of Open Access Journals (Sweden)

    Carlucci Filippo

    2009-01-01

    Full Text Available Abstract Rottlerin is a natural product isolated from Mallotus philippinensis. This polyphenolic compound, originally described as a selective inhibitor of PKCδ, can inhibit many other PKC-unrelated kinases and has a number of biological actions, including mitochondrial uncoupling effects. We recently found that Rottlerin inhibits the transcription factor nuclear factor κB in different cell types, causing downregulation of cyclin D1 and growth arrest. The present study was carried out to clarify the surprising lack of effect of Rottlerin on MCF-7 cell viability, assessed by the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT test. We found that Rottlerin causes overestimation of the MTT test, leading to inconsistent results between cell number and cell viability. Rottlerin, however, strongly differs from other antioxidant polyphenols, which directly reduce tetrazolium salts, since it does not exhibit any reactivity toward the tetrazolium salts in vitro nor does it modulate lactate dehydrogenase activity. The interference in the MTT assay occurred only in cultured cells, concomitantly with a decrease in the energy charge. Because the same MTT overestimation was observed in the presence of uncoupling agents, we conclude that the Rottlerin artifact is linked to its uncoupling action that, by accelerating oxidative chain, accidentally results in enhanced MTT reduction. These results suggest caution in the use of the MTT assay in the presence of Rottlerin and uncouplers in general.

  16. Vital dye reaction and granule localization in periplasm of Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Liyan Ping

    Full Text Available Tetrazolium salts are widely used in biology as indicators of metabolic activity - hence termed vital dyes - but their reduction site is still debated despite decades of intensive research. The prototype, 2,3,5- triphenyl tetrazolium chloride, which was first synthesized a century ago, often generates a single formazan granule at the old pole of Escherichia coli cells after reduction. So far, no explanation for their pole localization has been proposed.Here we provide evidence that the granules form in the periplasm of bacterial cells. A source of reducing power is deduced to be thiol groups destined to become disulfides, since deletion of dsbA, coding for thiol-oxidase, enhances the formation of reduced formazan. However, pervasive reduction did not result in a random distribution of formazan aggregates. In filamentous cells, large granules appear at regular intervals of about four normal cell-lengths, consistent with a diffusion-to-capture model. Computer simulations of a minimal biophysical model showed that the pole localization of granules is a spontaneous process, i.e. small granules in a normal size bacterium have lower energy at the poles. This biased their diffusion to the poles. They kept growing there and eventually became fixed.We observed that formazan granules formed in the periplasm after reduction of tetrazolium, which calls for re-evaluation of previous studies using cell-free systems that liberate inaccessible intracellular reductant and potentially generate artifacts. The localization of formazan granules in E. coli cells can now be understood. In living bacteria, the seeds formed at or migrated to the new pole would become visible only when that new pole already became an old pole, because of the relatively slow growth rate of granules relative to cell division.

  17. Phorbol myristate acetate stimulated NBT test: a simple method suitable for antenatal diagnosis of chronic granulomatous disease.

    Science.gov (United States)

    Levinsky, R J; Harvey, B A; Rodeck, C H; Soothill, J F

    1983-01-01

    When endotoxin was compared with phorbol myristate acetate (PMA) for stimulation of phagocytes in the nitroblue tetrazolium (NBT) test, both methods discriminated between affected patients with X-linked chronic granulomatous disease (CGD) and controls, but only the PMA NBT test distinguished female carriers of CGD. Endotoxin provided no stimulation of normal fetal blood whereas PMA was an effective stimulator. Our results indicate the superiority of the PMA NBT test for diagnosis of patients and carriers of CGD and should allow accurate antenatal diagnosis of the disease. Images Fig. 3 PMID:6360441

  18. Leukocytes respiratory burst activity as indicator of innate immunity of pacu Piaractus mesopotamicus

    Directory of Open Access Journals (Sweden)

    JD Biller-Takahashi

    Full Text Available The present study evaluated the assay to quantify the respiratory burst activity of blood leukocytes of pacu as an indicator of the innate immune system, using the reduction of nitroblue tetrazolium (NBT to formazan as a measure of the production of reactive oxygen species (ROS. In order to assess the accuracy of the assay, fish were challenged by Aeromonas hydrophila and sampled one week after challenge. The A. hydrophila infection increased the leukocyte respiratory burst activity. The protocol showed a reliable and easy assay, appropriate to determine the respiratory burst activity of blood leukocytes of pacu, a neotropical fish, in the present experimental conditions.

  19. The NBT test in burned patients.

    Science.gov (United States)

    Roe, E. A.; Jones, R. J.

    1979-01-01

    The number of polymorphs which stained with the dye nitro-blue tetrazolium (NBT "Positive") increased sharply during the first week after burning, reaching levels 4--5 times above values for healthy volunteers. In burns of more than 20% of the body surface a second, smaller increase in the number of NBT "positives" occurred 4 to 6 weeks after burning. The high levels of NBT "positive" polymorphs occurred independently of infection on the burns. A burned patient who died from septicaemia had very low numbers of NBT "positive" polymorphs for 3 weeks before death. PMID:444418

  20. Histochemical localization of glutathione dependent NBT-reductase in mouse skin.

    Science.gov (United States)

    Shukla, Y

    2001-09-01

    Localization of the glutathione dependent Nitroblue tetrazolium (NBT) reductase in fresh frozen sections of mouse skin and possible dependence of NBT reductase on tissue thiol levels has been investigated. The fresh frozen tissue sections (8 m thickness) were prepared and incubated in medium containing NBT, reduced glutathione (GSH) and phosphate buffer. The staining for GSH was performed with mercury orange. The activity of the NBT-reductase in mouse skin has been found to be localized in the areas rich in glutathione and actively proliferating area of the skin. The activity of the NBT-reductase seems to be dependent on the glutathione contents.

  1. Optimization of a Paper-Based ELISA for a Human Performance Biomarker

    Science.gov (United States)

    2013-11-11

    The antibody incubation solution contained 0.05% v/v Tween 20 in 1X PBS with the conjugated antibody (1:1000 ratio). The NBT /BCIP colorimetric...substrate consisted of 2.68 mM 5-bromo-4-chloro-3-indolylphosphate toluidine salt (BCIP), 1.8 mM nitro-blue tetrazolium chloride ( NBT ), 5 mM MgCl2, 100 mM...38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 7 the appropriate period of time. For the BCIP/ NBT , pNPP, and TMB substrates

  2. Podarcis lilfordi from the Balearic islands as a potential disperser of the rare Mediterranean plant Withania frutescens

    Science.gov (United States)

    Castilla, Aurora M.

    1999-04-01

    This study examines whether the lizard Podarcis lilfordi is a legitimate disperser of the rare Mediterranean plant Withania frutescens by using the biochemical test of triphenyl-2H-tetrazolium chloride for testing seed viability. This lizard eats the fresh fruits of the plants and defecates intact seeds which have been retained 1 to 3 d in their gut. Viability of seeds recovered from faeces was very high and comparable to the viability of fresh seeds. Seed dispersal by this lizard in the Balearic islands may facilitate population expansion of this rare plant in the Balearics.

  3. Surface coated polyurethane with improved bioactivity and cytocompatability

    CSIR Research Space (South Africa)

    Chetty, AS

    2006-02-01

    Full Text Available . The topographical morphologies induced during cell attachment and growth was assessed by employing SEM. Mitochondrial dehydrogenase activity (MTT) assay: Seeded cells were incubated with the MTT (3-[4,5 dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay... refer to the HA coated and uncoated PU substrates respectively. The cell culture plate was used as the 100% control. The asterisk indicate statistically significant P-values of P< 0.05 with n=5 Figure 6: Topographical morphologies of Graham 293...

  4. Simple and efficient method for isolation and cultivation of endoscopically obtained human colonocytes

    DEFF Research Database (Denmark)

    Seidelin, Jakob B; Horn, Thomas; Nielsen, Ole H

    2003-01-01

    -diphenyl-tetrazolium bromide assay, by flowcytometry, by phase contrast microscopy, and by transmission electron microscopy. Incubation at 21 degrees C for 75 min gave an optimal yield of 3 x 10(6) (2.0-3.8 x 10(6)) viable epithelial cells in intact crypts per seven biopsies. Embedding of crypts in a collagen gel put...... and where the diagnosis of irritable bowel syndrome was later reached, were included. Seven colon biopsies were taken and incubated at varying time periods of 10-120 min and temperatures of 4-37 degrees C in a chelating buffer. The epithelium was then harvested and cultivated under three different...

  5. The rapid inhibition of root respiration after exposure of bean ( Phaseolus vulgaris L.) plants to ozone

    Science.gov (United States)

    Hofstra, G.; Ali, A.; Wukasch, R. T.; Fletcher, R. A.

    The metabolic activity of the roots was very sensitive to the changes induced in the leaves by O 3. Respiratory activity began to decrease well before visible injury appeared on the leaves, and the per cent reduction of respiration was much greater than the per cent leaf injury. The triphenyl tetrazolium chloride (TTC) staining technique revealed changes in root tips very quickly, was generally more sensitive to changes in respiratory activity and was a convenient technique for handling large numbers of samples. Reducing foliar injury from O 3 with low levels of SO 2 reduced the effects on the roots indicating the effect of O 3 is on processes in the leaf.

  6. Surface modification of diamond-like carbon films with protein via polydopamine inspired coatings

    Energy Technology Data Exchange (ETDEWEB)

    Tao Caihong [State Key Laboratory of Solid Lubrication, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Tianshui Middle Road 18th, Lanzhou 730000 (China); China and Graduate University of Chinese Academy of Sciences, Beijing 100080 (China); Yang Shengrong [State Key Laboratory of Solid Lubrication, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Tianshui Middle Road 18th, Lanzhou 730000 (China); Zhang Junyan, E-mail: zhangjunyan@lzb.ac.cn [State Key Laboratory of Solid Lubrication, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Tianshui Middle Road 18th, Lanzhou 730000 (China); Wang Jinqing [State Key Laboratory of Solid Lubrication, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Tianshui Middle Road 18th, Lanzhou 730000 (China)

    2009-10-15

    In this paper, we report a facile two-step approach to immobilize proteins onto DLC surfaces. The first step was a simple immersion of DLC in a solution of dopamine. Polydopamine was deposited on DLC as a stable anchor to present protein molecules. Then the protein ad-layer was deposited on it. The chemical components of the modified DLC surfaces were characterized by Fourier transform infrared spectra and X-ray photoelectron spectroscopy. The biocompatibility of it was evaluated in vitro by the tetrazolium salt method. And it was indicated that the BSA modified surface had good haemocompatibility properties, and was cytocompatible to PC-12 cells.

  7. Spectrophotometric Determination of Carbon Disulphide in the Workplace Air

    Directory of Open Access Journals (Sweden)

    V. Pitschmann

    2013-01-01

    Full Text Available This papre describes a simple method of carbon disulphide determination in the air of working environment in the chemical companies and plants after its absorption into aprotic N,N-dimethylformamide solvent. Carbon disulphide absorbed into aprotic solvent was transformed by using ammonium hydroxide on sulphides which were determined by spectrophotometry. 5,5′-Dithiobis(2-nitrobenzoic acid and blue tetrazolium chloride were used as chromogenic sensing reagents. Colour-reducing products were measured at the wavelength of 500, respectively 520 nm. Detection limits for determination of carbon disulphide in the air are 0.2, respectively 0.4 mg·m−3.

  8. Evaluation of the leishmanicidal activity of plants used by Peruvian Chayahuita ethnic group.

    Science.gov (United States)

    Estevez, Y; Castillo, D; Pisango, M Tangoa; Arevalo, J; Rojas, R; Alban, J; Deharo, E; Bourdy, G; Sauvain, M

    2007-11-01

    A total of 27 ethanolic plant extracts from 27 species were screened for leishmanicidal activity in vitro against Leishmania amazonensis. Most of the selected species (19) are traditionally used by the Chayahuitas, an Amazonian Peruvian ethnic group, to treat skin affections and/or leishmaniasis. A colorimetric method based on the reduction of tetrazolium salt (MTT) was used to measure the viability of Leishmania amazonensis promastigote and amastigote stages. Only the leaves of two species of the Piperaceae family (Piper hispidum Sw., and Piper strigosum Trel.) showed good leishmanicidal activities (IC(50)literature.

  9. Chronic granulomatous disease

    Directory of Open Access Journals (Sweden)

    Nair Pradeep

    2005-01-01

    Full Text Available A 2½-year-old child presented with multiple discrete granulomatous lesions on the face and flexural regions since the age of 2 months along with lymphadenopathy. The patient also had recurrent bouts of pyodermas and respiratory tract infections. Biopsy of the lesion showed necrosis of tissue with suppuration and histiocytes but no evidence of tuberculosis, fungal infections or atypical mycobacteria. Lymph node biopsy also showed necrosis with suppuration but no infective organism. Nitroblue tetrazolium test was negative indicating that the neutrophils failed to oxidize the dye. We are reporting here a rare case of chronic granulomatous disease.

  10. The cytotoxic and embryotoxic effects of kaurenoic acid, a diterpene isolated from Copaifera langsdorffii oleo-resin.

    Science.gov (United States)

    Costa-Lotufo, L V; Cunha, G M A; Farias, P A M; Viana, G S B; Cunha, K M A; Pessoa, C; Moraes, M O; Silveira, E R; Gramosa, N V; Rao, V S N

    2002-08-01

    In this work, we studied the effects of kaurenoic acid, a diterpene isolated from the oleo-resin of Copaifera langsdorffii in developing sea urchin (Lytechinus variegatus) embryos, on tumor cell growth in microculture tetrazolium (MTT) test and on mouse and human erythrocytes in hemolysis assay. Continuous exposure of embryos to kaurenoic acid starting immediately after fertilization inhibited the first cleavage (IC(50): 84.2 microM) and progressively induced embryo destruction (IC(50): 44.7 microM and sea urchin embryos, the inhibition of tumor cell growth and the hemolysis of mouse and human erythrocytes indicate the potential cytotoxicity of kaurenoic acid.

  11. [Chronic granulomatosis in childhood].

    Science.gov (United States)

    Fakan, F

    1977-08-01

    In a boy aged 8 years suffering from chronic granulomatosis of childhood necrotizing pneumonie and small pulmonary tuberculoid granulomas containing filaments of moulds were found at autopsy. Necrotizing leucocytic granulomas were present in the liver, spleen and the lymph nodes. All the organs showed aggregates of histiocytes containing yellowish cytoplasmic deposits of lipogment surrounded by a high acid phosphatase activity. The NBT-reduction leucocytic tests was repeatedly negative in vivo. The activities of NADH- and NADPH-tetrazolium reductase and succinate dehydrogenase in the tissues were histochemically normal.

  12. Effects on cytotoxicity and antibacterial properties of the incorporations of silver nanoparticles into the surface coating of dental alloys*

    OpenAIRE

    Shen, Xiao-ting; Zhang, Yan-zhen; Xiao, Fang; Zhu, Jing; Zheng, Xiao-Dong

    2017-01-01

    The aim of this study was to research the changes in cytotoxicity and antibacterial properties after silver nanoparticles (AgNPs) were incorporated into the surface coating of dental alloys. AgNPs were attached to cobalt chromium alloys and pure titanium using a hydrothermal method, according to the reaction: AgNO3+NaBH4? Ag+1/2H2+1/2B2H6+NaNO3. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to evaluate the cytotoxicity of the alloys when in contact with ...

  13. Antiproliferative effects of abietane diterpenoids isolated from Hyptis martiusii Benth (Labiatae).

    Science.gov (United States)

    Costa-Lotufo, L V; Araújo, E C C; Lima, M A S; Moraes, M E A; Pessoa, C; Silviera, E R; Moraes, M O

    2004-01-01

    Two abietane diterpenes were isolated from a hexane extract of Hyptis martiusii roots and identified as carnasol 11,14-dihidroxy-8,11,13-abietatrien-7-one. These compounds were tested for their antiproliferative effects on tumor cell lines using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and on the sea urchin egg development. Both compounds displayed cytotoxic activity against tumor cell lines, but only carnasol was able to inhibit the sea urchin egg cleavages.

  14. Comparative study of cytotoxicity, oxidative stress and genotoxicity induced by four typical nanomaterials: the role of particle size, shape and composition.

    Science.gov (United States)

    Yang, Hui; Liu, Chao; Yang, Danfeng; Zhang, Huashan; Xi, Zhuge

    2009-01-01

    Although the biological effects of some nanomaterials have already been assessed, information on toxicity and possible mechanisms of various particle types are insufficient. Moreover, the role of particle properties in the toxic reaction remains to be fully understood. In this paper, we aimed to explore the interrelationship between particle size, shape, chemical composition and toxicological effects of four typical nanomaterials with comparable properties: carbon black (CB), single wall carbon nanotube, silicon dioxide (SiO(2)) and zinc dioxide (ZnO) nanoparticles. We investigated the cytotoxicity, genotoxicity and oxidative effects of particles on primary mouse embryo fibroblast cells. As observed in the methyl thiazolyl tetrazolium (MTT) and water-soluble tetrazolium (WST) assays, ZnO induced much greater cytotoxicity than non-metal nanoparticles. This was significantly in accordance with intracellular oxidative stress levels measured by glutathione depletion, malondialdehyde production, superoxide dismutase inhibition as well as reactive oxygen species generation. The results indicated that oxidative stress may be a key route in inducing the cytotoxicity of nanoparticles. Compared with ZnO nanoparticles, carbon nanotubes were moderately cytotoxic but induced more DNA damage determined by the comet assay. CB and SiO(2) seemed to be less effective. The comparative analysis demonstrated that particle composition probably played a primary role in the cytotoxic effects of different nanoparticles. However, the potential genotoxicity might be mostly attributed to particle shape. Copyright (c) 2008 John Wiley & Sons, Ltd.

  15. Quantitation of intracellular NAD(P)H can monitor an imbalance of DNA single strand break repair in base excision repair deficient cells in real time

    Science.gov (United States)

    Nakamura, Jun; Asakura, Shoji; Hester, Susan D.; de Murcia, Gilbert; Caldecott, Keith W.; Swenberg, James A.

    2003-01-01

    DNA single strand breaks (SSBs) are one of the most frequent DNA lesions in genomic DNA generated either by oxidative stress or during the base excision repair pathways. Here we established a new real-time assay to assess an imbalance of DNA SSB repair by indirectly measuring PARP-1 activation through the depletion of intracellular NAD(P)H. A water-soluble tetrazolium salt is used to monitor the amount of NAD(P)H in living cells through its reduction to a yellow colored water-soluble formazan dye. While this assay is not a direct method, it does not require DNA extraction or alkaline treatment, both of which could potentially cause an artifactual induction of SSBs. In addition, it takes only 4 h and requires less than a half million cells to perform this measurement. Using this assay, we demonstrated that the dose- and time-dependent depletion of NAD(P)H in XRCC1-deficient CHO cells exposed to methyl methanesulfonate. This decrease was almost completely blocked by a PARP inhibitor. Furthermore, methyl methanesulfonate reduced NAD(P)H in PARP-1+/+cells, whereas PARP-1–/– cells were more resistant to the decrease in NAD(P)H. These results indicate that the analysis of intracellular NAD(P)H level using water-soluble tetrazolium salt can assess an imbalance of SSB repair in living cells in real time. PMID:12930978

  16. Investigation of Total Phenolic and Flavonoid Contents, and Evaluation of Antimicrobial and Antioxidant Activities from Baeckea frutescens Extracts

    Science.gov (United States)

    Nisa, K.; Nurhayati, S.; Apriyana, W.; Indrianingsih, A. W.

    2017-12-01

    Baeckea frutescens L. is a medicinal plant endemic to the tropical area and it has been used by locals for topical and oral ailments. This study investigated total phenolic and flavonoid contents and also evaluated in vitro antimicrobial and antioxidant activities of of Baeckea frutescens crude extracts. These extracts were assessed for their antibacterial activities against strains of Escherichia coli, Staphylococcus aureus, Salmonella thypii, and Pseudomonas aureginosa by the broth micro-dilution methods using a modified tetrazolium-based colorimetric assay (3-(4, 5-dimethylthiazol)-2, 5-diphenyl tetrazolium bromide (MTT) assay). Baeckea frutescens crude extracts were also tested against the stable DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free-radical. The results indicated that Baeckea frutescens water and ethanol extracts possesed remarkable antibacterial activity with the minimum inhibitory concentration less than 100 μg/ml against Escherichia coli and Salmonella thypi. On the evaluation of the antioxidant activity via DPPH assay, Baeckea frutescens ethanol extracts exhibited a good antioxidant activity with IC50 less than 50 μg/ml and Baeckea frutescens water extracts showed a moderate antioxidant activity with IC50 less than 100 μg/ml.

  17. MORPHOPHYSIOLOGICAL ASPECTS OF SELENICEREUS MEGALANTHUS (K. SCHUM EX VAUPEL MORAN

    Directory of Open Access Journals (Sweden)

    M. Sorace

    2016-07-01

    Full Text Available Selenicereus megalanthus (K. Schumer Vaupel Moran is known as yellow Pitaya because of yellow peel color. Originated from Colombia, Peru, Ecuador and Bolivia, it belongs to the family Cactaceae and has climbing habit, besides being edible and currently grown. In Brazil the production of yellow pitaya is incipient. Pitaya propagates through cuttings, seed or grafting. Its seeds have sarcotesta mucilaginous, which may be a deterrent factor or decrease germination. This study aimed to study biometric aspects and germination of seeds with and without mucilage removal. The removal of mucilage was made by immersion in 25% sucrose solution and were evaluated biometric aspects of fruit and seed quality through tests of germination and tetrazolium, rate of germination speed and imbibition curve. Through biometrics establishes the relationship between the size of the fruit and seed number, where the number of seeds per unit mass is greater in smaller fruits. The continuous production of mucilage prevented the establishment of imbibition curve. The result obtained in the tetrazolium test was not consistent with the germination. Seeds with mucilage removal by pretreatment with sucrose solution showed better germination and IVG, producing stronger plants.

  18. Superoxide anion radical scavenging property of catecholamines.

    Science.gov (United States)

    Kładna, Aleksandra; Berczyński, Paweł; Kruk, Irena; Michalska, Teresa; Aboul-Enein, Hassan Y

    2013-01-01

    The direct effect of the four catecholamines (adrenaline, noradrenaline, dopamine and isoproterenol) on superoxide anion radicals (O2•) was investigated. The reaction between 18-crown-6-ether and potassium superoxide in dimethylsulfoxide was used as a source of O2•. The reactivity of catecholamines with O2• was examined using chemiluminescence, reduction of nitroblue tetrazolium and electron paramagnetic resonance spin-trapping techniques. 5,5-Dimethyl-1-pyrroline-N-oxide was included as the spin trap. The results showed that the four catecholamines were effective and efficient in inhibiting chemiluminescence accompanying the potassium superoxide/18-crown-6-ether system in a dose-dependent manner over the range 0.05-2 mM in the following order: adrenaline > noradrenaline > dopamine > isoproterenol, with, IC50 = 0.15 ± 0.02 mM 0.21 ± 0.03 mM, 0.27 ± 0.03 mM and 0.50 ± 0.04 mM, respectively. The catecholamines examined also exhibited a strong scavenging effect towards O2• when evaluated this property by the inhibition of nitroblue tetrazolium reduction (56-73% at 1 M concentration). A very similar capacity of O2• scavenging was monitored in the 5,5-dimethyl-1-pyrroline-N-oxide spin-trapping assay. The results suggest that catecholamines tested may involve a direct effect on scavenging O2- radicals. Copyright © 2013 John Wiley & Sons, Ltd.

  19. Thermotolerance of human myometrium: implications for minimally invasive uterine therapies

    Science.gov (United States)

    Thomas, Aaron C.; Grisez, Brian T.; McMillan, Kathleen; Chill, Nicholas; Harclerode, Tyler P.; Radabaugh, Rebecca; Jones, Ryan M.; Coad, James E.

    2013-02-01

    Endometrial ablation has gained significant clinical acceptance over the last decade as a minimally invasive treatment for abnormal uterine bleeding. To improve upon current thermal injury modeling, it is important to better characterize the myometrium's thermotolerance. The extent of myometrial thermal injury was determined across a spectrum of thermal histories/doses (time-temperature combinations). Fresh extirpated human myometrium was obtained from 13 subjects who underwent a previous scheduled benign hysterectomy. Within two hours of hysterectomy, the unfixed myometrium was treated in a stabilized saline bath with temperatures ranging from 45-70 °C and time intervals from 30- 150 seconds. The time-temperature combinations were selected to simulate treatment times under 2.5 minutes. A total of six such thermal matrices, each comprised of 45 time-temperature combinations, were prepared for evaluation. The treated myometrium was cryosectioned for nitro blue tetrazolium (NBT) staining to assess for thermal respiratory enzyme inactivation. Image analysis was subsequently used to quantitatively assess the stained myometrium's capacity to metabolize the tetrazolium at each time-temperature combination. This colorimetric data was then used as marker of cellular viability and determine survival parameters with implications for developing minimally invasive uterine therapies.

  20. Do Multiwell Plate High Throughput Assays Measure Loss of Cell Viability Following Exposure to Genotoxic Agents?

    Directory of Open Access Journals (Sweden)

    Razmik Mirzayans

    2017-08-01

    Full Text Available Cell-based assays in multiwell plates are widely used for radiosensitivity and chemosensitivity assessment with different mammalian cell types. Despite their relative ease of performance, such assays lack specificity as they do not distinguish between the cytostatic (reversible/sustained growth arrest and cytotoxic (loss of viability effects of genotoxic agents. We recently reported studies with solid tumor-derived cell lines demonstrating that radiosensitivity as measured by multiwell plate colorimetric (e.g., XTT and fluorimetric (e.g., CellTiter-Blue assays reflects growth arrest but not loss of viability. Herein we report similar observations with cancer cell lines expressing wild-type p53 (A549 lung carcinoma or mutant p53 (MDA–MB-231 breast carcinoma after treatment with the chemotherapeutic drug cisplatin. Importantly, we show that treatment of cancer cells with concentrations of cisplatin that result in 50% effect (i.e., IC50 in multiwell plate assays trigger the emergence of growth arrested cells that exhibit highly enlarged morphology, remain viable and adherent to the culture dish, and metabolize the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl-tetrazolium bromide (MTT to its formazan derivative. The emergence of markedly enlarged viable cells complicates the interpretation of chemosensitivity data obtained with multiwell plate high throughput assays. Relying solely on IC50 values could be misleading.

  1. Immune response of shrimp (Penaeus monodon against Vibrios furnissii pathogen

    Directory of Open Access Journals (Sweden)

    Kumaran Subramanian

    2014-04-01

    Full Text Available Objective: To analyse experimental infection and immune system of shrimp (Penaeus monodon against Vibrios furnissii (V. furnissii. Methods: Experimental animals were collected and acclimatized by maintaining specific temperature, pH and salinity to avoid mortality. Shrimps were experimentally infected with V. furnissii and their immune responses were monitored. After the infection all the shrimps were monitored for any symptoms, death rate in 0, 12, 24, 36, 48 h. Then haemolymph were collected and tetrahydrocannabinol, phenol oxidase, nitroblue tetrazolium and lysozyme were monitored in every 12 h at the interval of 48 h. Results: Shrimps infected by live V. furnissii had showed gradual increase in tetrahydrocannabinol, phenol oxidase activity, nitro-blue-tetrazolium and lysozyme activity comparing with the killed and control. Conclusions: The live V. furnissii had showed infection in the shrimp immune system. The live V. furnissii shows infection in experimental shrimps comparing with killed V. furnissii. So the V. furnissii in nature cause the infection in shrimp Penaeus monodon immune system. This report could be applied to control of the infection in shrimp hatchery.

  2. Konsentrasi Aman Kurkumin dan PGV-0 terhadap Sel Vero Berdasarkan Hasil Uji Sitotoksik

    Directory of Open Access Journals (Sweden)

    Dewi Marbawati

    2015-08-01

    Full Text Available Curcumin (1,7-bis(3-methoxyphenyl 4'hidroksi -1.6 heptadien, 3,5-dione is the yellow pigment of Curcuma longa. Curcumin has various biological activities such as antioxidant, antibacterial, antifungal, antiprotozoal and antivirus. Various benefits of curcumin can not be separated from the weakness which is not stable to pH and light. Pentagamavunon-0 (PGV-0 were made by changing the β diketone group on cluster analog of curcumin into monoketon while eliminating active methylene group so it is more stable to pH and light. PGV-0 also has potential as a stronger antioxidant and antiinflammatory agent than other curcumin analogues. The objective of this research was to determine the safe concentrations of curcumin and PGV-0 on vero cells due to the increased use of the two compounds through the cytotoxic test. This research includes experimental research. Cytotoxic test performed with microculture tetrazolium technique (MTT method. Use of MTT to evaluate the cytotoxic is based on changes of tetrazolium salt into formazan crystals by mitochondrial enzyme succinate dehydrogenase with the help of cellular NADH. The results showed that the safe concentrations of curcumin and PGV-0 on vero cells respectively are 6.25 and 1.5625 ppm. Based on the cytotoxic test the secure concentration of curcumin was higher than PGV-0.

  3. Superação da dormência e avaliação da qualidade fisiológica de sementes de Sesbania virgata Dormancy break and evaluation of physiological quality of Sesbania virgata seeds

    Directory of Open Access Journals (Sweden)

    Valquíria Nogueira Camargos

    2008-12-01

    Full Text Available A avaliação da qualidade fisiológica de lotes de sementes de Sesbania virgata é dificultada pela dormência inerente à espécie, bem como pela inadequação metodológica de testes disponíveis. Para desenvolver procedimentos adequados à avaliação da qualidade das sementes de S. virgata, foram avaliados métodos tanto para quebra de dormência como para realização do teste de tetrazólio. Em uma primeira fase, as sementes de cinco lotes foram submetidas a tratamentos para quebra de dormência: lixa, ácido e corte do tegumento, previamente ao teste de germinação. Para caracterização dos lotes e adequação da metodologia do teste de tetrazólio, na segunda fase, as sementes foram lixadas na região oposta ao eixo embrionário, e submetidas aos seguintes testes: tetrazólio, germinação, velocidade de emergência, peso de matéria fresca e de matéria seca de plântulas. Foram utilizados dois métodos de pré-condicionamento: embebição em papel por 18 h a 30 ºC e imersão em água por 12 h a 30 ºC e três períodos (uma, duas e três horas de imersão em solução de tetrazólio 0,5%. Na análise do perfil dos lotes de S. virgata foram detectadas variações na qualidade fisiológica pelos resultados dos testes de vigor e germinação. O método de escarificação com lixa possibilitou a quebra de dormência das sementes de S. virgata,e o pré-condicionamento das sementes em papel com embebição em solução de tetrazólio 0,5% por duas horas é um procedimento adequado para avaliação da viabilidade das sementes.The evaluation of Sesbania virgata seed physiological quality is hampered by its intrinsic seed dormancy and inadequate methodologies among available tests. To develop an adequate procedure to evaluate S. virgata seed quality, methods for dormancy breaking and tetrazolium test accomplishment were evaluated. In a first stage, seeds from five lots were submitted to dormancy breaking methods: sandpaper, acid and

  4. Viabilidade de sementes de acerola (Malpighia emarginata DC: avaliação da vitalidade dos tecidos Seed viability of acerola (Malpighia emarginata DC.: evaluation of the tissue vitality

    Directory of Open Access Journals (Sweden)

    Luciana Claudia Costa

    2003-12-01

    Full Text Available A morfologia interna e a viabilidade de sementes de acerola (Malpighia emarginata DC. foram estudadas utilizando-se o tetrazólio (cloreto de 2, 3, 5 trifenil tetrazólio. Dos clones testados, o Flórida Sweet foi o que apresentou a menor percentagem de sementes com embriões normais (10% como também em reação às sementes sem embriões (8% e o maior percentagem de sementes com embriões deformados (81%. O clone 07-OS apresentou maior percentagem de sementes com embriões normais (51% e um número considerado elevado de sementes sem embriões (34%. Os demais clones apresentaram valores intermediários. Para todos os clones avaliados, as sementes com embriões normais apresentaram 100% de embriões viáveis. Essas sementes submetidas ao teste de tetrazólio por um período de 12 horas, apresentaram-se com uma coloração vermelha intensa, considerada ideal para a avaliação positiva da viabilidade das sementes. Estes resultados não podem, entretanto, ser tomados para prognóstico e o cálculo da taxa de germinação e dormência, apenas indicando que as sementes estão vivas.The morphology and seed viability of acerola (Malpighia emarginata DC. were evaluated using the tetrazolium (2, 3, 5 triphenyl tetrazolium chloride. From all clones tested, Florida Sweet presented the smallest percentage of seeds with normal embryos (10% or without them (8% and the greatest percentage of seeds with malformed embryos, (81%. The clone 07-0S had the greatest percentage of seeds with normal embryos (5 l% and an elevated number of seeds without embryos (34%. For all other clones evaluated, the seeds with normal embryos were 100% viable. The seeds subjected to the tetrazolium test for 12 hours, had a deep red color, considered ideal for viability positive evaluation. These tests cannot be taken as prognosis to calculate the germination and dormancy rates, only indicating that the seeds are alive.

  5. Imaging of Cellular Oxidoreductase Activity Suggests Mixotrophic Metabolisms in Thiomargarita spp.

    Science.gov (United States)

    Bailey, Jake V; Flood, Beverly E; Ricci, Elizabeth; Delherbe, Nathalie

    2017-11-07

    The largest known bacteria, Thiomargarita spp., have yet to be isolated in pure culture, but their large size allows for individual cells to be monitored in time course experiments or to be individually sorted for omics-based investigations. Here we investigated the metabolism of individual cells of Thiomargarita spp. by using a novel application of a tetrazolium-based dye that measures oxidoreductase activity. When coupled with microscopy, staining of the cells with a tetrazolium-formazan dye allows metabolic responses in Thiomargarita spp. to be to be tracked in the absence of observable cell division. Additionally, the metabolic activity of Thiomargarita sp. cells can be differentiated from the metabolism of other microbes in specimens that contain adherent bacteria. The results of our redox dye-based assay suggest that Thiomargarita is the most metabolically versatile under anoxic conditions, where it appears to express cellular oxidoreductase activity in response to the electron donors succinate, acetate, citrate, formate, thiosulfate, H2, and H2S. Under hypoxic conditions, formazan staining results suggest the metabolism of succinate and likely acetate, citrate, and H2S. Cells incubated under oxic conditions showed the weakest formazan staining response, and then only to H2S, citrate, and perhaps succinate. These results provide experimental validation of recent genomic studies of Candidatus Thiomargarita nelsonii that suggest metabolic plasticity and mixotrophic metabolism. The cellular oxidoreductase response of bacteria attached to the exterior of Thiomargarita also supports the possibility of trophic interactions between these largest of known bacteria and attached epibionts.IMPORTANCE The metabolic potential of many microorganisms that cannot be grown in the laboratory is known only from genomic data. Genomes of Thiomargarita spp. suggest that these largest of known bacteria are mixotrophs, combining lithotrophic metabolism with organic carbon

  6. Applicability of a colorimetric method for evaluation of lactic acid bacteria with probiotic properties.

    Science.gov (United States)

    Jung, Min Young; Lee, Jina; Park, Boyeon; Hwang, Hyelyeon; Sohn, Sung-Oh; Lee, Se Hee; Lim, Hyeong In; Park, Hae Woong; Lee, Jong-Hee

    2017-06-01

    Rapid colorimetric methods using various indicator reagents have been developed to monitor bacterial viability. Here, we examined the applicability of a method based on the reduction of resazurin or water-soluble tetrazolium salt-8 (WST-8) to screen lactic acid bacteria (LAB) for growth, tolerance against bile acid and low pH. The resazurin reduction test proved unsuitable for screening LAB such as Lactobacillus plantarum and Leuconostoc mesenteroides since it reacted with acid present in the cultures. LAB growth could be indirectly quantified by measuring WST-8 reduction. This method proved more sensitive and quickly results than counting bacterial colony forming units and turbidity at 600 nm in the presence of bile and acid. Our results suggested that the WST-8-based method could be useful for the characterization of growth and tolerance of the lactic acid producing bacteria. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Acetonyltriphenylphosphonium 2,3,5-triphenyltetrazolium tetrachloridocuprate(II

    Directory of Open Access Journals (Sweden)

    Mouhamadou Birame Diop

    2018-01-01

    Full Text Available The title compound, (C21H20OP(C19H15N4[CuCl4], was obtained by reacting CuCl2·2H2O with a mixture of one equivalent of acetonyltriphenylphosphonium chloride and one equivalent of 2,3,5-triphenyltetrazolium chloride in acetonitrile. In the structure, the Cu centre in the dianion is bonded to four chloride ligands and adopts a distorted tetrahedral geometry. The phosphonium cation likewise adopts the expected tetrahedral geometry. The tetrazolium ring forms dihedral angles of 77.68 (10, 26.85 (11 and 66.48 (10° with the planes of the benzene rings of the substituent groups. In the crystal, weak C—H...Cl hydrogen-bonding interactions involving both cations and the anion give rise to a three-dimensional supramolecular structure.

  8. Cellular resolution expression profiling using confocal detection of NBT/BCIP precipitate by reflection microscopy.

    Science.gov (United States)

    Jékely, Gáspár; Arendt, Detlev

    2007-06-01

    The determination of gene expression patterns in three dimensions with cellular resolution is an important goal in developmental biology. However the most sensitive, efficient, and widely used staining technique for whole-mount in situ hybridization (WMISH), nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) precipitation by alkaline phosphatase, could not yet be combined with the most precise, high-resolution detection technique, confocal laser-scanning microscopy (CLSM). Here we report the efficient visualization of the NBT/BCIP precipitate using confocal reflection microscopy for WMISH samples of Drosophila, zebrafish, and the marine annelid worm, Platynereis dumerilii. In our simple WMISH protocol for reflection CLSM, NBT/BCIP staining can be combined with fluorescent WMISH, immunostainings, or transgenic green fluorescent protein (GFP) marker lines, allowing double labeling of cell types or of embryological structures of interest. Whole-mount reflection CLSM will thus greatly facilitate large-scale cellular resolution expression profiling in vertebrate and invertebrate model organisms.

  9. Experimental Infection of the Skin in the Hamster Simulating Human Impetigo IV. Cellular Responses after Streptococcal and Staphylococcal Infections

    Science.gov (United States)

    Dajani, Adnan S.; Wannamaker, Lewis W.

    1972-01-01

    Various cellular responses to skin infections in an experimental animal model were explored. Total leukocyte counts varied after group A streptococcal infections, but a depression was commonly seen after M type 12 impetigo. Staphylococcus aureus infections resulted in moderate leukocytosis. A marked neutrophilia was universal with streptococcal or staphylococcal disease. A positive nitroblue tetrazolium (NBT) response appeared 24 hr after infection, reached a peak in 48 hr, and then declined. This occurred in the absence of extensive cellulitis or bacteremia. An increase in the percentage and absolute number of NBT-positive neutrophils occurred. M type 57 streptococcus produced a more strongly positive NBT test than did M type 12. Cell-free filtrates of a broth culture of M type 57 streptococcus produced NBT responses in hamsters comparable to the responses seen after injection of live organisms. These studies indicate the usefulness of this animal model to study various parameters of the NBT test. PMID:4117885

  10. Studies on Cytotoxic Activity against HepG-2 Cells of Naphthoquinones from Green Walnut Husks of Juglans mandshurica Maxim

    Directory of Open Access Journals (Sweden)

    Yuanyuan Zhou

    2015-08-01

    Full Text Available Twenty-seven naphthoquinones and their derivatives, including four new naphthalenyl glucosides and twenty-three known compounds, were isolated from green walnut husks, which came from Juglans mandshurica Maxim. The structures of four new naphthalenyl glucosides were elucidated based on extensive spectroscopic analyses. All of these compounds were evaluated for their cytotoxic activities against the growth of human cancer cells lines HepG-2 by MTT [3-(4,5-dimethylthiazo l-2-yl-2,5 diphenyl tetrazolium bromide] assay. The results were shown that most naphthoquinones in an aglycone form exhibited better cytotoxicity in vitro than naphthalenyl glucosides with IC50 values in the range of 7.33–88.23 μM. Meanwhile, preliminary structure-activity relationships for these compounds were discussed.

  11. Fabrication, Characterization, and Evaluation of Bionanocomposites Based on Natural Polymers and Antibiotics for Wound Healing Applications

    Directory of Open Access Journals (Sweden)

    Marius Rădulescu

    2016-06-01

    Full Text Available The aim of our research activity was to obtain a biocompatible nanostructured composite based on naturally derived biopolymers (chitin and sodium alginate loaded with commercial antibiotics (either Cefuroxime or Cefepime with dual functions, namely promoting wound healing and assuring the local delivery of the loaded antibiotic. Compositional, structural, and morphological evaluations were performed by using the thermogravimetric analysis (TGA, scanning electron microscopy (SEM, and fourier transform infrared spectroscopy (FTIR analytical techniques. In order to quantitatively and qualitatively evaluate the biocompatibility of the obtained composites, we performed the tetrazolium-salt (MTT and agar diffusion in vitro assays on the L929 cell line. The evaluation of antimicrobial potential was evaluated by the viable cell count assay on strains belonging to two clinically relevant bacterial species (i.e., Escherichia coli and Staphylococcus aureus.

  12. Immunological aspects of the common food colorants, amaranth and tartrazine.

    Science.gov (United States)

    Koutsogeorgopoulou, L; Maravelias, C; Methenitou, G; Koutselinis, A

    1998-02-01

    We describe a sensitive and reproducible microassay model using human peripheral blood lymphocytes (PBL) for discrimination between the cytotoxic and immunosuppressive effects of food colorants such as amaranth and tartrazine. The cytotoxic effects of a wide range of concentrations of these substances were studied on human PBL by the colorimetric in vitro cytotoxicity assays, neutral red uptake (NR) and thiazolyl blue tetrazolium bromide (MTT). The immunotoxic properties of these 2 substances were determined by a [3H]-thymidine DNA incorporation assay on phytohemagglutinin stimulated or non-stimulated lymphocytes, as well as by a Cr51 release Natural Killer assays. The results showed clear immunosuppressive effects from the 2 substances tested, although the concentrations chosen for this study proved to be non-cytotoxic by NR and MTT cytotoxic endpoints.

  13. Study on decaying characteristics of activated sludge from a circular plug-flow reactor using response surface methodology.

    Science.gov (United States)

    Xie, En; Ding, Aizhong; Dou, Junfeng; Zheng, Lei; Yang, Jin

    2014-10-01

    Using pH values, temperature, and dissolved oxygen as the influencing factors, a decaying characteristics experiment of activated sludge was carried out by combining the LIVE/DEAD® Baclight technique with the 2,3,5-triphenyl tetrazolium chloride - dehydrogenase activity determination method. Using batch experiments, a response surface methodology was applied in the experimental design to determine the most important influential factor in the decay of activated sludge. The activated sludge mixed liquor for the experiment was generated in a laboratory-scale circular plug-flow reactor, which has already been approved for an invention patent. The analyzed results revealed that the most important influential factor in sludge activity decay is the pH, followed by temperature and then dissolved oxygen. After the decay experiment, 40.94-90.03% of sludge activity decay is caused by reduced cell activity, and the rest is due to cell death. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Atmospheric-pressure plasma jet characterization and applications on melanoma cancer treatment (B/16-F10)

    Energy Technology Data Exchange (ETDEWEB)

    Mashayekh, Shahriar [Physics Department, Shahid Beheshti University, G.C., Evin, 19839-63113 Tehran, Islamic Republic of Iran (Iran, Islamic Republic of); Rajaee, Hajar; Hassan, Zuhir M. [Imonology Department, Faculty of Medical Science, Tarbiat Modarres University, Tehran (Iran, Islamic Republic of); Akhlaghi, Morteza [Laser-Plasma Research Institute, Shahid Beheshti University, G.C., Evin, 19839-63113 Tehran, Islamic Republic of Iran (Iran, Islamic Republic of); Shokri, Babak [Physics Department and Laser-Plasma Research Institute, Shahid Beheshti University, G.C., Evin, 19839-63113 Tehran, Islamic Republic of Iran (Iran, Islamic Republic of)

    2015-09-15

    A new approach in medicine is the use of cold plasma for various applications such as sterilization blood coagulation and cancer cell treatment. In this paper, a pin-to-hole plasma jet for biological applications has been designed and manufactured and characterized. The characterization includes power consumption via Lissajous method, thermal behavior of atmospheric-pressure plasma jet by using Infra-red camera as a novel method and using Speicair software to determine vibrational and transitional temperatures, and optical emission spectroscopy to determine the generated species. Treatment of Melanoma cancer cells (B16/F10) was also implemented, and tetrazolium salt dye (MTT assay) and flow cytometry were used to evaluate viability. Effect of ultraviolet photons on cancerous cells was also observed using an MgF{sub 2} crystal with MTT assay. Finally, in-vivo studies on C57 type mice were also done in order to have a better understanding of the effects in real conditions.

  15. Cytotoxic Induction and Photoacoustic Imaging of Breast Cancer Cells Using Astaxanthin-Reduced Gold Nanoparticles

    Directory of Open Access Journals (Sweden)

    Subramaniyan Bharathiraja

    2016-04-01

    Full Text Available Astaxanthin, a kind of photosynthetic pigment, was employed for gold nanoparticle formation. Nanoparticles were characterized using Ulteraviolet-Visible (UV-Vis spectroscopy, transmission electron microscopy, and X-ray diffraction, and the possible presence of astaxanthin functional groups were analyzed by Fourier transform infrared spectroscopy (FTIR. The cytotoxic effect of synthesized nanoparticles was evaluated against MDA-MB-231 (human breast cancer cells using a tetrazolium-based assay, and synthesized nanoparticles exhibited dose-dependent toxicity. The morphology upon cell death was differentiated through fluorescent microscopy using different stains that predicted apoptosis. The synthesized nanoparticles were applied in ultrasound-coupled photoacoustic imaging to obtain good images of treated cells. Astaxanthin-reduced gold nanoparticle has the potential to act as a promising agent in the field of photo-based diagnosis and therapy.

  16. Structural characteristics and biological performance of silk fibroin nanofiber containing microalgae Spirulina extract.

    Science.gov (United States)

    Cha, Bum-Gyu; Kwak, Hyo Won; Park, A Reum; Kim, Shin Hwan; Park, Sook-Young; Kim, Hyun-Jeong; Kim, Ick-Soo; Lee, Ki Hoon; Park, Young Hwan

    2014-04-01

    Silk fibroin (SF) nanofiber scaffold containing microalgae Spirulina extract were prepared by electrospinning and the performance and functionality of the scaffold were evaluated. The viscosity and conductivity of the dope solution of Spirulina containing SF were examined for electrospinability and we found that the morphological structure of SF nanofiber is affected by the concentration of Spirulina extract added. The platelet adhesion and coagulation time test confirmed that the Spirulina containing SF nanofiber scaffold had excellent ability to prevent blood clotting or antithrombogenicity that is comparable to heparin. Low cytotoxicity and excellent cell adhesion and proliferation were also observed for Sprulina containing SF nanofiber scaffold by methylthiazolyldiphenyl-tetrazolium bromide assay and confocal fluorescence microscope using fibroblast and human umbilical vein endothelial cells. Based on these results, we believe SF nanofiber scaffold containing Spirulina extract has the potential to be used as tissue engineering scaffold that requires high hemocompatibility. Copyright © 2013 Wiley Periodicals, Inc.

  17. Differential Stimulatory Activities of Smooth and Rough Brucella abortus Lipopolysaccharide in Murine Macrophages

    Directory of Open Access Journals (Sweden)

    Raheela Akhtar1,2*, Yongqun O. He2, Charles B. Larson2, Zafar I. Chaudhary3 and Mansur ud-Din Ahmad4

    2012-06-01

    Full Text Available Brucella abortus lipopolysaccharide (LPS was isolated and purified from rough (RB51 and smooth (S2308 strains of Brucella. The LPS preparations were used to treat murine (RAW 264.7 macrophages in order to study their differential effects. Treated macrophages were tested by lysozyme release test (LRT, nitroblue tetrazolium test (NBT and nitric oxide (NO assay, respectively. Rough Brucella LPS induced significantly higher levels of lysozyme release, oxidative stress, and nitric oxide in murine macrophages than smooth Brucella LPS or combined LPS (rough + smooth LPS. These responses were dose-dependent. Macrophages treated with rough LPS were more Brucellacidal than those treated with smooth LPS. The minimal stimulation of murine macrophages by Brucella smooth LPS may provide basis for less active immune responses against smooth strains.

  18. Neutral Red versus MTT assay of cell viability in the presence of copper compounds.

    Science.gov (United States)

    Gomez Perez, Mariela; Fourcade, Lyvia; Mateescu, Mircea Alexandru; Paquin, Joanne

    2017-10-15

    Copper is essential for numerous physiological functions, and copper compounds may display therapeutic as well as cytotoxic effects. The MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay is a standard test largely used in cytotoxicity studies. This report shows that low micromolar levels of copper compounds such as Cu(II)Urea2, Cu(II)Ser2 and CuCl2 can interfere with the MTT assay making improper the detection of formazan product of MTT reduction. Comparatively, the Neutral Red assay appears to be sensitive and showing no interference with these compounds. The lactate dehydrogenase alternative assay cannot be used because of inhibitory effect of these copper compounds on the enzyme activity. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Preparation and characterization of aminoethyl hydroxypropyl methyl cellulose modified with nisin.

    Science.gov (United States)

    Fan, Lihong; Hu, Jin; Hu, Zhihai; Peng, Min; Wen, Huigao; Li, Ya; Wang, Tan

    2016-08-01

    Nisin grafted aminoethyl hydroxypropyl methyl cellulose (AEHPMC) was prepared by an enzyme-catalyzed reaction in the presence of microbial transglutaminase (MTGase). AEHPMC was synthesized with 2-chloroethylamine hydrochloride (CEH) which was as an intermediate reactant. The parameters, which influenced the NH2% and the degree of substitution (DS), including reaction time, reaction temperature and the mass ratio of the reactants were investigated. Antioxidant activities of AEHPMC-nisin were evaluated by the scavenging activity of hydroxyl and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. With the DS and concentration increasing of AEHPMC-nisin, the radical scavenging activity increased. The Methylthiazol tetrazolium (MTT) assay indicated that AEHPMC-nisin had low-toxicity to L929 cells. Therefore, the derivative of HPMC may show a promising potential application in biomedical, food and pharmaceutical fields. Copyright © 2016. Published by Elsevier B.V.

  20. Cytotoxicity and genotoxicity of gliotoxin on human lymphocytes in vitro

    Directory of Open Access Journals (Sweden)

    Mohammed Adel Nouri

    2015-07-01

    Full Text Available The cytotoxic effects on human lymphocytes of two gliotoxin samples (one pure sample produced in the laboratory for this study, and one sample purchased from a standard source were assessed at four different concentrations (25, 50, 100 and 200 ng/ml using the methylthiazol tetrazolium (MTT bioassay. The results showed that growth was inhibited by 21, 39.10, 61.99 and 87.45% for each of the four concentrations of the pure sample, respectively, and by 17.89, 34.92, 58.34 and 85.22% respectively, in the case of the standard purchased sample. Deoxyribonucleic acid (DNA was extracted from the lymphocytes and analysed by electrophoresis on a 1% agarose gel. Gliotoxin appeared to have the ability to degrade or damage the DNA. The present study showed that both the growth inhibition and DNA damage experienced by the human lymphocytes increased linearly with increasing concentrations of toxin.

  1. Sensitive detection of Escherichia coli O157:H7 in food and water by immunomagnetic separation and solid-phase laser cytometry

    Science.gov (United States)

    Pyle, B. H.; Broadaway, S. C.; McFeters, G. A.

    1999-01-01

    Rapid, direct methods are needed to assess active bacterial populations in water and foods. Our objective was to determine the efficiency of bacterial detection by immunomagnetic separation (IMS) and the compatibility of IMS with cyanoditolyl tetrazolium chloride (CTC) incubation to determine respiratory activity, using the pathogen Escherichia coli O157:H7. Counterstaining with a specific fluorescein-conjugated anti-O157 antibody (FAb) following CTC incubation was used to allow confirmation and visualization of bacteria by epifluorescence microscopy. Broth-grown E. coli O157:H7 was used to inoculate fresh ground beef (laser cytometry. Enumeration by laser scanning permitted detection of ca. 10 CFU/g of ground beef or laser scanning cytometry gave results that compared favorably with plating following IMS.

  2. Arsenic trioxide-mediated oxidative stress and genotoxicity in human hepatocellular carcinoma cells.

    Science.gov (United States)

    Alarifi, Saud; Ali, Daoud; Alkahtani, Saad; Siddiqui, Maqsood A; Ali, Bahy A

    2013-01-01

    Arsenic is a ubiquitous environmental toxicant, and abnormalities of the skin, lung, kidney, and liver are the most common outcomes of long-term arsenic exposure. This study was designed to investigate the possible mechanisms of genotoxicity induced by arsenic trioxide in human hepatocellular carcinoma cells. A mild cytotoxic response of arsenic trioxide was observed in human hepatocellular carcinoma cells, as evident by (3-(4,5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) and lactate dehydrogenase assays after 24 and 48 hours of exposure. Arsenic trioxide elicited a significant (P hepatocellular carcinoma cells exposed to arsenic trioxide. The results demonstrate that arsenic trioxide induces apoptosis and genotoxicity in human hepatocellular carcinoma cells through reactive oxygen species and oxidative stress.

  3. [Effect of astragalus membranaceus on the proliferation, osteogenic capacity and structure of periodontal ligament cells in vitro].

    Science.gov (United States)

    Zhang, Chao-liang; Kong, Xiang-li; Chen, Si-xiu; Li, Xiao-yu

    2010-10-01

    To investigate the effect of Astragalus membranaceus (APS) on the proliferation, osteogenic capacity and structure of periodontal ligament cells (PDLCs) in vitro. PDLCs were cultured in vitro with APS of 0.08, 0.1, 0.2, 0.4 mg x mL(-1). Methyl thiazolyl tetrazolium (MTr), alkaline phosphatase (ALP) and cell structure were detected to determine the proliferation and differentiation of PDLCs proliferation and differentiation. When the APS was 0.2 mg x mL(-1), the absorbance of MTT and ALP exhibit significantly increased as compared to the control (P structure. APS with proper concentration in short-term culture may promote the proliferation and differentiation of PDLCs.

  4. A tip-high, Ca(2+) -interdependent, reactive oxygen species gradient is associated with polarized growth in Fucus serratus zygotes.

    Science.gov (United States)

    Coelho, Susana M B; Brownlee, Colin; Bothwell, John H F

    2008-04-01

    We report the existence of a tip-high reactive oxygen species (ROS) gradient in growing Fucus serratus zygotes, using both 5-(and 6-) chloromethyl-2',7'-dichlorodihydrofluorescein and nitroblue tetrazolium staining to report ROS generation. Suppression of the ROS gradient inhibits polarized zygotic growth; conversely, exogenous ROS generation can redirect zygotic polarization following inhibition of endogenous ROS. Confocal imaging of fluo-4 dextran distributions suggests that the ROS gradient is interdependent on the tip-high [Ca(2+)](cyt) gradient which is known to be associated with polarized growth. Our data support a model in which localized production of ROS at the rhizoid tip stimulates formation of a localized tip-high [Ca(2+)](cyt) gradient. Such modulation of intracellular [Ca(2+)](cyt) signals by ROS is a common motif in many plant and algal systems and this study extends this mechanism to embryogenesis.

  5. Simultaneous immobilization of dehydrogenases on polyvinylidene difluoride resin after separation by non-denaturing two-dimensional electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Shimazaki, Youji [Graduate School of Science and Engineering (Science Section) and Venture Business Laboratory, Ehime University, Bunkyo-cho 2-5, Matsuyama City 790-8577 (Japan)], E-mail: yoji@dpc.ehime-u.ac.jp; Kadota, Mariko [Faculty of Science, Ehime University, Matsuyama (Japan)

    2008-06-16

    We detected mouse liver malate, sorbitol and aldehyde dehydrogenases by negative staining, analysis of malate and sorbitol dehydrogenase activities using each substrate, and electron transfers including nicotinamide adenine dinucleotide (NAD) and nitroblue tetrazolium in non-denaturing two-dimensional electrophoresis (2-DE) gel. Dehydrogenases were also identified by electrospray ionization tandem mass spectrometry (ESI-MS/MS) after 2-DE separation and protein detection by negative staining. Spots of dehydrogenases separated by 2-DE were excised, and simultaneously transferred and immobilized on polyvinylidene difuoride (PVDF) resin by electrophoresis. The dehydrogenase activities remained intact after immobilization. In conclusion, resin-immobilized dehydrogenases can be simultaneously obtained after separation by non-denaturing 2-DE, detection by negative staining and transferring to resins.

  6. Sensitive detection of PDT-induced cell damages with luminescent oxygen nanosensors.

    Science.gov (United States)

    Ma, Hong-Ru; Peng, Hong-Shang; You, Fang-Tian; Ping, Jian-Tao; Zhou, Chao; Guo, Lan-Ying

    2016-07-28

    In this work luminescent nanosensors specifically created for intracellular oxygen (ic-O 2 ) were utilized to assess photodynamic therapy (PDT) -induced cell damages. Firstly, ic-O 2 was demonstrated to be consumed much faster than extracellular O 2 with respective O 2 nanosensors. Using the ic-O 2 nanosensors, PDT-treated cells with different degree of impairment were then resolved according to the oxygen consumption rate (OCR). The evolving trend of cytotoxicity derived from OCRs was in agreement with cell viability obtained from 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Moreover, the direct damage of PDT on cell mitochondria was successfully detected by monitoring respiration instantly after PDT treatment, which is actually beyond the scope of MTT assay. These results suggest that fluorescence sensing of ic-O 2 -associated cell respiration is promising and even may become a standardized method, complementary to MTT assay, to evaluate PDT-induced cytotoxicity.

  7. The use of a water-soluble formazan complex to quantitate the cell number and mitochondrial function of Leishmania major promastigotes

    DEFF Research Database (Denmark)

    Berg, K; Zhai, L; Chen, M

    1994-01-01

    in that the formazan complex precipitates inside the parasites and has to be extracted by denaturants before measurements can be performed. By using a new synthetic substrate, 3-(4,5-dimethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2-(4-sulfonyl)-2H-tetrazolium (MTS), the extraction procedure is eliminated...... radioactive thymidine uptake and cell counting, respectively. The method is simple, fast, and highly reproducible and is suitable for drug screening, identification of drug-resistant isolates, and growth-kinetics studies. It is therefore contemplated that the MTS method will be a general and useful technique......One of the methods to quantitate Leishmania major promastigotes (LmP) has been to utilize the formation of a formazan dye, which in turn is produced via conversion of an artificial substrate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The method has one major drawback...

  8. Cellular uptake and cytotoxicity of positively charged chitosan gold nanoparticles in human lung adenocarcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Seon Young; Jang, Soo Hwa [Seoul National University, Laboratory of Veterinary Pharmacology, College of Veterinary Medicine and Institute for Veterinary Science (Korea, Republic of); Park, Jin; Jeong, Saeromi; Park, Jin Ho; Ock, Kwang Su [Soongsil University, Department of Chemistry (Korea, Republic of); Lee, Kangtaek [Yonsei University, Department of Chemical and Biomolecular Engineering (Korea, Republic of); Yang, Sung Ik [Kyung Hee University, College of Environment and Applied Chemistry (Korea, Republic of); Joo, Sang-Woo, E-mail: sjoo@ssu.ac.kr [Soongsil University, Department of Chemistry (Korea, Republic of); Ryu, Pan Dong; Lee, So Yeong, E-mail: leeso@snu.ac.kr [Seoul National University, Laboratory of Veterinary Pharmacology, College of Veterinary Medicine and Institute for Veterinary Science (Korea, Republic of)

    2012-12-15

    Cellular uptake, cytotoxicity, and mechanisms of cytotoxicity of the positively charged Au nanoparticles (NPs) were examined in A549 cells, which are one of the most characterized pulmonary cellular systems. Positively charged Au NPs were prepared by chemical reduction using chitosan. The dimension and surface charge of Au NPs were examined by transmission electron microscopy (TEM), dynamic light scattering, and zeta potential measurements. The uptake of Au NPs into A549 cells was also monitored using TEM and dark-field microscopy (DFM) and z-stack confocal microRaman spectroscopy. DFM live cell imaging was also performed to monitor the entry of chitosan Au NPs in real time. The cytotoxic assay, using both methylthiazol tetrazolium and lactate dehydrogenase assays revealed that positively charged Au NPs decreased cell viability. Flow cytometry, DNA fragmentation, real-time PCR, and western blot analysis suggest that positively charged chitosan Au NPs provoke cell damage through both apoptotic and necrotic pathways.

  9. Sound waves effectively assist tobramycin in elimination of Pseudomonas aeruginosa biofilms in vitro.

    Science.gov (United States)

    Bandara, H M H N; Harb, A; Kolacny, D; Martins, P; Smyth, H D C

    2014-12-01

    Microbial biofilms are highly refractory to antimicrobials. The aim of this study was to investigate the use of low-frequency vibration therapy (20-20 kHz) on antibiotic-mediated Pseudomonas aeruginosa biofilm eradication. In screening studies, low-frequency vibrations were applied on model biofilm compositions to identify conditions in which surface standing waves were observed. Alginate surface tension and viscosity were also measured. The effect of vibration on P. aeruginosa biofilms was studied using a standard biofilm assay. Subminimal inhibitory concentrations (sub-MIC) of tobramycin (5 μg/ml) were added to biofilms 3 h prior, during, and immediately after vibration and quantitatively assessed by (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) reduction assay (XTT) and, qualitatively, by confocal laser scanning microscopy (CLSM). The standing waves occurred at frequencies sound waves together with antibiotics are a promising approach in eliminating pathogenic biofilms.

  10. Acetylsalicylic acid and acetaminophen protect against oxidative neurotoxicity.

    Science.gov (United States)

    Maharaj, H; Maharaj, D S; Daya, S

    2006-09-01

    Due to the implication of oxidative stress in neurodegenerative disorders we decided to investigate the antioxidant properties of acetylsalicylic acid and acetaminophen either alone or in combination. The thiobarbituric acid assay (TBA) and the nitroblue tetrazolium (NBT) assay were used to investigate quinolinic acid (QA)-induced: lipid peroxidation and superoxide anion generation in the rat hippocampus, in vivo. The study also shows, using cresyl violet staining, the preservation of structural integrity of neuronal cells following treatment with acetylsalicylic acid and acetaminophen in QA-lesioned rat hippocampus. Furthermore the study sought to determine whether these agents have any effect on endogenous (QA) formation. This study shows that acetylsalicylic acid and acetaminophen inhibit QA-induced superoxide anion generation, lipid peroxidation and cell damage, in vivo, in the rat hippocampus. In addition these agents inhibit the enzyme, 3-hydroxyanthranilic acid oxygenase (3-HAO), responsible for the synthesis of endogenous QA.

  11. Biofilm Formation by Pseudomonas Species Onto Graphene Oxide-TiO2 Nanocomposite-Coated Catheters: In vitro Analysis

    Science.gov (United States)

    Deb, Ananya; Vimala, R.

    The present study focuses on the development of an in vitro model system for biofilm growth by Pseudomonas aerouginosa onto small discs of foley catheter. Catheter disc used for the study was coated with graphene oxide-titanium oxide composite (GO-TiO2) and titanium oxide (TiO2) and characterized through XRD, UV-visible spectroscopy. Morphological analysis was done by scanning electron microscopy (SEM). The biofilm formed on the catheter surface was quantified by crystal violet (CV) staining method and a colorimetric assay (MTT assay) which involves the reduction of tetrazolium salt. The catheter coated with GO-TiO2 showed reduced biofilm growth in comparison to the TiO2-coated and uncoated catheter, thus indicating that it could be successfully used in coating biomedical devices to prevent biofilm formation which is a major cause of nosocomial infection.

  12. Comparative analysis of quantitative methodologies for Vibrionaceae biofilms.

    Science.gov (United States)

    Chavez-Dozal, Alba A; Nourabadi, Neda; Erken, Martina; McDougald, Diane; Nishiguchi, Michele K

    2016-11-01

    Multiple symbiotic and free-living Vibrio spp. grow as a form of microbial community known as a biofilm. In the laboratory, methods to quantify Vibrio biofilm mass include crystal violet staining, direct colony-forming unit (CFU) counting, dry biofilm cell mass measurement, and observation of development of wrinkled colonies. Another approach for bacterial biofilms also involves the use of tetrazolium (XTT) assays (used widely in studies of fungi) that are an appropriate measure of metabolic activity and vitality of cells within the biofilm matrix. This study systematically tested five techniques, among which the XTT assay and wrinkled colony measurement provided the most reproducible, accurate, and efficient methods for the quantitative estimation of Vibrionaceae biofilms.

  13. STUDY ON POLLEN VIABILITY AS BIOINDICATOR OF AIR QUALITY

    Directory of Open Access Journals (Sweden)

    Florentina ŞTEFLEA

    2012-01-01

    Full Text Available The aim of this research is to estimate the relationship between pollen viability and atmospheric pollution (in polluted and non-polluted conditions. The study was carried out in the city of Timisoara. Two areas, with different intensity of road traffic (very high and absent but all characterized by the presence of the same plant species, were selected. The pollen of herbaceous spontaneous species, arboreal species and a shrub species was used (Robinia pseudacacia, Aesculus x carnea, Catalpa bignonioides, Albizzia julibrissin, Rosa canina, Sambucus nigra, Malva neglecta, Ranunculus acer, Trifolium repens, Cichorium intybus. The pollen of these species was treated with TTC (2, 3, 5 Tryphenil-Tetrazolium-Chloride staining solution and viability was then estimated by light microscopy. The results of the mean pollen viability percentage of the examined species are reported. Pollen viability of herbaceous plants is significantly different between the two environments.

  14. Evidence for Detrimental Cross Interactions between Reactive Oxygen and Nitrogen Species in Leber's Hereditary Optic Neuropathy Cells

    Science.gov (United States)

    Santini, Paolo

    2016-01-01

    Here we have collected evidence suggesting that chronic changes in the NO homeostasis and the rise of reactive oxygen species bioavailability can contribute to cell dysfunction in Leber's hereditary optic neuropathy (LHON) patients. We report that peripheral blood mononuclear cells (PBMCs), derived from a female LHON patient with bilateral reduced vision and carrying the pathogenic mutation 11778/ND4, display increased levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS), as revealed by flow cytometry, fluorometric measurements of nitrite/nitrate, and 3-nitrotyrosine immunodetection. Moreover, viability assays with the tetrazolium dye MTT showed that lymphoblasts from the same patient are more sensitive to prolonged NO exposure, leading to cell death. Taken together these findings suggest that oxidative and nitrosative stress cooperatively play an important role in driving LHON pathology when excess NO remains available over time in the cell environment. PMID:26881022

  15. Conjunctival leishmaniasis in a case of disseminated cutaneous leishmaniasis.

    Science.gov (United States)

    Razeghinejad, M Reza; Monabati, Ahmad; Kadivar, Mohammad Rahim; Alborzi, Abdolvahab

    2017-01-01

    A 15-year-old female patient presented with numerous, small, papulonodular skin lesions, and hepatosplenomegaly 9 months after a treated biopsy proved cutaneous leishmaniasis. In ocular examination there were two yellowish, raised gelatinous conjunctival lesions in the left eye. The exisional conjunctival lesion biopsy revealed many Leishman bodies inside tissue histiocytes. The patient had no systemic immunologic problems (normal serum immunoglobulins, nitroblue-tetrazolium test, complement CH50 test and flow cytometry of leukocytes). The indirect immunofluorescent antibody test for Leishmania tropica (titre of 1:1024) and the leishmanin skin test were positive. DNA of L. tropica was detected by a specific polymerase chain reaction on whole blood, bone marrow and skin biopsy specimens. The skin and conjunctival lesions disappeared with miltefosine and no intraocular tissue penetration of organism happened. Conjunctival leishmaniasis should be considered in the differential diagnosis of raised conjunctival lesions in a disseminated cutaneous leishmaniasis patient and needs proper systemic therapy. © The Author(s) 2016.

  16. Antimutagenic and antioxidant potentials of Teucrium ramosissimum essential oil.

    Science.gov (United States)

    Sghaier, Mohamed Ben; Boubaker, Jihed; Neffati, Aicha; Limem, Ilef; Skandrani, Ines; Bhouri, Wissem; Bouhlel, Ines; Kilani, Soumaya; Chekir-Ghedira, Leila; Ghedira, Kamel

    2010-07-01

    The mutagenic and antimutagenic effects of the essential oil extracted from the aerial parts of Teucrium ramosissimum were evaluated by the bacterial reverse mutation assay in Salmonella typhimurium TA98, TA100, and TA1535, with and without exogenous metabolic activation (S9 fraction). The T. ramosissimum essential oil showed no mutagenic effect. In contrast, our results established that it possessed antimutagenic effects against sodium azide (SA), aflatoxin B1 (AFB1), benzo[a]pyrene (B[a]P), and 4-nitro-o-phenylenediamine (NOPD). The antioxidant capacity of the tested essential oil was evaluated using enzymatic, i.e., the xanthine/xanthine oxidase (X/XOD) assay, and nonenzymatic systems, i.e., the nitro-blue tetrazolium (NBT)/riboflavin and the DPPH assays. A moderate free radical-scavenging activity was observed towards DPPH(.) and O2(.-). In contrast, T. ramosissimum essential oil showed no effect for all the tested concentrations in the X/XOD assay.

  17. Determination of fecal bacteriophages fecales from pre-potable waters using a simple membrane filtration technique; Determinacion de bacteriofagos fecales en aguas pre-potables por la tecnica de filtracion por membrana

    Energy Technology Data Exchange (ETDEWEB)

    Luque, A.; Basanta, A. [EMASESA. Sevilla (Spain); Fuentes, M. V.; Alonso, M. C.; Borrego, J. J. [Universidad de Malaga (Spain)

    2000-07-01

    A modification of the simple filtration technique used for the detection and enumeration of fecal bacteriophages from pre-treated and treated drinking water is described. The filters and equipments used are similar to those employed for the classical bacterial indicator analysis, and the titer of bacteriophages may be recorded in only 6 hours. The proposed modification is based on the elimination of the bacterial decontamination of the water sample by use the selective medium mFC agar; and, in addition, an optimal visualization of the lysis plaques is obtained in this medium, which avoid the use of specific stains like triphenyl tetrazolium chloride (TTC). On the other hand, the proposed modification of the technique presents a high accuracy, since bacteriophage titers of<10 pfu/100 ml of water are detected. (Author) 46 refs.

  18. Chemical Constituents of Caesalpinia decapetala (Roth Alston

    Directory of Open Access Journals (Sweden)

    Song Yang

    2013-01-01

    Full Text Available The current study targets the chemical constituents of Caesalpinia decapetala (Roth Alston and investigates the bioactivities of the isolated compounds. Fourteen known compounds were isolated using column chromatography, and structural identification was performed by physical and spectral analyses. The biological activities of the compounds were also evaluated by 3-(4,5-dimethythiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT and 2,2-diphenlyl-1-picrylhydrazyl (DPPH assays. Emodin (6, baicalein (9, and apigenin (12 displayed antitumor activities against the MGC-803 cell line, while quercetin (2, rutin (5, baicalein (9, and epicatechin (13 showed stronger DPPH scavenging activities compared with ascorbic acid. Andrographolide (1, quercetin (2, bergenin (4, rutin (5, emodin (6, betulin (7, baicalein (9, polydatin (10, salicin (11, and apigenin (12, were obtained from C. decapetala (Roth Alston for the first time.

  19. Preliminary viability studies of fibroblastic cells cultured on microcrystalline and nanocrystalline diamonds produced by chemical vapour deposition method

    Directory of Open Access Journals (Sweden)

    Ana Amélia Rodrigues

    2013-02-01

    Full Text Available Implant materials used in orthopedics surgery have demonstrated some disadvantages, such as metallic corrosion processes, generation of wear particles, inflammation reactions and bone reabsorption in the implant region. The diamond produced through hot-filament chemical vapour deposition method is a new potential biomedical material due to its chemical inertness, extreme hardness and low coefficient of friction. In the present study we analysis two samples: the microcrystalline diamond and the nanocrystalline diamond. The aim of this study was to evaluate the surface properties of the diamond samples by scanning electron microscopy, Raman spectroscopy and atomic force microscopy. Cell viability and morphology were assessed using thiazolyl blue tetrazolium bromide, cytochemical assay and scanning electron microscopy, respectively. The results revealed that the two samples did not interfere in the cell viability, however the proliferation of fibroblasts cells observed was comparatively higher with the nanocrystalline diamond.

  20. Protective Effect of Ethanol Extracts of the Chinese Caterpillar Mushroom, Ophiocordyceps sinensis (Ascomycetes), on the Experimental Middle Cerebral Artery Occlusion/Reperfusion (MCAO/R) Model.

    Science.gov (United States)

    Kong, Ran; Zhang, Ying; Zhang, Shuofeng; Liu, Min; Sun, Wenyan; Xing, Yue; Guan, Yalan; Han, Chunchao; Liu, Zhenquan

    2015-01-01

    In this study, we investigated the effects of ethanol extracts of Ophiocordyceps sinensis (EEOS) on neuroprotective efficacy in a rat model of focal cerebral ischemia/reperfusion (IR). The effects of EEOS on mortality rate, neurobehavior, grip strength, polymorphonuclear (PMN) cells, interleukin (IL)-1β, inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF)-α, intracellular adhesion molecule 1 (ICAM-1), and cyclooxygenase-2 (COX-2) were determined by enzyme-linked immunosorbent assay and immunohistochemistry. The cerebral infarction was examined through tetrazolium chloride staining. EEOS significantly inhibited IR-induced brain production of IL-1β, TNF-α, iNOS, ICAM-1, and COX-2. Moreover, EEOS suppressed infiltration of PMN cells. EEOS caused a significant reduction in the infarct size compared with the middle cerebral artery occlusion group. The study demonstrates the neuroprotective potential of EEOS inhibition of IR through anti-inflammatory activity in a rat model of IR.

  1. Cellular uptake and cytotoxicity of positively charged chitosan gold nanoparticles in human lung adenocarcinoma cells

    Science.gov (United States)

    Choi, Seon Young; Jang, Soo Hwa; Park, Jin; Jeong, Saeromi; Park, Jin Ho; Ock, Kwang Su; Lee, Kangtaek; Yang, Sung Ik; Joo, Sang-Woo; Ryu, Pan Dong; Lee, So Yeong

    2012-12-01

    Cellular uptake, cytotoxicity, and mechanisms of cytotoxicity of the positively charged Au nanoparticles (NPs) were examined in A549 cells, which are one of the most characterized pulmonary cellular systems. Positively charged Au NPs were prepared by chemical reduction using chitosan. The dimension and surface charge of Au NPs were examined by transmission electron microscopy (TEM), dynamic light scattering, and zeta potential measurements. The uptake of Au NPs into A549 cells was also monitored using TEM and dark-field microscopy (DFM) and z-stack confocal microRaman spectroscopy. DFM live cell imaging was also performed to monitor the entry of chitosan Au NPs in real time. The cytotoxic assay, using both methylthiazol tetrazolium and lactate dehydrogenase assays revealed that positively charged Au NPs decreased cell viability. Flow cytometry, DNA fragmentation, real-time PCR, and western blot analysis suggest that positively charged chitosan Au NPs provoke cell damage through both apoptotic and necrotic pathways.

  2. Assessment of Geographical Variation in the Respiratory Toxicity of Desert Dust Particles

    Science.gov (United States)

    2013-01-01

    diphenyltetrazolium bromide assay and assessment of lactate dehydrogenase leakage. The relative in vitro cytotoxicity of the sand extracts was Taji...before the impact on cell metabolism was evaluated using an MTT [3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ] assay. This tetrazolium...124.13 0.7 0.4 0.2 0.6 Copper 36.5 26.0 27.33 0.1 15.3 2.8 7.1 Iron 34 275.1 33 550.8 24 414.6 2.8 4.3 5LOD 12.0 Lithium 20.2 24.9 17.9 5LOD 34.0 8.9

  3. Synergistic cytotoxicity induced by α-solanine and α-chaconine.

    Science.gov (United States)

    Yamashoji, Shiro; Matsuda, Takako

    2013-11-15

    α-Solanine and α-chaconine are well-known potato toxins, but the mechanism of the synergistic cytotoxic effect of these alkaloids has been little clarified. This study confirmed their synergistic cytotoxic effects on C6 rat glioma cells by three different cell viability tests, namely WST-1 (water-soluble tetrazolium) assay sensitive to intracellular NADH concentration, menadione-catalysed chemiluminescent assay depending on both NAD(P)H concentration and NAD(P)H:quinone reductase activity, and LDH (lactate dehydrogenase) assay sensitive to the release of LDH from damaged cells. The maximum cytotoxic effect was observed at a ratio of 1:1 between α-solanine and α-chaconine at micromolar concentrations. The cytotoxic effects of these alkaloids were observed immediately after incubation and were constant after 30min, suggesting that rapid damage of plasma membrane causes the lethal disorder of metabolism. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. Synthesis of acyl-hydrazone from usnic acid and isoniazid and its anti-Mycobacterium tuberculosis activity

    Directory of Open Access Journals (Sweden)

    Melissa Barrera Tomas

    2017-09-01

    Full Text Available Compound usnic acid (1, isolated from lichen Evernia prunastri (Cajamarca-Peru and the synthesis and characterization of its acyl-hydrazone (2, from the condensation reaction between usnic acid and isoniazid in an ethanol solution under reflux, giving an overall yield of 95%, were evaluated. Both compounds were evaluated and compared with isoniazid according to its anti-Mycobacterium tuberculosis activity based on the tetrazolium microplate assay (TEMA. Compound 1 had MIC (minimal inhibitory concentration value of 16.0 μg/mL in each test of H37Rv (susceptible type, TB DM 97 (resistant wild type and MDR DM 1098 (multi drug resistances type strains. In similar tests, compound 2 MIC values were 2.0, 64.0 and 64.0 μg/mL respectively.

  5. The effect of ethylene oxide sterilization on the surface chemistry and in vitro cytotoxicity of several kinds of chitosan.

    Science.gov (United States)

    França, Rodrigo; Mbeh, Doris A; Samani, Taraneh Djavanbakht; Le Tien, Canh; Mateescu, Mircea A; Yahia, L'Hocine; Sacher, Edward

    2013-11-01

    The surfaces of three chitosan samples, differing only in their degrees of deacetylation and of carboxyethyl chitosan were chemically characterized by X-ray photoelectron spectroscopy, time-of-flight secondary ion mass spectroscopy, X-ray diffraction, and Fourier transform infrared, both before and after sterilization with ethylene oxide. Unexpected elemental ratios suggest that surface chemical modification occurred during the processing of the original chitin, with further surface modification on subsequent sterilization, despite previous reports to the contrary. Cell viability was evaluated by direct contact methyl thiazole tetrazolium and lactate dehydrogenase assays between the chitosan particles and A549 human epithelial cells, which demonstrated that the modifications incurred on sterilization are reflected in biocompatibility changes. All the samples were found to be biocompatible and nontoxic before sterilization and remained so subsequently. Copyright © 2013 Wiley Periodicals, Inc.

  6. The in vitro biomineralization and cytocompatibility of polydopamine coated carbon nanotubes

    Energy Technology Data Exchange (ETDEWEB)

    Yan Penghua [State Key Laboratory of Solid Lubrication, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, 18 Tianshui Middle Road, Lanzhou 730000 (China); Key Laboratory of Polymer Materials of Gansu Province, College of Chemistry and Chemical Engineering, Northwest Normal University, Lanzhou 730070 (China); Wang Jinqing, E-mail: jqwang@licp.cas.cn [State Key Laboratory of Solid Lubrication, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, 18 Tianshui Middle Road, Lanzhou 730000 (China); Wang Lin; Liu Bin [School of Stomatology, Lanzhou University, Lanzhou 730000 (China); Lei Ziqiang [Key Laboratory of Polymer Materials of Gansu Province, College of Chemistry and Chemical Engineering, Northwest Normal University, Lanzhou 730070 (China); Yang Shengrong, E-mail: sryang@lzb.ac.cn [State Key Laboratory of Solid Lubrication, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, 18 Tianshui Middle Road, Lanzhou 730000 (China); Key Laboratory of Polymer Materials of Gansu Province, College of Chemistry and Chemical Engineering, Northwest Normal University, Lanzhou 730070 (China)

    2011-03-15

    In this work, polydopamine coated carbon nanotubes were firstly prepared by a simple and feasible route. Then, for comparison, the in vitro bioactivity and cytocompatibility of the carbon nanotubes and the polydopamine coated carbon nanotubes were assessed by immersion study in simulated body fluids and 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide test using osteoblast cells (MC3T3-E1), respectively. As a result, it has been demonstrated that the introduction of polydopamine coating can greatly enhance the bioactivity and promote cell proliferation of the carbon nanotubes. The improvement of bioactive behavior is attributed to the good combination of catecholamines structure of the polydopamine and the structural advantages of carbon nanotubes as a framework material. It is anticipated that the polydopamine coated carbon nanotubes would find potential applications in bone tissue engineering and other biomedical areas.

  7. [Analysis of Pseudomonas aeruginosa biofilm production in relation to cultivation media and biofilm staining methods].

    Science.gov (United States)

    Kalicińska, Agnieszka; Tyski, Stefan

    2009-01-01

    The aim of this study was to analyse P aeruginosa biofilm growing in three media, using three methods of biofilm staining: with 2% crystal violet solution (CV), with 1% 2,3,5-triphenyltetrazolium chloride solution (TTC) and 3-(4,5-dimethyl-2-thiazolyl)-2,5 diphenyl-2H-tetrazolium bromide (MTT). Using the method with MTT solution we could observe growth of strains forming biofilm both slowly or quickly. Achieved values of absorbation allow to observe dynamic of growing selected strains in the period of time. Among three groups of analysed strains, the biggest percentage of medium growing strains were achieved, while in the fourth group (strains isolated from spit of patients with cystic fibrosis) the biggest percentage of low growing strains was observed. Achieved results allow to select the strains quickly forming biofilm, the best media (LB, TSB2%glu) and method of biofilm staining (MTT), which will be used in the further studies.

  8. Avidin-biotin-immunoglucose oxidase: use in single and double labeling procedures.

    Science.gov (United States)

    Gown, A M; Garcia, R; Ferguson, M; Yamanaka, E; Tippens, D

    1986-03-01

    We have investigated the use of an avidin-biotin-immunoglucose oxidase (AB-GO) technique for single and double antigen localization in conjunction with the avidin-biotin-immunoperoxidase (AB-P) technique in fixed, embedded specimens, using sequential monoclonal and polyclonal antibodies of the same species. The optimal technique for double labeling requires the first antibody to be applied and localized with the AB-P technique using 3,3'-diaminobenzidine (DAB) as the chromogen, followed by an optional elution step and/or incubation with mild detergent (0.01% Triton). The second antigen is localized with the AB-GO technique with nitro blue tetrazolium (NBT) as a chromogen. Effects of antigen concentration, intermediate elution steps, and the relative efficiency of the two methodologies are described.

  9. Preliminary viability studies of fibroblastic cells cultured on microcrystalline and nanocrystalline diamonds produced by chemical vapour deposition method

    Directory of Open Access Journals (Sweden)

    Ana Amélia Rodrigues

    2012-01-01

    Full Text Available Implant materials used in orthopedics surgery have demonstrated some disadvantages, such as metallic corrosion processes, generation of wear particles, inflammation reactions and bone reabsorption in the implant region. The diamond produced through hot-filament chemical vapour deposition method is a new potential biomedical material due to its chemical inertness, extreme hardness and low coefficient of friction. In the present study we analysis two samples: the microcrystalline diamond and the nanocrystalline diamond. The aim of this study was to evaluate the surface properties of the diamond samples by scanning electron microscopy, Raman spectroscopy and atomic force microscopy. Cell viability and morphology were assessed using thiazolyl blue tetrazolium bromide, cytochemical assay and scanning electron microscopy, respectively. The results revealed that the two samples did not interfere in the cell viability, however the proliferation of fibroblasts cells observed was comparatively higher with the nanocrystalline diamond.

  10. [Effect of PRX-2 gene transferred by lipofectamine on the proliferation of human skin fibroblasts].

    Science.gov (United States)

    Song, Hui-feng; Chai, Jia-ke; Lin, Zi-hao

    2011-10-11

    To explore the effects of PRX-2 gene transferred by lipofectamine on the human skin fibroblasts. Normal human skin fibroblasts were cultured and PRX-2 gene was transferred by lipofectamine. The proliferation of fibroblasts was detected by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and flow cytometry. The proliferation of PRX-2-transfected fibroblasts was stronger than that of normal counterparts. There were fewer cells during G0-G1 period and more cells during S and G2-M periods. The proliferative index increased. The proliferation of fibroblasts may be modified by transfected PRX-2. Thus PRX-2 plays an important role during the healing of human skin wound.

  11. Synthesis, characterization, antibacterial activity, SOD mimic and interaction with DNA of drug based copper(II) complexes

    Science.gov (United States)

    Patel, Mohan N.; Dosi, Promise A.; Bhatt, Bhupesh S.; Thakkar, Vasudev R.

    2011-02-01

    Novel metal complexes of the second-generation quinolone antibacterial agent enrofloxacin with copper(II) and neutral bidentate ligands have been prepared and characterized with elemental analysis reflectance, IR and mass spectroscopy. Complexes have been screened for their in-vitro antibacterial activity against two Gram (+ve)Staphylococcus aureus, Bacillus subtilis, and three Gram (-ve)Serratia marcescens, Escherichia coli and Pseudomonas aeruginosa organisms using the double dilution technique. The binding of this complex with CT-DNA has been investigated by absorption titration, salt effect and viscosity measurements. Binding constant is ranging from 1.3 × 10 4-3.7 × 10 4. The cleavage ability of complexes has been assessed by gel electrophoresis using pUC19 DNA. The catalytic activity of the copper(II) complexes towards the superoxide anion (O 2rad -) dismutation was assayed by their ability to inhibit the reduction of nitroblue tetrazolium (NBT).

  12. The effect of –hydroxybutyrate and estradiol on the process of phagocytosis of ovine neutrophils in vitro

    Directory of Open Access Journals (Sweden)

    Ali Rezapour

    2008-11-01

    Full Text Available The object of this study was to evaluate the independent effect different concentration of beta-hydroxybutyrate (2.4 & 4.8 mmol/ml and estradiol (20&400 pg/ml on the functions phagocytosis and Nitroblue tetrazolium reduction test or respiratory burst of isolated ovine neutrophils in vitro. Isolated neutrophils of sheep were incubated for one hour in room temperature with the previously treatments. The study consisted of 7 treatments in 5 replicates and both phagocytosis and NBT tests of tests were performed in every treatment. The Results of this study indicated that 2.4 & 4.8 mmol/ml concentrations of beta-hydroxybutyrate and estrodiol concentrations of 20 & 400 pg/ml significantly (p

  13. Fingerprint deposition on nitrocellulose and polyvinylidene difluoride membranes using alkaline phosphatase.

    Science.gov (United States)

    Kurien, Biji T; Danda, Debashish; Scofield, R Hal

    2015-01-01

    Dactyloscopy or fingerprint identification is a vital part of forensic evidence. Identification with fingerprints has been known since the finding of finger impressions on the clay surface of Babylonian legal contracts almost 4,000 years ago. The skin on the fingers and palms appears as grooves and ridges when observed under a microscope. A unique fingerprint is produced by the patterns of these friction skin ridges. Visible fingerprints can be deposited on solid surfaces. Colored inks have been used to deposit fingermarks on documents. Herein, we show that alkaline phosphatase can be used to transfer prints from fingers or palm to nitrocellulose or polyvinylidene difluoride membranes. The prints can be detected by using the nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate method of detection.

  14. Fabrication of Gold Nanochains with Octreotide Acetate Template

    Directory of Open Access Journals (Sweden)

    Jing Zhou

    2013-01-01

    Full Text Available We described a facile method for assembly gold nanochains by using octreotide acetate as template in aqueous environment. In acidic solution, octreotide acetate was conferred positive charges and its structure changed to chain-like. The monodisperse negative gold nanoparticles were bound to the surface of octreotide acetate template by electrostatic attraction and the interaction of gold nanoparticles with amino acid residues (tryptophan and lysine. The fabricated gold nanostructure presented chain-like observed by transmission electron microscopy. The cytotoxicity of gold nanochains was examined by tetrazolium dye-based microtitration (MTT assay, which demonstrated significantly less toxicity than that of octreotide acetate alone. The MTT assay also reflected the combinative action between the gold nanoparticles with octreotide acetate. Our work lays the groundwork for developing octreotide acetate-templated nanomaterials that can be used as a building block for the creation of nanomaterials. Meanwhile, the harmfulless gold nanochains have great application prospects in the biomedical filed.

  15. Effects of cobalt nanoparticles on human T cells in vitro.

    Science.gov (United States)

    Jiang, Haitao; Liu, Fan; Yang, Huilin; Li, Yang

    2012-04-01

    Limited information is available on the potential risk of degradation products of metal-on-metal bearings in joint arthroplasty. The aim of this study was to investigate the cytotoxicity and genotoxicity of orthopedic-related cobalt nanoparticles on human T cells in vitro. T cells were collected using magnetic CD3 microbeads and exposed to different concentrations of cobalt nanoparticles and cobalt chloride. Cytotoxicity was evaluated by methyl thiazolyl tetrazolium and lactate dehydrogenase release assay. Cobalt nanoparticles dissolution in culture medium was determined by inductively coupled plasma-mass spectrometry. To study the probable mechanism of cobalt nanoparticles effects on T cells, superoxide dismutase, catalase, and glutathione peroxidase level was measured. Cobalt nanoparticles and cobalt ions could inhibit cell viability and enhance lactate dehydrogenase release in a concentration- and time-dependent manner (P metal-on-metal total hip arthroplasty, and the inhibition of antioxidant capacity may play important role in cobalt nanoparticles effects on T cells.

  16. Evidence for Detrimental Cross Interactions between Reactive Oxygen and Nitrogen Species in Leber’s Hereditary Optic Neuropathy Cells

    Directory of Open Access Journals (Sweden)

    Micol Falabella

    2016-01-01

    Full Text Available Here we have collected evidence suggesting that chronic changes in the NO homeostasis and the rise of reactive oxygen species bioavailability can contribute to cell dysfunction in Leber’s hereditary optic neuropathy (LHON patients. We report that peripheral blood mononuclear cells (PBMCs, derived from a female LHON patient with bilateral reduced vision and carrying the pathogenic mutation 11778/ND4, display increased levels of reactive oxygen species (ROS and reactive nitrogen species (RNS, as revealed by flow cytometry, fluorometric measurements of nitrite/nitrate, and 3-nitrotyrosine immunodetection. Moreover, viability assays with the tetrazolium dye MTT showed that lymphoblasts from the same patient are more sensitive to prolonged NO exposure, leading to cell death. Taken together these findings suggest that oxidative and nitrosative stress cooperatively play an important role in driving LHON pathology when excess NO remains available over time in the cell environment.

  17. A Cytochemical Study of the Dehydrogenases of Mitochondria and Mitochondrial Particulates by a Monotetrazolium-Cobalt Chelation Method

    Science.gov (United States)

    Pearse, A. G. E.; Scarpelli, D. G.; Hess, R.

    1960-01-01

    In one of the current histochemical methods for dehydrogenases and diaphorases the final product is a metal-formazan dye derived from reduction of an N-thiazolyl-substituted tetrazolium. Sites of enzymic activity consistently appear as intramitochondrial dots 0.2 to 0.3 µ in diameter. When applied to active particles from disrupted mitochondria (Keilin-Hartree preparation, electron transport particle, Cooper-Lehninger particle) the individual particles appear as black dots 0.1 to 0.3 µ in diameter. It is clear that formazan is deposited progressively upon the particles and the results suggest that the latter may be spatially arranged in mitochondria so that areas of activity are separated by quiescent regions. PMID:14431240

  18. Comparing different methods for fast screening of microbiological quality of beach sand aimed at rapid-response remediation.

    Science.gov (United States)

    Testolin, Renan C; Almeida, Tito C M; Polette, Marcus; Branco, Joaquim O; Fischer, Larissa L; Niero, Guilherme; Poyer-Radetski, Gabriel; Silva, Valéria C; Somensi, Cleder A; Corrêa, Albertina X R; Corrêa, Rogério; Rörig, Leonardo R; Itokazu, Ana Gabriela; Férard, Jean-François; Cotelle, Sylvie; Radetski, Claudemir M

    2017-05-15

    There is scientific evidence that beach sands are a significant contributor to the pathogen load to which visitors are exposed. To develop beach quality guidelines all beach zones must be included in microbiological evaluations, but monitoring methods for beach sand quality are relatively longstanding, expensive, laborious and require moderate laboratory infrastructure. This paper aimed to evaluate the microorganism activity in different beach zones applying and comparing a classical method of membrane filtration (MF) with two colorimetric screening methods based on fluorescein (FDA) and tetrazolium (TTC) salt biotransformation to evaluate a new rapid and low-cost method for beach sand microbiological contamination assessments. The colorimetric results can help beach managers to evaluate rapidly and at low cost the microbiological quality of different beach zones in order to decide whether remedial actions need to be adopted to prevent exposure of the public to microbes due to beach sand and/or water contamination. Copyright © 2017. Published by Elsevier Ltd.

  19. Bacteroides fragilis induce necrosis on mice peritoneal macrophages: In vitro and in vivo assays

    Energy Technology Data Exchange (ETDEWEB)

    Vieira, J.M.B.D., E-mail: jmanya@terra.com.br [Laboratorio de Tecnologia em Cultura de Celulas, UEZO, Rio de Janeiro (Brazil); Laboratorio de Biologia de Anaerobios, IMPPG, UFRJ, Rio de Janeiro (Brazil); Seabra, S.H. [Laboratorio de Tecnologia em Cultura de Celulas, UEZO, Rio de Janeiro (Brazil); Vallim, D.C. [Instituto Oswaldo Cruz, Rio de Janeiro (Brazil); Americo, M.A.; Fracallanza, S.E.L. [Laboratorio de Bacteriologia Medica, IMPPG, UFRJ, Rio de Janeiro (Brazil); Vommaro, R.C. [Laboratorio de Ultra-estrutura Celular Hertha Meyer, IBCCF, UFRJ (Brazil); Domingues, R.M.C.P. [Laboratorio de Biologia de Anaerobios, IMPPG, UFRJ, Rio de Janeiro (Brazil)

    2009-10-02

    Bacteroides fragilis is an anaerobic bacteria component of human intestinal microbiota and agent of infections. In the host B. fragilis interacts with macrophages, which produces toxic radicals like NO. The interaction of activated mice peritoneal macrophages with four strains of B. fragilis was evaluated on this study. Previously was shown that such strains could cause metabolic and morphologic alterations related to macrophage death. In this work propidium iodide staining showed the strains inducing macrophage necrosis in that the labeling was evident. Besides nitroblue tetrazolium test showed that B. fragilis stimulates macrophage to produce oxygen radicals. In vivo assays performed in BalbC mice have results similar to those for in vitro tests as well as scanning electron microscopy, which showed the same surface pore-like structures observed in vitro before. The results revealed that B. fragilis strains studied lead to macrophage death by a process similar to necrosis.

  20. Sensitive detection of PDT-induced cell damages with luminescent oxygen nanosensors

    Science.gov (United States)

    Ma, Hong-Ru; Peng, Hong-shang; You, Fang-tian; Ping, Jian-tao; Zhou, Chao; Guo, Lan-ying

    2016-09-01

    In this work luminescent nanosensors specifically created for intracellular oxygen (ic-O2) were utilized to assess photodynamic therapy (PDT) -induced cell damages. Firstly, ic-O2 was demonstrated to be consumed much faster than extracellular O2 with respective O2 nanosensors. Using the ic-O2 nanosensors, PDT-treated cells with different degree of impairment were then resolved according to the oxygen consumption rate (OCR). The evolving trend of cytotoxicity derived from OCRs was in agreement with cell viability obtained from 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Moreover, the direct damage of PDT on cell mitochondria was successfully detected by monitoring respiration instantly after PDT treatment, which is actually beyond the scope of MTT assay. These results suggest that fluorescence sensing of ic-O2-associated cell respiration is promising and even may become a standardized method, complementary to MTT assay, to evaluate PDT-induced cytotoxicity.

  1. Synergistic Effect of Eicosapentaenoic Acid on Antiproliferative Action of Anticancer Drugs in a Cancer Cell Line Model.

    Science.gov (United States)

    Ogo, Ayako; Miyake, Sachi; Kubota, Hisako; Higashida, Masaharu; Matsumoto, Hideo; Teramoto, Fusako; Hirai, Toshihiro

    2017-11-14

    It has been found experimentally and clinically that eicosapentaenoic acid (EPA) exerts an anticancer effect and that it has a minimal adverse event profile relative to other anticancer drugs. Any synergy between EPA and other anticancer drugs could be of therapeutic relevance, especially in elderly or high-risk patients. Therefore, we investigated the synergism between anticancer drugs and EPA experimentally. EPA was coadministered in vitro with various anticancer drugs (paclitaxel, docetaxel, 5-fluorouracil and cis-diamminedichloridoplatinum[II]) to TE-1 cells, which were derived from human esophageal cancer tumors. Cell proliferation was measured by the water soluble tetrazolium-1 method. Sub-threshold concentrations of EPA, which alone produced no anticancer effect, caused a synergistic suppressive effect on TE-1 cell proliferation when combined with other anticancer agents. Coadministration of EPA with other anticancer drugs may represent a new therapeutic paradigm offering a reduced side effect profile. © 2017 S. Karger AG, Basel.

  2. Non-destructive monitoring of viability in an ex vivo organ culture model of osteochondral tissue.

    Science.gov (United States)

    Elson, K M; Fox, N; Tipper, J L; Kirkham, J; Hall, R M; Fisher, J; Ingham, E

    2015-06-30

    Organ culture is an increasingly important tool in research, with advantages over monolayer cell culture due to the inherent natural environment of tissues. Successful organ cultures must retain cell viability. The aim of this study was to produce viable and non-viable osteochondral organ cultures, to assess the accumulation of soluble markers in the conditioned medium for predicting tissue viability. Porcine femoral osteochondral plugs were cultured for 20 days, with the addition of Triton X-100 on day 6 (to induce necrosis), camptothecin (to induce apoptosis) or no toxic additives. Tissue viability was assessed by the tissue destructive XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide tetrazolium salt) assay method and LIVE/DEAD® staining of the cartilage at days 0, 6 and 20. Tissue structure was assessed by histological evaluation using haematoxylin & eosin and safranin O. Conditioned medium was assessed every 3-4 days for glucose depletion, and levels of lactate dehydrogenase (LDH), alkaline phosphatase (AP), glycosaminoglycans (GAGs), and matrix metalloproteinase (MMP)-2 and MMP-9. Necrotic cultures immediately showed a reduction in glucose consumption, and an immediate increase in LDH, GAG, MMP-2 and MMP-9 levels. Apoptotic cultures showed a delayed reduction in glucose consumption and delayed increase in LDH, a small rise in MMP-2 and MMP-9, but no significant effect on GAGs released into the conditioned medium. The data showed that tissue viability could be monitored by assessing the conditioned medium for the aforementioned markers, negating the need for tissue destructive assays. Physiologically relevant whole- or part-joint organ culture models, necessary for research and pre-clinical assessment of therapies, could be monitored this way, reducing the need to sacrifice tissues to determine viability, and hence reducing the sample numbers necessary.

  3. Benzylidene derivatives of andrographolide inhibit growth of breast and colon cancer cells in vitro by inducing G(1) arrest and apoptosis.

    Science.gov (United States)

    Jada, S R; Matthews, C; Saad, M S; Hamzah, A S; Lajis, N H; Stevens, M F G; Stanslas, J

    2008-11-01

    Andrographolide, the major phytoconstituent of Andrographis paniculata, was previously shown by us to have activity against breast cancer. This led to synthesis of new andrographolide analogues to find compounds with better activity than the parent compound. Selected benzylidene derivatives were investigated for their mechanisms of action by studying their effects on the cell cycle progression and cell death. Microculture tetrazolium, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and sulphorhodamine B (SRB) assays were utilized in assessing the in vitro growth inhibition and cytotoxicity of compounds. Flow cytometry was used to analyse the cell cycle distribution of control and treated cells. CDK1 and CDK4 levels were determined by western blotting. Apoptotic cell death was assessed by fluorescence microscopy and flow cytometry. Compounds, in nanomolar to micromolar concentrations, exhibited growth inhibition and cytotoxicity in MCF-7 (breast) and HCT-116 (colon) cancer cells. In the NCI screen, 3,19-(2-bromobenzylidene) andrographolide (SRJ09) and 3,19-(3-chloro-4-fluorobenzylidene) andrographolide (SRJ23) showed greater cytotoxic potency and selectivity than andrographolide. SRJ09 and SRJ23 induced G(1) arrest and apoptosis in MCF-7 and HCT-116 cells, respectively. SRJ09 downregulated CDK4 but not CDK1 level in MCF-7 cells. Apoptosis induced by SRJ09 and SRJ23 in HCT-116 cells was confirmed by annexin V-FITC/PI flow cytometry analysis. The new benzylidene derivatives of andrographolide are potential anticancer agents. SRJ09 emerged as the lead compound in this study, exhibiting anticancer activity by downregulating CDK4 to promote a G(1) phase cell cycle arrest, coupled with induction of apoptosis.

  4. Cytoprotective effect of trolox against oxidative damage and apoptosis in the NRK-52e cells induced by melamine.

    Science.gov (United States)

    Guo, Chengzhi; He, Zuping; Wen, Lixin; Zhu, Li; Lu, Yin; Deng, Sijun; Yang, Yang; Wei, Qiang; Yuan, Hui

    2012-02-01

    An outbreak of urinary stones associated with consumption of melamine-tainted milk products occurred in 2008 in China, leading to serious illness of many infants and even death. However, the toxicity of melamine in kidney epithelial cells remains unclear. We have explored the effects of melamine and trolox on renal NRK-52e (normal rat kidney 52e) cells. The IC(50) of melamine was measured by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay. Total SOD (superoxide dismutase) was determined by NBT (Nitro Blue Tetrazolium) staining method. GSH-Px (glutathione peroxidase) activity was detected by UV colorimetric assay, and MDA (malondialdehyde) content was determined by thiobarbituric acid assay. Apoptosis induced by melamine was determined by flow cytometry. The IC(50) increased when NRK-52e cells were treated with both melamine and trolox compared with melamine only. SOD and GSH-Px activities were decreased, but MDA content was increased by melamine in a dose-dependent manner. Trolox significantly enhanced SOD and GSH-Px activity in melamine-treated NRK-52e cells, but it decreased their MDA content. LDH (lactate dehydrogenase) activity and the level of ROS (reactive oxygen species) of the NRK-52e cells were enhanced by melamine compared with the control. Furthermore, the apoptosis rate increased in NRK-52e cells treated with melamine, whereas trolox was protective. These results show that melamine has an obvious adverse effect on proliferation of NRK-52e cells, causing oxidative damage and apoptosis, thus providing a novel insight into renal cytotoxicology of melamine. Trolox ameliorates the effect on melamine toxicity.

  5. Measurement of microbial activity in soil by colorimetric observation of in situ dye reduction: an approach to detection of extraterrestrial life

    Directory of Open Access Journals (Sweden)

    Barnes Bruce

    2002-07-01

    Full Text Available Abstract Background Detecting microbial life in extraterrestrial locations is a goal of space exploration because of ecological and health concerns about possible contamination of other planets with earthly organisms, and vice versa. Previously we suggested a method for life detection based on the fact that living entities require a continual input of energy accessed through coupled oxidations and reductions (an electron transport chain. We demonstrated using earthly soils that the identification of extracted components of electron transport chains is useful for remote detection of a chemical signature of life. The instrument package developed used supercritical carbon dioxide for soil extraction, followed by chromatography or electrophoresis to separate extracted compounds, with final detection by voltammetry and tandem mass-spectrometry. Results Here we used Earth-derived soils to develop a related life detection system based on direct observation of a biological redox signature. We measured the ability of soil microbial communities to reduce artificial electron acceptors. Living organisms in pure culture and those naturally found in soil were shown to reduce 2,3-dichlorophenol indophenol (DCIP and the tetrazolium dye 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl-2H-tetrazolium-5-carboxanilide inner salt (XTT. Uninoculated or sterilized controls did not reduce the dyes. A soil from Antarctica that was determined by chemical signature and DNA analysis to be sterile also did not reduce the dyes. Conclusion Observation of dye reduction, supplemented with extraction and identification of only a few specific signature redox-active biochemicals such as porphyrins or quinones, provides a simplified means to detect a signature of life in the soils of other planets or their moons.

  6. Effects of asymmetric dimethylarginine on bovine retinal capillary endothelial cell proliferation, reactive oxygen species production, permeability, intercellular adhesion molecule-1, and occludin expression.

    Science.gov (United States)

    Chen, Yi-Hui; Xu, Xun; Sheng, Min-Jie; Zheng, Zhi; Gu, Qing

    2011-02-01

    Asymmetric dimethylarginine (ADMA), an endogenous competitive inhibitor of nitric oxide synthase, is associated with impaired endothelial dysfunction, such as chronic heart failure, hypertension, diabetes, and pulmonary hypertension. The effects of ADMA on cell proliferation, reactive oxygen species (ROS) production, cell permeability, intercellular adhesion molecule-1 (ICAM-1), and tight-junction protein occludin levels in bovine retinal capillary endothelial cells (BRCECs) were investigated. A cell proliferation assay was performed using the novel tetrazolium compound 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and an electron coupling reagent. Intracellular ROS levels were determined using the fluorescent probe CM-H(2)DCFDA. Horseradish peroxidase was used for a permeability assay. ICAM-1 and tight-junction protein occludin were assessed by western blotting and quantitative real-time PCR. Cell proliferation was significantly inhibited by ADMA. ADMA increased intracellular ROS generation in BRCECs. The increased ROS production induced by ADMA was markedly inhibited by the angiotensin II receptor-blocker telmisartan, the angiotensin-converting enzyme inhibitor benazepril, the reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenyliodonium (DPI), or the antioxidant and free-radical scavenger N-acetyl-L-cysteine (NAC). ADMA significantly increased horseradish peroxidase (HRP) permeability in BRCECs. Benazepril, telmisartan, DPI, and NAC downregulated cell permeability. ADMA markedly upregulated ICAM-1 expression in BRCECs, which were downregulated by telmisartan, DPI, and NAC. ADMA significantly downregulated occludin expression in BRCECs. Benazepril and telmisartan upregulated occludin expression in BRCECs exposed to ADMA. Our results provide the first reported evidence that ADMA has potent adverse effects on cell proliferation, intracellular ROS generation, cell permeability

  7. Real-time monitoring of superoxide anion radical generation in response to wounding: electrochemical study

    Directory of Open Access Journals (Sweden)

    Ankush Prasad

    2017-07-01

    Full Text Available Background The growth and development of plants is deleteriously affected by various biotic and abiotic stress factors. Wounding in plants is caused by exposure to environmental stress, mechanical stress, and via herbivory. Typically, oxidative burst in response to wounding is associated with the formation of reactive oxygen species, such as the superoxide anion radical (O2•−, hydrogen peroxide (H2O2 and singlet oxygen; however, few experimental studies have provided direct evidence of their detection in plants. Detection of O2•− formation in plant tissues have been performed using various techniques including electron paramagnetic resonance spin-trap spectroscopy, epinephrine-adrenochrome acceptor methods, staining with dyes such as tetrazolium dye and nitro blue tetrazolium (NBT; however, kinetic measurements have not been performed. In the current study, we provide evidence of O2•− generation and its kinetics in the leaves of spinach (Spinacia oleracea subjected to wounding. Methods Real-time monitoring of O2•− generation was performed using catalytic amperometry. Changes in oxidation current for O2•− was monitored using polymeric iron-porphyrin-based modified carbon electrodes (φ = 1 mm as working electrode with Ag/AgCl as the reference electrode. Result The results obtained show continuous generation of O2•− for minutes after wounding, followed by a decline. The exogenous addition of superoxide dismutase, which is known to dismutate O2•− to H2O2, significantly suppressed the oxidation current. Conclusion Catalytic amperometric measurements were performed using polymeric iron-porphyrin based modified carbon electrode. We claim it to be a useful tool and a direct method for real-time monitoring and precise detection of O2•− in biological samples, with the potential for wide application in plant research for specific and sensitive detection of O2•−.

  8. Chemical composition and evaluation of the antibacterial and Cytotoxic activities of the essential oil from the leaves of Myracrodruon urundeuva.

    Science.gov (United States)

    Rebouças de Araújo, Ítalo Diego; Coriolano de Aquino, Nayara; Véras de Aguiar Guerra, Andreza Conceição; Ferreira de Almeida Júnior, Renato; Mendonça Araújo, Renata; Fernandes de Araújo Júnior, Raimundo; Silva Farias, Kléber Juvenal; Fernandes, José Veríssimo; Sousa Andrade, Vânia

    2017-08-22

    This study evaluated the in vitro activity of essential oil extracted from the leaves of Myracrodruon urundeuva. The oil was obtained by hydro-distillation and characterized by Gas Chromatography coupled to Mass Spectrometry (GC-MS) and Gas Chromatography with Flame Ionization Detector (GC-FID). The antibacterial activity was evaluated by the broth microdilution technique and the MIF was determined by using growth indicator CTT (2,3,5-triphenyl-tetrazolium) and CBM in BHI agar. The oil's cytotoxicity was evaluated in HeLa, HEK-293, and Vero E6 cells using MTT, 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium. The oil showed chemical markers, including α-pinene (87.85%), trans-caryophyllene (1.57%), limonene (1.49%) and β -pinene (1.42%), and activity against all strains: Staphylococcus aureus (MIC = MBC = 0.22 mg/mL), Staphylococcus epidermidis (MIC = 0.11 mg/mL and MBC = 0.22 mg/mL), Escherichia coli (MIC = 0.88 mg/mL and MBC = 1.75 mg/mL), Pseudomonas aeruginosa (MIC = MBC = 7 mg/mL) and Salmonella Enteritidis (MIC = MBC = 0.44 mg/mL). In vitro cytotoxicity tests showed that the oil is not toxic and has slight antitumor activity. We conclude that the M. urundeuva oil results are promising, with prospects of being pharmacologically viable.

  9. Cryopreservation of coffee zygotic embryos: dehydration and osmotic rehydration

    Directory of Open Access Journals (Sweden)

    Maísa de Siqueira Pinto

    Full Text Available ABSTRACT Conservation of plant genetic resources is important to prevent genetic erosion. Seed banks are the most common method of ex situ conservation; however, coffee seeds can not be stored by conventional methods. Cryopreservation is a viable alternative for long-term conservation of species that produce intermediate or recalcitrant seeds, as coffee. The aim of this work was to cryopreserve Coffea arabica L. cv Catuaí Vermelho IAC 144 zygotic embryos, and analyse the effects of dehydration prior cryopreservation and osmotic rehydration after thawing, in embryos germination and seedlings formation after cryopreservation. Prior to cryopreservation, different dehydration times (0, 15, 30, 60 and 120 min were tested. Dehydrated embryos were cryopreserved in liquid nitrogen for 1 hour, and after thawing were rehydrated by osmotic solutions. Dehydrated and non-cryopreserved embryos were also analysed. The test with 2,3,5 triphenyl tetrazolium chloride (TTC was used to evaluate the embryos viability. Non-dehydrated embryos did not survive after freezing. Embryos that were dehydrated until 20% of the moisture content did not germinate when osmotic rehydration was not performed. In contrast, cryopreserved embryos with the same moisture content presented 98% germination when they were rehydrated slowly in osmotic solution. According to tetrazolium tests, embryos presented maximum viability (75% after dehydration for 60 minutes (23% moisture content. Therefore, coffee zygotic embryos (Coffea arabica L. cv. Catuaí Vermelho can be successfully cryopreserved using physical dehydration in silica gel for 60 minutes (23% moisture content, followed by osmotic rehydration after thawing. This method allowed a germination of 98% of cryopreserved zygotic embryos.

  10. Asymbiotic germination response to photoperiod and nutritional media in six populations of Calopogon tuberosus var. tuberosus (Orchidaceae): evidence for ecotypic differentiation.

    Science.gov (United States)

    Kauth, Philip J; Kane, Michael E; Vendrame, Wagner A; Reinhardt-Adams, Carrie

    2008-11-01

    Ecotypic differentiation has been explored in numerous plant species, but has been largely ignored in the Orchidaceae. Applying a specific germination protocol for widespread seed sources may be unreliable due to inherent physiological or genetic differences in localized populations. It is crucial to determine whether ecotypic differentiation exists for restoration and conservation programmes. Calopogon tuberosus var. tuberosus, a widespread terrestrial orchid of eastern North America, is a model species to explore ecotypic differences in germination requirements, as this species occupies diverse habitats spanning a wide geographical range. Mature seeds were collected from south Florida, north central Florida, three locations in South Carolina, and the upper Michigan peninsula. Effects of three photoperiods (8/16, 12/12, 16/8 h L/D) were examined on asymbiotic in vitro seed germination and seedling development of C. tuberosus. Germination and early development was monitored for 8 weeks, while advanced development was monitored for an additional 8 weeks. In an additional experiment, asymbiotic seed germination and development was monitored for 8 weeks on six culture media (BM-1 terrestrial orchid medium, Knudson C, Malmgrem, half-strength MS, P723, and Vacin and Went). A tetrazolium test for embryo viability was performed. Short days promoted the highest germination among Florida populations, but few differences among photoperiods in other seed sources existed. Different media had little effect on the germination of Michigan and Florida populations, but germination of South Carolina seeds was higher on media with higher calcium and magnesium. Tetrazolium testing confirmed that South Carolina seeds exhibited low viability while viability was higher in Florida seeds. Seed germination and corm formation was rapid in Michigan seeds across all treatments. Michigan seedlings allocated more biomass to corms compared with other seed sources. Rapid germination and corm

  11. Antimycobacterial and cytotoxic activity of selected medicinal plant extracts.

    Science.gov (United States)

    Nguta, Joseph M; Appiah-Opong, Regina; Nyarko, Alexander K; Yeboah-Manu, Dorothy; Addo, Phyllis G A; Otchere, Isaac; Kissi-Twum, Abena

    2016-04-22

    Tuberculosis (TB) caused by Mycobacterium tuberculosis remains an ongoing threat to human health. Several medicinal plants are used traditionally to treat tuberculosis in Ghana. The current study was designed to investigate the antimycobacterial activity and cytotoxicity of crude extracts from five selected medicinal plants. The microplate alamar blue assay (MABA) was used for antimycobacterial studies while the CellTiter 96® AQueous Assay, which is composed of solutions of a novel tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine methosulfate) PMS, was used for cytotoxic studies. Correlation coefficients were used to compare the activity of crude extracts against nonpathogenic strains and the pathogenic Mycobacterium tuberculosis subsp.tuberculosis. Results of the MIC determinations indicated that all the crude extracts were active on all the three tested mycobacterial strains. Minimum inhibitory concentration values as low as 156.3µg/mL against M. tuberculosis; Strain H37Ra (ATCC® 25,177™) were recorded from the leaves of Solanum torvum Sw. (Solanaceae). Cytotoxicity of the extracts varied, and the leaves from S. torvum had the most promising selectivity index. Activity against M. tuberculosis; Strain H37Ra was the best predictor of activity against pathogenic Mycobacterium tuberculosis subsp.tuberculosis (correlation coefficient=0.8). The overall results of the present study provide supportive data on the use of some medicinal plants for tuberculosis treatment. The leaves of Solanum torvum are a potential source of anti-TB natural products and deserve further investigations to develop novel anti-TB agents against sensitive and drug resistant strains of M. tuberculosis. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  12. In vitro antimycobacterial activity and toxicity of eight medicinal plants against pathogenic and nonpathogenic mycobacterial strains

    Directory of Open Access Journals (Sweden)

    Joseph M Nguta

    2016-01-01

    Full Text Available Tuberculosis (TB caused by Mycobacterium tuberculosis remains a serious public health challenge towards which new hits are urgently needed. Medicinal plants remains a major source of new ligands against global infectious illnesses. In our laboratories, we are currently investigating locally used ethnobotanicals for novel compounds against zoonotic tuberculosis. The microplate alamar blue assay (MABA was used to study the anti-TB activity while the CellTiter 96® AQueous Assay, which is composed of solutions of a novel tetrazolium compound [3-(4,5-dimethylthiazol-2-yl-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine methosulfate PMS, was used for cytotoxic studies. Correlation coefficients (R2 were used to compare the relationship between antimycobacterial activity of the eight crude extracts against nonpathogenic strains and the pathogenic Mycobacterium bovis. Minimum inhibitory concentration (MICs values indicated that all the eight tested medicinal plant species had activity against all the three tested mycobacterial strains. Minimum inhibitory concentration value as low as 19.5 μg/mL was observed against non-pathogenic strains M. bovis. Activity of the crude extracts against M. aurum was the best predictor of natural product activity against the pathogenic Mycobacterium bovis strain, with a correlation coefficient value (R2 of 0.1371. Results obtained from the current study validate, in part, the traditional utilization of the tested medicinal plants against tuberculosis. The unripe fruits from Solanum torvum are a potential source of safe and efficacious anti-TB crude drugs as well as a source for natural compounds that act as new anti-infection agents, and thus deserve further investigation towards development of a new class of molecules with activity against sensitive and drug resistant strains of M. bovis.

  13. In vitro antimycobacterial activity and toxicity of eight medicinal plants against pathogenic and nonpathogenic mycobacterial strains.

    Science.gov (United States)

    Nguta, Joseph M; Appiah-Opong, Regina; Nyarko, Alexander K; Yeboah-Manu, Dorothy; Addo, Phyllis G A; Otchere, Isaac Darko; Kissi-Twum, Abena

    2016-12-01

    Tuberculosis (TB) caused by Mycobacterium tuberculosis remains a serious public health challenge towards which new hits are urgently needed. Medicinal plants remains a major source of new ligands against global infectious illnesses. In our laboratories, we are currently investigating locally used ethnobotanicals for novel compounds against zoonotic tuberculosis. The microplate alamar blue assay (MABA) was used to study the anti-TB activity while the CellTiter 96® AQ ueous Assay, which is composed of solutions of a novel tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine methosulfate) PMS, was used for cytotoxic studies. Correlation coefficients (R 2 ) were used to compare the relationship between antimycobacterial activity of the eight crude extracts against nonpathogenic strains and the pathogenic Mycobacterium bovis. Minimum inhibitory concentration (MICs) values indicated that all the eight tested medicinal plant species had activity against all the three tested mycobacterial strains. Minimum inhibitory concentration value as low as 19.5µg/mL was observed against non-pathogenic strains M. bovis. Activity of the crude extracts against M. aurum was the best predictor of natural product activity against the pathogenic Mycobacterium bovis strain, with a correlation coefficient value (R 2 ) of 0.1371. Results obtained from the current study validate, in part, the traditional utilization of the tested medicinal plants against tuberculosis. The unripe fruits from Solanum torvum are a potential source of safe and efficacious anti-TB crude drugs as well as a source for natural compounds that act as new anti-infection agents, and thus deserve further investigation towards development of a new class of molecules with activity against sensitive and drug resistant strains of M. bovis. Copyright © 2016.

  14. Combinatorial cytotoxic effects of Curcuma longa and Zingiber officinale on the PC-3M prostate cancer cell line

    Science.gov (United States)

    Kurapati, Kesava Rao V.; Samikkannu, Thangavel; Kadiyala, Dakshayani B.; Zainulabedin, Saiyed M.; Gandhi, Nimisha; Sathaye, Sadhana S.; Indap, Manohar A.; Boukli, Nawal; Rodriguez, Jose W.; Nair, Madhavan P.N.

    2015-01-01

    Background Many plant-derived products exhibit potent chemopreventive activity against animal tumor models as well as rodent and human cancer cell lines. They have low side effects and toxicity and presumably modulate the factors that are critical for cell proliferation, differentiation, senescence and apoptosis. The present study investigates the effects of some medicinal plant extracts from generally recognized as safe plants that may be useful in the prevention and treatment of cancer. Methods Clonogenic assays using logarithmically-growing cells were performed to test the effect. The cytotoxic effects of Curcuma longa and Zingiber officinale were studied using sulforhodamine B assay, tetrazolium dye assay, colony morphology and microscopic analysis. Results Out of the 13 lyophilized plant-derived extracts evaluated for growth-inhibitory effects on the PC-3M prostate cancer cell line, two extracts derived from C. longa and Z. officinale showed significant inhibitory effects on colony-forming ability. The individual and augmentative effects of these two extracts were tested for their narrow range effective lower concentration on PC-3M in clonogenic assays. At relatively lower concentrations, C. longa showed significant inhibition of colony formation in clonogenic assays; whereas at same concentrations Z. officinale showed only moderate inhibitory effects. However, when both the agents were tested together at the same concentrations, the combined effects were much more significant than their individual ones. On normal prostate epithelial cells both C. longa and Z. officinale had similar effects but at a lower magnitude. These observations were confirmed by several cytotoxicity assays involving the morphological appearance of the colonies, microscopic observations, per cent inhibition in comparison to control by sulforhodamine B and tetrazolium dye assay. Conclusions From these observations, it was concluded that the combined effects of C. longa and Z. officinale

  15. Asymbiotic Germination Response to Photoperiod and Nutritional Media in Six Populations of Calopogon tuberosus var. tuberosus (Orchidaceae): Evidence for Ecotypic Differentiation

    Science.gov (United States)

    Kauth, Philip J.; Kane, Michael E.; Vendrame, Wagner A.; Reinhardt-Adams, Carrie

    2008-01-01

    Background and Aims Ecotypic differentiation has been explored in numerous plant species, but has been largely ignored in the Orchidaceae. Applying a specific germination protocol for widespread seed sources may be unreliable due to inherent physiological or genetic differences in localized populations. It is crucial to determine whether ecotypic differentiation exists for restoration and conservation programmes. Calopogon tuberosus var. tuberosus, a widespread terrestrial orchid of eastern North America, is a model species to explore ecotypic differences in germination requirements, as this species occupies diverse habitats spanning a wide geographical range. Methods Mature seeds were collected from south Florida, north central Florida, three locations in South Carolina, and the upper Michigan peninsula. Effects of three photoperiods (8/16, 12/12, 16/8 h L/D) were examined on asymbiotic in vitro seed germination and seedling development of C. tuberosus. Germination and early development was monitored for 8 weeks, while advanced development was monitored for an additional 8 weeks. In an additional experiment, asymbiotic seed germination and development was monitored for 8 weeks on six culture media (BM-1 terrestrial orchid medium, Knudson C, Malmgrem, half-strength MS, P723, and Vacin and Went). A tetrazolium test for embryo viability was performed. Key Results Short days promoted the highest germination among Florida populations, but few differences among photoperiods in other seed sources existed. Different media had little effect on the germination of Michigan and Florida populations, but germination of South Carolina seeds was higher on media with higher calcium and magnesium. Tetrazolium testing confirmed that South Carolina seeds exhibited low viability while viability was higher in Florida seeds. Seed germination and corm formation was rapid in Michigan seeds across all treatments. Michigan seedlings allocated more biomass to corms compared with other seed

  16. Measurement of microbial activity in soil by colorimetric observation of in situ dye reduction: an approach to detection of extraterrestrial life.

    Science.gov (United States)

    Crawford, Ronald; Paszczynski, Andrzej; Lang, Qingyong; Erwin, Daniel; Allenbach, Lisa; Corti, Giancarlo; Anderson, Tony; Cheng, I; Wai, Chien; Barnes, Bruce; Wells, Richard; Assefi, Touraj; Mojarradi, Mohammad

    2002-07-31

    Detecting microbial life in extraterrestrial locations is a goal of space exploration because of ecological and health concerns about possible contamination of other planets with earthly organisms, and vice versa. Previously we suggested a method for life detection based on the fact that living entities require a continual input of energy accessed through coupled oxidations and reductions (an electron transport chain). We demonstrated using earthly soils that the identification of extracted components of electron transport chains is useful for remote detection of a chemical signature of life. The instrument package developed used supercritical carbon dioxide for soil extraction, followed by chromatography or electrophoresis to separate extracted compounds, with final detection by voltammetry and tandem mass-spectrometry. Here we used Earth-derived soils to develop a related life detection system based on direct observation of a biological redox signature. We measured the ability of soil microbial communities to reduce artificial electron acceptors. Living organisms in pure culture and those naturally found in soil were shown to reduce 2,3-dichlorophenol indophenol (DCIP) and the tetrazolium dye 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT). Uninoculated or sterilized controls did not reduce the dyes. A soil from Antarctica that was determined by chemical signature and DNA analysis to be sterile also did not reduce the dyes. Observation of dye reduction, supplemented with extraction and identification of only a few specific signature redox-active biochemicals such as porphyrins or quinones, provides a simplified means to detect a signature of life in the soils of other planets or their moons.

  17. Totally Organic Redox-Active pH-Sensitive Nanoparticles Stabilized by Amphiphilic Aromatic Polyketones.

    Science.gov (United States)

    Araya-Hermosilla, Esteban; Catalán-Toledo, José; Muñoz-Suescun, Fabián; Oyarzun-Ampuero, Felipe; Raffa, Patrizio; Polgar, Lorenzo Massimo; Picchioni, Francesco; Moreno-Villoslada, Ignacio

    2018-02-08

    Amphiphilic aromatic polymers have been synthesized by grafting aliphatic polyketones with 4-(aminomethyl)benzoic acid at different molar ratios via the Paal-Knorr reaction. The resulting polymers, showing diketone conversion degree of 16%, 37%, 53%, and 69%, have been complexed with the redox-active 2,3,5-triphenyl-2H-tetrazolium chloride, a precursor molecule with which aromatic-aromatic interactions are held. Upon addition of ascorbic acid to the complexes, in situ reduction of the tetrazolium salt produced 1,3,5-triphenylformazan nanoparticles stabilized by the amphiphilic polymers. The stabilized nanoparticles display highly negative zeta potential [-(35-70) mV] and hydrodynamic diameters in the submicron range (100-400 nm). Nonaromatic polyelectrolytes or hydrophilic aromatic copolymers showing low linear aromatic density and high linear charge density such as acrylate/maleate and sulfonate/maleate-containing polymers were unable to stabilize formazan nanoparticles synthesized by the same method. The copolymers studied here bear uncharged nonaromatic comonomers (unreacted diketone units) as well as charged aromatic comonomers, which furnish amphiphilia. Thus, the linear aromatic density and the maximum linear charge density have the same value for each copolymer, and the hydrophilic/hydrophobic balance varies with the diketone conversion degree. The amphiphilia of the copolymers allows the stabilization of the nanoparticles, even with the copolymers showing a low linear aromatic density. The method of nanoparticle synthesis constitutes a simple, cheap, and green method for the production of switchable totally organic, redox-active, pH-sensitive nanoparticles that can be reversibly turned into macroprecipitates upon pH changing.

  18. Recombinant Nox4 cytosolic domain produced by a cell or cell-free base systems exhibits constitutive diaphorase activity

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, Minh Vu Chuong, E-mail: mvchuong@yahoo.fr [GREPI AGIM FRE 3405 CNRS-UJF, Universite Joseph Fourier, Grenoble (France); Zhang, Leilei [GREPI AGIM FRE 3405 CNRS-UJF, Universite Joseph Fourier, Grenoble (France); Lhomme, Stanislas; Mouz, Nicolas [PX' Therapeutics, MINATEC/Batiment de Haute Technologie, Grenoble (France); Lenormand, Jean-Luc [HumProTher Laboratory, TheReX/TIMC-IMAG UMR 5525 CNRS UJF, Universite Joseph Fourier, UFR de Medecine, Domaine de la Merci, 38706 La Tronche (France); Lardy, Bernard; Morel, Francoise [GREPI AGIM FRE 3405 CNRS-UJF, Universite Joseph Fourier, Grenoble (France)

    2012-03-16

    Highlights: Black-Right-Pointing-Pointer A comparison of two bacterial cell and cell-free protein expression systems is presented. Black-Right-Pointing-Pointer Soluble and active truncated Nox4 proteins are produced. Black-Right-Pointing-Pointer Nox4 has a constitutive diaphorase activity which is independent of cytosolic factors. Black-Right-Pointing-Pointer Isoform Nox4B is unable to initiate the first electronic transfer step. Black-Right-Pointing-Pointer Findings contribute to the understanding of the mechanism of Nox4 oxidase activity. -- Abstract: The membrane protein NADPH (nicotinamide adenine dinucleotide phosphate) oxidase Nox4 constitutively generates reactive oxygen species differing from other NADPH oxidases activity, particularly in Nox2 which needs a stimulus to be active. Although the precise mechanism of production of reactive oxygen species by Nox2 is well characterized, the electronic transfer throughout Nox4 remains unclear. Our study aims to investigate the initial electronic transfer step (diaphorase activity) of the cytosolic tail of Nox4. For this purpose, we developed two different approaches to produce soluble and active truncated Nox4 proteins. We synthesized soluble recombinant proteins either by in vitro translation or by bacteria induction. While proteins obtained by bacteria induction demonstrate an activity of 4.4 {+-} 1.7 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 20.5 {+-} 2.8 nmol/min/nmol with cytochrome c, the soluble proteins produced by cell-free expression system exhibit a diaphorase activity with a turn-over of 26 {+-} 2.6 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 48 {+-} 20.2 nmol/min/nmol with cytochrome c. Furthermore, the activity of the soluble proteins is constitutive and does not need any stimulus. We also show that the cytosolic tail of the isoform Nox4B lacking the first NADPH binding site is unable to demonstrate any diaphorase activity pointing out the

  19. Arecoline is cytotoxic for human endothelial cells.

    Science.gov (United States)

    Ullah, Mafaz; Cox, Stephen; Kelly, Elizabeth; Boadle, Ross; Zoellner, Hans

    2014-11-01

    Oral submucous fibrosis is a pre-malignant fibrotic condition caused by areca nut use and involves reduced mucosal vascularity. Arecoline is the principal areca nut alkaloid and is cytotoxic for epithelium and fibroblasts. Endothelial cell cycle arrest is reported on exposure to arecoline, as is cytotoxicity for endothelial-lung carcinoma hybrid cells. We here describe cytotoxicity for primary human endothelial cultures from seven separate donors. Human umbilical vein endothelial cells were exposed to increasing concentrations of arecoline and examined by: phase-contrast microscopy, haemocytometer counts, transmission electron microscopy, lactate dehydrogenase release and the methyl-thiazol-tetrazolium assay. Vacuolation and detachment of endothelium were observed at and above arecoline concentrations of 333 μg/ml or more. Ultrastructural features of cellular stress were seen after 24-h treatment with 111 μg/ml arecoline and included reduced ribosomal studding of endoplasmic reticulum, increased autophagolysosomal structures, increased vacuolation and reduced mitochondrial cristae with slight swelling. Similar changes were seen at 4 h with arecoline at 333 μg/ml or above, but with more severe mitochondrial changes including increased electron density of mitochondrial matrix and greater cristal swelling, while by 24 h, these cells were frankly necrotic. Haemocytometer counts were paralleled by both lactate dehydrogenase release and the methyl-thiazol-tetrazolium assays. Arecoline is cytotoxic via necrosis for endothelium, while biochemical assays indicate no appreciable cellular leakage before death and detachment, as well as no clear effect on mitochondrial function in viable cells. Arecoline toxicity may thus contribute to reduced vascularity in oral submucous fibrosis. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  20. A simple high-content cell cycle assay reveals frequent discrepancies between cell number and ATP and MTS proliferation assays.

    Science.gov (United States)

    Chan, Grace Ka Yan; Kleinheinz, Tracy L; Peterson, David; Moffat, John G

    2013-01-01

    In order to efficiently characterize both antiproliferative potency and mechanism of action of small molecules targeting the cell cycle, we developed a high-throughput image-based assay to determine cell number and cell cycle phase distribution. Using this we profiled the effects of experimental and approved anti-cancer agents with a range mechanisms of action on a set of cell lines, comparing direct cell counting versus two metabolism-based cell viability/proliferation assay formats, ATP-dependent bioluminescence, MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) reduction, and a whole-well DNA-binding dye fluorescence assay. We show that, depending on compound mechanisms of action, the metabolism-based proxy assays are frequently prone to 1) significant underestimation of compound potency and efficacy, and 2) non-monotonic dose-response curves due to concentration-dependent phenotypic 'switching'. In particular, potency and efficacy of DNA synthesis-targeting agents such as gemcitabine and etoposide could be profoundly underestimated by ATP and MTS-reduction assays. In the same image-based assay we showed that drug-induced increases in ATP content were associated with increased cell size and proportionate increases in mitochondrial content and respiratory flux concomitant with cell cycle arrest. Therefore, differences in compound mechanism of action and cell line-specific responses can yield significantly misleading results when using ATP or tetrazolium-reduction assays as a proxy for cell number when screening compounds for antiproliferative activity or profiling panels of cell lines for drug sensitivity.

  1. Combinatorial cytotoxic effects of Curcuma longa and Zingiber officinale on the PC-3M prostate cancer cell line.

    Science.gov (United States)

    Kurapati, Kesava Rao V; Samikkannu, Thangavel; Kadiyala, Dakshayani B; Zainulabedin, Saiyed M; Gandhi, Nimisha; Sathaye, Sadhana S; Indap, Manohar A; Boukli, Nawal; Rodriguez, Jose W; Nair, Madhavan P N

    2012-01-01

    Many plant-derived products exhibit potent chemopreventive activity against animal tumor models as well as rodent and human cancer cell lines. They have low side effects and toxicity and presumably modulate the factors that are critical for cell proliferation, differentiation, senescence and apoptosis. The present study investigates the effects of some medicinal plant extracts from generally recognized as safe plants that may be useful in the prevention and treatment of cancer. Clonogenic assays using logarithmically-growing cells were performed to test the effect. The cytotoxic effects of Curcuma longa and Zingiber officinale were studied using sulforhodamine B assay, tetrazolium dye assay, colony morphology and microscopic analysis. Out of the 13 lyophilized plant-derived extracts evaluated for growth-inhibitory effects on the PC-3M prostate cancer cell line, two extracts derived from C. longa and Z. officinale showed significant inhibitory effects on colony-forming ability. The individual and augmentative effects of these two extracts were tested for their narrow range effective lower concentration on PC-3M in clonogenic assays. At relatively lower concentrations, C. longa showed significant inhibition of colony formation in clonogenic assays; whereas at same concentrations Z. officinale showed only moderate inhibitory effects. However, when both the agents were tested together at the same concentrations, the combined effects were much more significant than their individual ones. On normal prostate epithelial cells both C. longa and Z. officinale had similar effects but at a lower magnitude. These observations were confirmed by several cytotoxicity assays involving the morphological appearance of the colonies, microscopic observations, per cent inhibition in comparison to control by sulforhodamine B and tetrazolium dye assay. From these observations, it was concluded that the combined effects of C. longa and Z. officinale are much greater than their individual

  2. Liposomes and pulmonary alveolar macrophages: functional and morphologic interactions.

    Science.gov (United States)

    Gonzalez-Rothi, R J; Straub, L; Cacace, J L; Schreier, H

    1991-01-01

    In vitro toxicity of liposomes and their functional and morphologic interactions with rat pulmonary alveolar macrophage (AMs) were investigated using viability (trypan blue exclusion), phagocytic and killing activity (uptake and digestion of live S. cerevisiae), surface adherence, respiratory burst (nitro-blue tetrazolium reduction), and morphometry (computerized image analysis) as indicators. Liposome stability in physiologic solutions and uptake of liposome-encapsulated carboxyfluorescein (CF) by AMs was assessed by fluorescence spectroscopy and microscopy. Liposomes made from saturated phospholipids and cholesterol were stable, whereas liposomes consisting of unsaturated phospholipids without cholesterol lost 30% to 40% of their content over 24 h. However, CF uptake was highest with unsaturated phospholipid preparations, whereas uptake of the three other formulations was comparable. Although liposome exposure did not affect macrophage viability, a reduction in the number of phagocytizing macrophages to 73% of control was noted after 24-h incubation with the highest lipid concentration tested (10 mumol/ml). Phagocytic killing was similar under all circumstances observed. The fraction of intracellularly killed yeast ranged from 32% to 42% for both control and experimental samples. An increase in cell surface area from 166.1 +/- 39.9 microns 2 on day O (n = 709) to 196.3 +/- 57.6 microns 2 on day 1 (n = 516) and 211.2 +/- 48.0 microns 2 on day 4 (n = 834) was observed after liposome treatment. The corresponding average cell areas of control samples did not change during the observation period. There was no net cell loss of adherence from monolayers as determined by protein assay. The respiratory burst, indicating generation of intracellular superoxide, was also similar--84% to 92% of experimental and control cells under all conditions showed a strong nitro-blue tetrazolium reduction. In summary, in vitro exposure of AMs to large concentrations of liposomes

  3. In vitro germination of zygotic embryos of hybrid BRS Manicoré (E. guineensis X E. oleifera

    Directory of Open Access Journals (Sweden)

    KEILA A.P. BONETTI

    Full Text Available ABSTRACT The interspecific oil palm hybrid BRS Manicoré (E. guineensis x E. oleifera has superior agronomic characteristics. However, the germination rate is low (30% and the process is slow when the seeds are sown in a conventional form. The purpose of this study was to optimize the in vitro germination of zygotic embryos of this hybrid comparing seed lots. The viability of zygotic embryos was evaluated by the tetrazolium test (0.075% for 4 h. The embryos were cultured on MS and Y3 culture media, with and without the addition of NaH2PO4, as well as on MS, MS1/2 and N6 medium. In MS medium containing NaH2PO4, the germination rate was increased from 40 to 70% in comparison with the medium without sodium phosphate. The comparison between the culture media MS, MS 1/2, N6 and Y3 showed that 75% of zygotic embryos cultured in the Y3 medium formed whole plants (with roots and shoots defined, a higher percentage than embryos cultured on MS, MS 1/2 and N6 media (46, 35 and 17% respectively. In the same Y3 culture medium, the embryos were larger (36% ≥ 2 cm and 30% ≥ 5 cm than in the other media. Results obtained by the tetrazolium test were similar to those of germination, showing the effect of the genotype of each seed lot. For the germination and development of plantlets it is essential to add NaH2PO4 to a culture medium containing no phosphate or with a low phosphate concentration.

  4. Hemograma e metabolismo oxidativo dos neutrófilos de bovinos da raça Holandesa preta e branca - Influência dos fatores etários.

    Directory of Open Access Journals (Sweden)

    A. Kohayagawa

    2005-03-01

    Full Text Available RESUMO: O objetivo deste trabalho foi avaliar o quadro hematológico e o metabolismo oxidativo dos neutrófilos de bezerros da raça Holandesa preta e branca analisando a influência dos fatores etários. Foram colhidas amostras de sangue da veia jugular de 20 bezerros com idade entre 0 e 90 dias em dois diferentes tubos: 5 mL de sangue total em EDTA e 500 mL em heparina para a determinação respectivamente, do hemograma e do teste da função oxidativa neutrofílica. Os animais foram divididos em dois grupos de 10 bezerros: o Grupo 1 com idade inferior a 30 dias e o Grupo 2 com idades entre 30 e 90 dias. Os valores obtidos para o número total de eritrócitos e de leucócitos foram, respectivamente: 7,61 ± 1,32 x 106 mL e 11,0 ± 3,87 x 103 mL para animais do Grupo 1 e de 7,88 ± 1,22 x 106 mL e 11,9 ± 2,92 x 103 mL para os animais do Grupo 2. A função oxidativa neutrofílica determinada pelo teste de redução do NBT (Nitroblue Tetrazolium foi superior no Grupo 2. PALAVRAS CHAVE: bovinos, hemograma, NBT, função de neutrófilos. SUMMARY: The objective of this study was to evaluate the hematologic picture and the oxidative metabolism of the neutrophils of calves of Holstein-friesan, analyzing the influence of the age factors. Blood was colleted of 20 calves with age among 0 and 90 days by jugular venipuncture into two different tubes : 5 mL of total blood in EDTA and 500 mL in heparina for the determination of the hemogram and for the test of the oxidative function of the neutrophils. The animals were divided in two groups of 10 calves, the Group 1 with age inferior to 30 days and the Group 2 between 30 and 90 days. The values obtained for RBC (red blood cells and WBC ( white blood cells counts were 7,61 ± 1,32 x106 mL and 11,0 ± 3,87 x103 mL respectively for animals of the Group 1 and 7,88 ± 1,22 x106 mL and 11,9 ± 2,92 x103 mL for the animals of the Group 2. The oxidative

  5. Lymphocyte subpopulations and neutrophil function in chronic human Chagas' disease Subpopulações linfocitárias e função neutrofílica na doença de Chagas humana

    Directory of Open Access Journals (Sweden)

    J. C. Voltarelli

    1990-08-01

    Full Text Available The absolute numbers of total leukocytes, lymphocytes, T cells, helper/inducer, suppressor/cytotoxic and B cells were decreased in the peripheral blood of patients with chronic Chagas' disease. Since antilymphocyte antibodies were present only in a minority of patients they probably cannot account for the abnormalities in lymphocyte subsets. Patient neutrophils stimulated with endotoxin-treated autologous plasma showed depressed chemotactic activity and this seems to be an intrinsic cellular defect rather than plasma inhibition. Random migration of neutrophils was normal. Reduction of nitroblue tetrazolium by endotoxin- stimulated neutrophils was also decreased. These findings further document the presence of immunosuppression in human Chagas' disease. They may be relevant to autoimmunity, defense against microorganisms and against tumor cells at least in a subset of patients with more severe abnormalities.Números absolutos de leucócitos e linfócitos, de células T totais, indutoras/auxiliares, supressoras/citotóxicas e de células B estavam diminuídos no sangue periférico de pacientes com doença de Chagas crônica. Como anticorpos antilinfocitários estavam presentes em apenas uma minoria de pacientes, eles provavelmente não são responsáveis pelas anormalidades das subpopulações de linfócitos. Neutrófilos de pacientes estimulados por plasma autólogo tratado por endotoxina mostravam atividade quimiotática diminuída que deve ser devida a um defeito celular intrínseco e não a inibição plasmática. A migração aleatória dos neutrófilos estava normal. A redução do corante "nitroblue tetrazolium" (NBT por neutrófilos estimulados por endotoxina também estava diminuída nos pacientes. Estes achados estendem a documentação da imunossupressão na doença de Chagas humana. Eles podem ser relevantes para autoimunidade e para defesa contra microorganismos e células tumoráis, pelo menos em um subgrupo de pacientes com

  6. Anticancer Activity of Chloroform Extract and Sub-fractions of Nepeta deflersiana on Human Breast and Lung Cancer Cells: An In vitro Cytotoxicity Assessment.

    Science.gov (United States)

    Al-Oqail, Mai M; Al-Sheddi, Ebtesam S; Siddiqui, Maqsood A; Musarrat, Javed; Al-Khedhairy, Abdulaziz A; Farshori, Nida N

    2015-10-01

    Cancer is one of the major causes of death worldwide. The plant-derived natural products have received considerable attention in recent years due to their diverse pharmacological properties including anticancer effects. Nepeta deflersiana (ND) is used in the folk medicine as antiseptic, carminative, antimicrobial, antioxidant, and for treating rheumatic disorders. However, the anticancer activity of ND chloroform extract has not been explored so far. The present study was aimed to investigate the anticancer activities of chloroform Nepeta deflersiana extract and various sub-fractions (ND-1-ND-15) of ND against human breast cancer cells (MCF-7) and human lung cancer cells (A-549). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and neutral red uptake assays, and cellular morphological alterations using phase contrast light microscope were studied. Cells were exposed with 10-1000 μg/ml of sub-fractions of ND for 24 h. Results showed that selected sub-fractions of the chloroform extract significantly reduced the cell viability of MCF-7 and A-549 cells, and altered the cellular morphology in a concentration-dependent manner. Among the sub-fractions, ND-10 fraction showed relatively higher cytotoxicity compared to other fractions whereas, ND-1 did not cause any cytotoxicity even at higher concentrations. The A-549 cells were found to be more sensitive to growth inhibition by all the extracts as compared to the MCF-7 cells. The present study provides preliminary screening of anticancer activities of chloroform extract and sub-fractions of ND, which can be further used for the development of a potential therapeutic anticancer agent. Nepeta deflersiana extract exhibit cytotoxicity and altered the cellular morphology. Sub-fractions of the chloroform extract of Nepeta deflersiana reduced the cell viability of MCF-7 and A-549 cells. Among the sub-fractions, ND-10 fraction showed relatively higher cytotoxicity. The A-549 cells were found to be more sensitive

  7. Quebra de dormência, viabilidade e conservação de sementes de buriti (Mauritia flexuosa Dormancy breaking, viability and conservation of Mauritia flexuosa seeds

    Directory of Open Access Journals (Sweden)

    Maria Roseli Nicoli Spera

    2001-12-01

    Full Text Available Os objetivos deste trabalho foram avaliar a viabilidade de embriões de buriti (Mauritia flexuosa L. pelo teste de tetrazólio e cultivo in vitro, o tratamento térmico na quebra de dormência das sementes, e o efeito do armazenamento em duas temperaturas na conservação das sementes. A viabilidade de embriões de sementes recém-colhidas foi condizente com os resultados de germinação de embriões in vitro, com valores superiores a 90%. O estudo da quebra de dormência foi feito com tratamento das sementes em temperaturas de 30, 35 e 40ºC por períodos de 15, 30 e 45 dias. Até 15 e 30 dias, os resultados obtidos foram maiores que os do controle. Sementes armazenadas em saco de plástico por um período de quatro meses e meio, sob temperatura de 20ºC, apresentaram resultados de germinação de embrião superiores a 90%, e sob temperatura de 30ºC houve perda total da viabilidade.The present work was conducted to evaluate the viability of embryos of Mauritia flexuosa L. by tetrazolium test and in vitro culture, high temperature treatment on dormancy breaking, and the effect of storage at different temperatures on seed conservation. The viability of newly harvested embryos presented a high correlation with the rate of embryo germination in vitro. The rate of viability was higher than 90% either when evaluated by the tetrazolium method or by in vitro embryo culture. Dormancy breaking of seeds was tested by treating the seeds under temperatures of 30, 35 and 40ºC for 15, 30, and 45 days. Great variation was observed among treatments, but, at least on day 15 and on day 30, the germination was higher than in the control. Seeds stored in plastic bags for four and a half months and temperature of 20ºC presented a germination rate higher than 90% while in the seeds stored at 30ºC the viability was zero.

  8. The effects of myocyte enhancer factor 2A gene on the proliferation, migration and phenotype of vascular smooth muscle cells.

    Science.gov (United States)

    Zhao, Wang; Zhao, Shui-ping; Peng, Dao-quan

    2012-03-01

    The genetic basis for the phenotypic switching of vascular smooth muscle cells (VSMCs) is unclear in atherosclerosis. Recent studies showed that the 21-base pair deletion mutation (Δ21) in myocyte enhancer factor 2A (MEF2A) gene could be an inherited marker for coronary artery disease. MEF2A mutation may affect the phenotypic switching of VSMCs. Human aortic VSMCs were used. Four groups of VSMCs transfected with green fluorescent protein plasmid (control group), MEF2A wild-type (WT) plasmid (WT group), MEF2A Δ21 plasmid (Δ21 group) or MEF2A siRNA (siRNA group) were studied. The proliferation of VSMCs was determined by methylthiazolyldiphenyl-tetrazolium bromide, and the migration of VSMCs was measured by Millicell chamber. The protein expressions of MEF2A, smooth muscle α-actin, SM22α, osteopontin and p38 mitogen-activated protein kinase signaling pathway were detected by Western blotting. MEF2A protein expression was knockdown by siRNA transfection. MEF2A protein was overexpressed in WT and Δ21 groups. Δ21 and siRNA groups obviously showed more proliferation (methylthiazolyldiphenyl-tetrazolium bromide, 0.63 vs 0.66 vs 0.31, P < 0.01) and migration (52.6 vs 58.0 vs 21.2, P < 0.01) of VSMCs as compared with the WT group. In addition, the transfection of Δ21 and siRNA could induce the down-regulation of smooth muscle α-actin and SM22α (P < 0.01) and the up-regulation of osteopontin (P < 0.01) in VSMCs. The phosphorylated p38 signaling pathway expression was significantly enhanced in the Δ21 and siRNA groups as compared with that of the WT group (P < 0.01). These results suggest that MEF2A dominant negative mutation and RNA silence could induce the phenotypic switching of VSMCs, leading to its increased proliferation and migration, and p38 mitogen-activated protein kinase signaling pathway may participate in it. Copyright © 2011 John Wiley & Sons, Ltd.

  9. Treatment with Rutin - A Therapeutic Strategy for Neutrophil-Mediated Inflammatory and Autoimmune Diseases - Anti-inflammatory Effects of Rutin on Neutrophils -

    Directory of Open Access Journals (Sweden)

    Bahareh Abd Nikfarjam

    2017-03-01

    Full Text Available Objectives: Neutrophils represent the front line of human defense against infections. Immediately after stimulation, neutrophilic enzymes are activated and produce toxic mediators such as pro-inflammatory cytokines, nitric oxide (NO and myeloperoxidase (MPO. These mediators can be toxic not only to infectious agents but also to host tissues. Because flavonoids exhibit antioxidant and anti-inflammatory effects, they are subjects of interest for pharmacological modulation of inflammation. In the present study, the effects of rutin on stimulus-induced NO and tumor necrosis factor (TNF-α productions and MPO activity in human neutrophils were investigated. Methods: Human peripheral blood neutrophils were isolated using Ficoll-Hypaque density gradient centrifugation coupled with dextran T500 sedimentation. The cell preparations containing > 98% granulocytes were determined by morphological examination through Giemsa staining. Neutrophils were cultured in complete Roswell Park Memorial Institute (RPMI medium, pre-incubated with or without rutin (25 μM for 45 minutes, and stimulated with phorbol 12-myristate 13-acetate (PMA. Then, the TNF-α, NO and MPO productions were analyzed using enzyme-linked immunosorbent assay (ELISA, Griess Reagent, and MPO assay kits, respectively. Also, the viability of human neutrophils was assessed using tetrazolium salt 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT, and neutrophils were treated with various concentrations of rutin (1 - 100 μM, after which MTT was appended and incubated at 37ºC for 4 hour. Results: Rutin at concentrations up to 100 μM did not affect neutrophil viability during the 4-hour incubation period. Rutin significantly decreased the NO and TNF-α productions in human peripheral blood neutrophils compared to PMA-control cells (P < 0.001. Also, MPO activity was significantly reduced by rutin (P < 0.001. Conclusion: In this in vitro study, rutin had an anti-inflammatory effect

  10. Pruebas de vigor y calidad fisiológica de semillas de soja Vigor tests and physiological quality of soybean seeds

    Directory of Open Access Journals (Sweden)

    Adriana Rita Salinas

    2001-02-01

    Full Text Available El objetivo de este trabajo fue estudiar las alteraciones producidas por el deterioro en semillas de soja (Glycine max L. Merrill utilizando diferentes pruebas de vigor. Se utilizaron semillas de soja de diferentes cultivares en 1997 y 1998, las cuales fueron sometidas a la prueba de germinación y diferentes pruebas de vigor: envejecimiento acelerado; conductividad eléctrica; deterioro controlado y tetrazolio. El deterioro controlado en semillas con 22% de humedad mostró ser un mejor indicador de la disminución del vigor de la semilla de soja que el envejecimiento acelerado. La conductividad eléctrica en semillas individuales podría ser considerada una buena prueba de vigor para detectar alteraciones producidas en las membranas citoplasmáticas en un estadio temprano del deterioro de las semillas. La clasificación de la viabilidad y el vigor a través de la prueba de tetrazolio está dada por el lugar donde se produce el daño y no por el tipo de daño que sufre la semilla.The objective of this work was to study soybean (Glycine max L. Merrill seed alterations produced by deterioration using different vigor tests. Soybean seeds of different cultivars were used in 1997 and 1998, which were submitted to standard germination test and the vigor tests: accelerated aging; electric conductivity; controlled deterioration and tetrazolium. The controlled deterioration with an elevation of the seed humidity to 22%, showed to be a better indicator of the diminishing of soybean seed vigor than the accelerated aging. The electric conductivity in individual seeds would be considered a good vigor test to detect the alterations produced in the cytoplasmic membranes in an early stage of the seed deterioration. The tetrazolium test by the evaluation of the different kinds of injuries suffered by the seeds, depends on the place where the seed is damaged not on the kind of injury suffered by the seed.

  11. Rapid glycation with D-ribose induces globular amyloid-like aggregations of BSA with high cytotoxicity to SH-SY5Y cells

    Directory of Open Access Journals (Sweden)

    Ge Lin

    2009-02-01

    Full Text Available Abstract Background D-ribose in cells and human serum participates in glycation of proteins resulting in advanced glycation end products (AGEs that affect cell metabolism and induce cell death. However, the mechanism by which D-ribose-glycated proteins induce cell death is still unclear. Results Here, we incubated D-ribose with bovine serum albumin (BSA and observed changes in the intensity of fluorescence at 410 nm and 425 nm to monitor the formation of D-ribose-glycated BSA. Comparing glycation of BSA with xylose (a control for furanose, glucose and fructose (controls for pyranose, the rate of glycation with D-ribose was the most rapid. Protein intrinsic fluorescence (335 nm, Nitroblue tetrazolium (NBT assays and Western blotting with anti-AGEs showed that glycation of BSA incubated with D-ribose occurred faster than for the other reducing sugars. Protein intrinsic fluorescence showed marked conformational changes when BSA was incubated with D-ribose. Importantly, observations with atomic force microscopy showed that D-ribose-glycated BSA appeared in globular polymers. Furthermore, a fluorescent assay with Thioflavin T (ThT showed a remarkable increase in fluorescence at 485 nm in the presence of D-ribose-glycated BSA. However, ThT fluorescence did not show the same marked increase in the presence of xylose or glucose. This suggests that glycation with D-ribose induced BSA to aggregate into globular amyloid-like deposits. As observed by Hoechst 33258 staining, 3-(4, 5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT and cell counting kit-8 (CCK-8 assay, lactate dehydrogenase (LDH activity assay, flow cytometry using Annexin V and Propidium Iodide staining and reactive oxygen species (ROS measurements, the amyloid-like aggregation of glycated BSA induced apoptosis in the neurotypic cell line SH-SY5Y. Conclusion Glycation with D-ribose induces BSA to misfold rapidly and form globular amyloid-like aggregations which play an important

  12. Nuclear factor-kappa B inhibition can enhance apoptosis of differentiated thyroid cancer cells induced by 131I.

    Directory of Open Access Journals (Sweden)

    Zhaowei Meng

    Full Text Available OBJECTIVE: To evaluate changes of nuclear factor-kappa B (NF-κB during radioiodine 131 ((131I therapy and whether NF-κB inhibition could enhance (131I-induced apoptosis in differentiated thyroid cancer (DTC cells in a synergistic manner. METHODS: Three human DTC cell lines were used. NF-κB inhibition was achieved by using a NF-κB inhibitor (Bay 11-7082 or by p65 siRNA transfection. Methyl-thiazolyl-tetrazolium assay was performed for cell viability assessment. DNA-binding assay, luciferase reporter assay, and Western blot were adopted to determine function and expression changes of NF-κB. Then NF-κB regulated anti-apoptotic factors XIAP, cIAP1, and Bcl-xL were measured. Apoptosis was analyzed by Western blot for caspase 3 and PARP, and by flow cytometry as well. An iodide uptake assay was performed to determine whether NF-κB inhibition could influence radioactive iodide uptake. RESULTS: The methyl-thiazolyl-tetrazolium assay showed significant decrease of viable cells by combination therapy than by mono-therapies. The DNA-binding assay and luciferase reporter assay showed enhanced NF-κB function and reporter gene activities due to (131I, yet significant suppression was achieved by NF-κB inhibition. Western blot proved (131I could increase nuclear NF-κB concentration, while NF-κB inhibition reduced NF-κB concentration. Western blot also demonstrated significant up-regulation of XIAP, cIAP1, and Bcl-xL after (131I therapy. And inhibition of NF-κB could significantly down-regulate these factors. Finally, synergism induced by combined therapy was displayed by significant enhancements of cleaved caspase 3 and PARP from Western blot, and of Annexin V positively staining from flow cytometry. The iodine uptake assay did not show significant changes when NF-κB was inhibited. CONCLUSION: We demonstrated that (131I could induce NF-κB activation, which would attenuate (131I efficacy in DTC cells. NF-κB inhibition by Bay 11-7082 or by p65 si

  13. Evaluation of the antibacterial and anticancer activities of some South African medicinal plants.

    Science.gov (United States)

    Bisi-Johnson, Mary A; Obi, Chikwelu L; Hattori, Toshio; Oshima, Yoshiteru; Li, Shenwei; Kambizi, Learnmore; Eloff, Jacobus N; Vasaikar, Sandeep D

    2011-02-17

    Several herbs are traditionally used in the treatment of a variety of ailments particularly in the rural areas of South Africa where herbal medicine is mainly the source of health care system. Many of these herbs have not been assessed for safety or toxicity to tissue or organs of the mammalian recipients. This study evaluated the cytotoxicity of some medicinal plants used, inter alia, in the treatment of diarrhoea, and stomach disorders. Six selected medicinal plants were assessed for their antibacterial activities against ampicillin-resistant and kanamycin-resistant strains of Escherichia coli by the broth micro-dilution methods. The cytotoxicities of methanol extracts and fractions of the six selected plants were determined using a modified tetrazolium-based colorimetric assay (3-(4, 5-dimethylthiazol)-2, 5-diphenyl tetrazolium bromide (MTT) assay). The average minimum inhibitory concentration (MIC) values of the plants extracts ranged from 0.027 mg/mℓ to 2.5 mg/mℓ after 24 h of incubation. Eucomis autumnalis and Cyathula uncinulata had the most significant biological activity with the least MIC values. The in vitro cytotoxicity assay on human hepatocarcinoma cell line (Huh-7) revealed that the methanol extract of E. autumnalis had the strongest cytotoxicity with IC(50) of 7.8 μg/mℓ. Ethyl acetate and butanol fractions of C. uncinulata, Hypoxis latifolia, E. autumnalis and Lantana camara had lower cytotoxic effects on the cancer cell lines tested with IC(50) values ranging from 24.8 to 44.1 μg/mℓ; while all the fractions of Aloe arborescens and A. striatula had insignificant or no cytotoxic effects after 72 h of treatment. Our results indicate that the methanol fraction of E. autumnalis had a profound cytotoxic effect even though it possessed very significant antibacterial activity. This puts a query on its safety and hence a call for caution in its usage, thus a product being natural is not tantamount to being entirely safe. However, the antibacterial

  14. Antimicrobial activity, toxicity and selectivity index of two biflavonoids and a flavone isolated from Podocarpus henkelii (Podocarpaceae) leaves.

    Science.gov (United States)

    Bagla, Victor P; McGaw, Lyndy J; Elgorashi, Esam E; Eloff, Jacobus N

    2014-10-08

    Different parts of Podocarpus henkelii have been used in many cultures around the world to treat ailments such as cholera, stomach diseases, rheumatism, cancer, canine distemper in dogs and gall sickness in cattle. The aim of this study was to evaluate the biological activity and toxicity of isolated compounds from Podocarpus henkelii after an earlier study indicated a promising activity in crude extracts against viral pathogens of veterinary importance. The antibacterial and antifungal activity of two biflavonoids 7, 4', 7", 4"'-tetramethoxy amentoflavone (TMA), isoginkgetin (IGG) and podocarpus flavone-A (PFA) isolated from the leaves of Podocarpus henkelii were determined using a serial microplate dilution method with tetrazolium violet as growth indicator. The cytotoxicity of compounds TMA and IGG were determined on different cell types using a tetrazolium-based colorimetric cellular assay (MTT). The Ames test was used to determine their mutagenic activities. TMA had reasonable antifungal activity against Aspergillus fumigatus (MIC = 30 μg/ml). IGG had a wide spectrum of activity against four bacterial and two fungal pathogens with much higher selectivity index values obtained for A. fumigatus and Cryptococcus neoformans (SI > 30). PFA had a broad spectrum of activity against Enterococcus faecalis and Pseudomonas aeruginosa (SI > 15) and less activity against the two fungal pathogens. In both the cytotoxicity assays and Ames mutagenicity test using Salmonella typhimurium strains TA98 and TA100, TMA and IGG had no deleterious effect on the different cell types and did not induce mutations in the Ames test. Although the antimicrobial activities of the isolated compounds were not that exciting, the compounds had no cytotoxic activity at the highest concentration (1000 μg/ml) tested against all three cell lines. IGG was the most active against E. coli, S. aureus, A. fumigatus and C. neoformans, exhibiting both antibacterial and antifungal activity with good

  15. Theranostic reduction-sensitive gemcitabine prodrug micelles for near-infrared imaging and pancreatic cancer therapy

    Science.gov (United States)

    Han, Haijie; Wang, Haibo; Chen, Yangjun; Li, Zuhong; Wang, Yin; Jin, Qiao; Ji, Jian

    2015-12-01

    A biodegradable and reduction-cleavable gemcitabine (GEM) polymeric prodrug with in vivo near-infrared (NIR) imaging ability was reported. This theranostic GEM prodrug PEG-b-[PLA-co-PMAC-graft-(IR820-co-GEM)] was synthesized by ring-opening polymerization and ``click'' reaction. The as-prepared reduction-sensitive prodrug could self-assemble into prodrug micelles in aqueous solution confirmed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). In vitro drug release studies showed that these prodrug micelles were able to release GEM in an intracellular-mimicking reductive environment. These prodrug micelles could be effectively internalized by BxPC-3 pancreatic cancer cells, which were observed by confocal laser scanning microscopy (CLSM). Meanwhile, a methyl thiazolyl tetrazolium (MTT) assay demonstrated that this prodrug exhibited high cytotoxicity against BxPC-3 cells. The in vivo whole-animal near-infrared (NIR) imaging results showed that these prodrug micelles could be effectively accumulated in tumor tissue and had a longer blood circulation time than IR820-COOH. The endogenous reduction-sensitive gemcitabine prodrug micelles with the in vivo NIR imaging ability might have great potential in image-guided pancreatic cancer therapy.A biodegradable and reduction-cleavable gemcitabine (GEM) polymeric prodrug with in vivo near-infrared (NIR) imaging ability was reported. This theranostic GEM prodrug PEG-b-[PLA-co-PMAC-graft-(IR820-co-GEM)] was synthesized by ring-opening polymerization and ``click'' reaction. The as-prepared reduction-sensitive prodrug could self-assemble into prodrug micelles in aqueous solution confirmed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). In vitro drug release studies showed that these prodrug micelles were able to release GEM in an intracellular-mimicking reductive environment. These prodrug micelles could be effectively internalized by BxPC-3 pancreatic cancer cells, which

  16. PKI-587 and sorafenib alone and in combination on inhibition of liver cancer stem cell proliferation.

    Science.gov (United States)

    Gedaly, Roberto; Galuppo, Roberto; Musgrave, Yolanda; Angulo, Paul; Hundley, Jonathan; Shah, Malay; Daily, Michael F; Chen, Changguo; Cohen, Donald A; Spear, Brett T; Evers, B Mark

    2013-11-01

    Deregulated Ras/Raf/mitogen-activated protein kinase and PI3 K/AKT/mTOR signaling pathways are significant in hepatocellular carcinoma proliferation (HCC). In this study we evaluated differences in the antiproliferative effect of dual PI3 K/Akt/mTOR and Ras/Raf/mitogen-activated protein kinase inhibition of non liver cancer stem cell lines (PLC and HuH7) and liver cancer stem cell (LCSC) lines (CD133, CD44, CD24, and aldehyde dehydrogenase 1-positive cells). Flow cytometry was performed on the resulting tumors to identify the LCSC markers CD133, CD44, CD24, and aldehyde dehydrogenase 1. Methylthiazol tetrazolium assay was used to assess cellular proliferation. Finally, a Western blot assay was used to evaluate for inhibition of specific enzymes in these two signaling pathways. Using flow cytometry, we found that LCSC contain 64.4% CD133 + cells, 83.2% CD44 + cells, and 96.4% CD24 + cells. PKI-587 and sorafenib caused inhibiton of LCSC and HCC cell proliferation. PLC cells were more sensitive to PKI-587 than LCSC or Huh7 (P PKI-587 and sorafenib caused significantly more inhibition than monotherapy in HuH7, PLC, and LCSC. Using the methylthiazol tetrazolium assay, we found that the LCSC proliferation was inhibited with sorafenib monotherapy 39% at 5 μM (P PKI-587 at 0.1 μM (P = 0.002, n = 12) compared with control. The combination of PKI-587 and sorafenib, however, synergistically inhibited LCSC proliferation by 86% (P = 0.002; n = 12). LCSC (CD133+, CD44+, CD24+) were able to develop very aggressive tumors with low cell concentrations at 4 to 6 wk. Cells CD133+, CD44+, CD24+, which demonstrated at least moderate resistance to therapy in vitro. The combination of PKI-587 and sorafenib was better than either drug alone at inhibiting of LCSC and on HCC cell proliferation. Copyright © 2013 Elsevier Inc. All rights reserved.

  17. Evaluation of the antibacterial and anticancer activities of some South African medicinal plants

    Science.gov (United States)

    2011-01-01

    Background Several herbs are traditionally used in the treatment of a variety of ailments particularly in the rural areas of South Africa where herbal medicine is mainly the source of health care system. Many of these herbs have not been assessed for safety or toxicity to tissue or organs of the mammalian recipients. Methods This study evaluated the cytotoxicity of some medicinal plants used, inter alia, in the treatment of diarrhoea, and stomach disorders. Six selected medicinal plants were assessed for their antibacterial activities against ampicillin-resistant and kanamycin-resistant strains of Escherichia coli by the broth micro-dilution methods. The cytotoxicities of methanol extracts and fractions of the six selected plants were determined using a modified tetrazolium-based colorimetric assay (3-(4, 5-dimethylthiazol)-2, 5-diphenyl tetrazolium bromide (MTT) assay). Results The average minimum inhibitory concentration (MIC) values of the plants extracts ranged from 0.027 mg/mℓ to 2.5 mg/mℓ after 24 h of incubation. Eucomis autumnalis and Cyathula uncinulata had the most significant biological activity with the least MIC values. The in vitro cytotoxicity assay on human hepatocarcinoma cell line (Huh-7) revealed that the methanol extract of E. autumnalis had the strongest cytotoxicity with IC50 of 7.8 μg/mℓ. Ethyl acetate and butanol fractions of C. uncinulata, Hypoxis latifolia, E. autumnalis and Lantana camara had lower cytotoxic effects on the cancer cell lines tested with IC50 values ranging from 24.8 to 44.1 μg/mℓ; while all the fractions of Aloe arborescens and A. striatula had insignificant or no cytotoxic effects after 72 h of treatment. Conclusions Our results indicate that the methanol fraction of E. autumnalis had a profound cytotoxic effect even though it possessed very significant antibacterial activity. This puts a query on its safety and hence a call for caution in its usage, thus a product being natural is not tantamount to being

  18. Evaluation of the antibacterial and anticancer activities of some South African medicinal plants

    Directory of Open Access Journals (Sweden)

    Kambizi Learnmore

    2011-02-01

    Full Text Available Abstract Background Several herbs are traditionally used in the treatment of a variety of ailments particularly in the rural areas of South Africa where herbal medicine is mainly the source of health care system. Many of these herbs have not been assessed for safety or toxicity to tissue or organs of the mammalian recipients. Methods This study evaluated the cytotoxicity of some medicinal plants used, inter alia, in the treatment of diarrhoea, and stomach disorders. Six selected medicinal plants were assessed for their antibacterial activities against ampicillin-resistant and kanamycin-resistant strains of Escherichia coli by the broth micro-dilution methods. The cytotoxicities of methanol extracts and fractions of the six selected plants were determined using a modified tetrazolium-based colorimetric assay (3-(4, 5-dimethylthiazol-2, 5-diphenyl tetrazolium bromide (MTT assay. Results The average minimum inhibitory concentration (MIC values of the plants extracts ranged from 0.027 mg/mℓ to 2.5 mg/mℓ after 24 h of incubation. Eucomis autumnalis and Cyathula uncinulata had the most significant biological activity with the least MIC values. The in vitro cytotoxicity assay on human hepatocarcinoma cell line (Huh-7 revealed that the methanol extract of E. autumnalis had the strongest cytotoxicity with IC50 of 7.8 μg/mℓ. Ethyl acetate and butanol fractions of C. uncinulata, Hypoxis latifolia, E. autumnalis and Lantana camara had lower cytotoxic effects on the cancer cell lines tested with IC50 values ranging from 24.8 to 44.1 μg/mℓ; while all the fractions of Aloe arborescens and A. striatula had insignificant or no cytotoxic effects after 72 h of treatment. Conclusions Our results indicate that the methanol fraction of E. autumnalis had a profound cytotoxic effect even though it possessed very significant antibacterial activity. This puts a query on its safety and hence a call for caution in its usage, thus a product being natural is not

  19. Variable Efficacy of the Proteinaceous Antifungal YvgO in Select Fruit Juices and Teas as a Complement with UV Methods of Food Protection.

    Science.gov (United States)

    Manns, David C; Churey, John J; Worobo, Randy W

    2015-10-01

    Heat-resistant fungal spores present a processing challenge for beverages and fruit juices, as thermal and UV strategies are often inadequate in reducing heat-resistant fungal burdens to acceptable levels. While effective against pathogenic or invasive bacteria, germicidal UV light treatments also fail to achieve an appreciable reduction of heat-resistant fungal spores. As an alternative, the efficacy of the antifungal protein YvgO was examined across a selection of fruit juices and teas, as well as solid model matrices. Compared with its efficacy in analogous liquid matrices, the apparent efficacy of YvgO was diminished on acidified solid matrices due to a reduction in YvgO diffusion. Using an XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] tetrazolium dye cytotoxicity assay, the effective concentrations to reduce growth by 50% were elucidated in samples challenged with Byssochlamys fulva H25. The MICs were determined and ranged from 2 ppm in apple juice and acidified teas to approximately 3 to 12 ppm for lemonade and orange, white cranberry, blueberry, prune, cherry, and grape juices. Apple cider and nonacidified teas showed reduced efficacy, with MICs exceeding 100 ppm. Tannin-rich products readily removed YvgO from the product, impairing its efficacy. Adding bovine serum albumin as a competitive inhibitor effectively reversed the YvgO-tannin association and restored efficacy in black but not green tea matrices. When challenged with a 5-log CFU inoculum of B. fulva, the shelf lives of the products were extended for various times up to 28 days in a concentrationdependent manner. However, initial efficacy was not predictive of shelf life extension, as some products exhibited improved protection at just two- and fourfold concentrations above the MIC, while others only exhibited long-term stability when concentrations exceeded 20 times the MIC. As such, YvgO may be an attractive alternative to currently available protection strategies

  20. Viability of Macrotyloma axillare cv. Guatá seeds under chemical scarification

    Directory of Open Access Journals (Sweden)

    Suleize Rocha Terra

    2012-12-01

    Full Text Available The use of the associacion of grasses and legumes results in a good forage yield as well as a good animal performance due to maintenance of adequate levels of crude protein (CP in the animal diet, both the direct effect of the ingestion of legumes, or even through indirect effect of the introduction of nitrogen by legumes in the pasture, thereby increasing the production of forage mass. However, the seeds of legumines have the so-called hardness seeds or dormant according to their coats impermeable to water. The hardness of the seed prevents the germination of the same since these seeds have a hard shell impervious to water and gas exchange, not allowing the imbibition of water by the seed, or embryo oxygenation. This dormancy may be broken by various known methods such as scarification, promoting cracks in the seed coat allowing the entry of water therein. In the case of Macrotyloma axillare exposure of seeds to scarification method has proven to be an efficient way to break dormancy and promoting seed germination. The aim of the present work was to evaluate the viability of seeds after suffering dormancy by immersion seeds in sulfuric acid. The experiment to break dormancy Macrotyloma axillares cv. Guatá was conducted in the laboratory of Plant Physiology at the Intituto de Zootecnia in Nova Odessa, São Paulo State, using the seeds that were stored in a cold room with temperatures between 10-12 ºC, relative humidity (RH 30-33% and no light. The treatments studied were: control, chemical scarification with sulfuric acid for 5, 10, 15, 20, 30 and 40 minutes. We used a completely randomized design with two replications, being 100 seeds each repetition. After treatment the seeds were under to tetrazolium test resulting in viable seeds and seed viability. So the result of the tetrazolium test was obtained by average percentage of viable seeds, found in the repeats tested, respecting the maximum tolerances set out in Brazilian Rules for Seed

  1. Muscle biopsy in Pompe disease Biópsia muscular na doença de Pompe

    Directory of Open Access Journals (Sweden)

    Lineu Cesar Werneck

    2013-05-01

    Full Text Available Pompe disease (PD can be diagnosed by measuring alpha-glucosidase levels or by identifying mutations in the gene enzyme. Muscle biopsies can aid diagnosis in doubtful cases. Methods: A review of muscle biopsy from 19 cases of PD (infantile, 6 cases; childhood, 4 cases; and juvenile/adult, 9 cases. Results: Vacuoles with or without glycogen storage were found in 18 cases. All cases had increased acid phosphatase activity. The vacuole frequency varied (almost all fibers in the infantile form to only a few in the juvenile/adult form. Atrophy of type 1 and 2 fibers was frequent in all forms. Atrophic angular fibers in the NADH-tetrazolium reductase and nonspecific esterase activity were observed in 4/9 of the juvenile/adult cases. Conclusion: Increased acid phosphatase activity and vacuoles were the primary findings. Most vacuoles were filled with glycogen, and the adult form of the disease had fewer fibers with vacuoles than the infantile or childhood forms.O diagnóstico da doença de Pompe (PD pode ser feito pela dosagem da enzima alfa-glicosidase ou pela mutação do seu gene codificador. A biópsia muscular pode ajudar em casos duvidosos. Métodos: Revisão das biópsias musculares de 19 casos de PD (forma infantil, 6 casos; infantil tardia, 4; e juvenil/adulto, 9. Resultados: Encontrados vacúolos em 18 casos, com ou sem depósito de glicogênio. Todos mostraram aumento da fosfatase ácida. Os vacúolos estavam presentes na maioria das fibras nas formas infantis, menos frequentes nas formas juvenil e mais raros nas formas do adulto. A atrofia de fibras dos tipos 1 e 2 ocorreram em todas as formas. Fibras atróficas na NADH-tetrazolium redutase e esterase não específica foram observadas em 4/9 das formas infantil tardia/adulta. Conclusões: Os dados mais frequentes foram vacúolos, preenchidos por glicogênio com atividade aumentada da fosfatase ácida. A forma adulta apresenta menor número de vacúolos que as formas infantil e infantil

  2. Exposure to sub-inhibitory concentrations of gentamicin, ciprofloxacin and cefotaxime induces multidrug resistance and reactive oxygen species generation in meticillin-sensitive Staphylococcus aureus.

    Science.gov (United States)

    Bhattacharya, Gargi; Dey, Diganta; Das, Satadal; Banerjee, Abhijit

    2017-06-01

    The role of antibiotics below their MIC in the development of bacterial drug resistance is becoming increasingly important. We investigated the effect of sub-MICs of bactericidal antibiotics on the susceptibility pattern of Staphylococcus aureus and evaluated the role of free radicals. A total of 12 S. aureus strains were recovered from pus samples and their antibiograms determined. The test isolates were treated with sub-MIC levels of tetracycline, gentamicin, ciprofloxacin and cefotaxime. Alterations in their respective breakpoints were observed along with measurements of free radical generation by nitro blue tetrazolium test.Results/Key findings. Gentamicin, ciprofloxacin and cefotaxime exposure significantly altered the breakpoints of exposed isolates against several tested antibiotics and higher levels of free radicals were generated after antibiotic exposure. Our study demonstrates that sub-MIC levels of antimicrobials can lead to resistance and cross-resistance across several classes of antibiotics in wild strains of S. aureus, possibly by free radical production. The molecular mechanisms behind the acquisition of drug resistance at low antibiotic concentrations and the specific target genes of reactive oxygen speciesneed to be explored further.

  3. Assessment of phagocytic activity of neutrophils in chronic obstructive pulmonary disease

    Directory of Open Access Journals (Sweden)

    Lalitha Shanmugam

    2015-01-01

    Full Text Available Aim: To assess the phagocytic activity of neutrophils in subjects with chronic obstructive pulmonary disease (COPD. Background/Need of Study: There is a paucity of data in relation to phagocytic function in COPD. By this multidisciplinary study, a better understanding about the etiology of lung destruction among COPD patients is being sought. Materials and Methods: The study was conducted among 28 subjects with COPD and 25 controls in a private tertiary hospital in Chennai after obtaining Institutional Ethical Clearance. Known cases of COPD as proven by clinical findings and spirometry were included in the study, and subjects with any other source of infection, recent surgery, or chronic granulomatous disease were excluded. The study subjects were divided into three groups based on the severity of COPD as determined by spirometry, and healthy volunteers were taken as Group 4. After obtaining informed consent, validated respiratory health questionnaire was administered. The phagocytic function was assessed by Candida phagocytic test and Nitroblue Tetrazolium (NBT Reduction Test. Results: Significantly impaired phagocytic function as indicated by lower phagocytic, lytic indices and decreased NBT reduction of neutrophils was seen in COPD subjects compared to normal healthy controls (P <.001. Conclusion: This study showed that there is phagocytic dysfunction in COPD subjects when compared with normal subjects. This could be due to underlying inflammation in human airway. Understanding the role of neutrophils may lead to improved understanding of the pathogenesis of COPD, which in turn may pave way for implementing modified therapeutic intervention strategies.

  4. Osteogenic effect of inflammatory macrophages loaded onto mineral bone substitute in subcutaneous implants

    Directory of Open Access Journals (Sweden)

    Živković Jelena M.

    2015-01-01

    Full Text Available We analyzed the influence of inflammatory macrophages on the osteogenic process in subcutaneous implants composed of mineral bone substitute. Thioglycollate-elicited peritoneal macrophages (TEPMs were characterized as inflammatory. This was confirmed microscopically by the nitroblue tetrazolium (NBT test and the production of tumor necrosis factor α (TNF-α. The implants (M-type were made of mineral bone substitute (Bio-Oss® mixed with TEPMs and blood clot. Implants without macrophages served as the control (C-type. Subcutaneous implantation in the interscapular area was performed on BALB/c mice. Implants were extracted after 2 and 8 weeks. In M-type implants, phagocytosis and angiogenesis were more pronounced, and osteoblast-like cells aligned onto granules of implanted material and osteoid structures can be seen. The observed higher osteocalcin and lower osteopontin immunoexpression in M-type implants when compared to the control after 8 weeks suggest a more advanced osteogenic process. Our results indicate that the presence of inflammatory macrophages in the composition of an implant may have a beneficial effect on the osteogenic process. [Projekat Ministarstva nauke Republike Srbije, br. III41017

  5. Night Light-Adaptation Strategies for Photosynthetic Apparatus in Yellow-Poplar (Liriodendron tulipifera L. Exposed to Artificial Night Lighting

    Directory of Open Access Journals (Sweden)

    Myeong Ja Kwak

    2018-02-01

    Full Text Available Plants can undergo external fluctuations in the natural light and dark cycle. The photosynthetic apparatus needs to operate in an appropriate manner to fluctuating environmental factors, especially in light. Yellow-poplar seedlings were exposed to nighttime artificial high-pressure sodium (HPS lighting to evaluate night light-adaptation strategies for photosynthetic apparatus fitness relative to pigment contents, photosystem II photochemistry, photosynthetic parameters, histochemical analysis of reactive oxygen species, and plant biomass. As a result, seedlings exhibited dynamic changes including the enhancement of accessory pigments, the reduction of photosystem II photochemistry, increased stomatal limitation, downregulation of photosynthesis, and the decreased aboveground and belowground biomass under artificial night lighting. Histochemical analysis with 3,3′-diaminobenzidine (DAB and nitroblue tetrazolium (NBT staining indicates the accumulation of in situ superoxide radicals (O2− and hydrogen peroxide (H2O2 in leaves exposed to the lowest level of artificial night lighting compared to control. Moreover, these leaves exposed to artificial night lighting had a lower nighttime respiration rate. These results indicated that HPS lighting during the night may act as a major factor as repressors of the fitness of photosynthesis and growth patterns, via a modification of the photosynthetic light harvesting apparatus.

  6. Simultaneous Detection of Multiple Fish Pathogens Using a Naked-Eye Readable DNA Microarray

    Directory of Open Access Journals (Sweden)

    King-Jung Lin

    2012-02-01

    Full Text Available We coupled 16S rDNA PCR and DNA hybridization technology to construct a microarray for simultaneous detection and discrimination of eight fish pathogens (Aeromonas hydrophila, Edwardsiella tarda, Flavobacterium columnare, Lactococcus garvieae, Photobacterium damselae, Pseudomonas anguilliseptica, Streptococcus iniae and Vibrio anguillarum commonly encountered in aquaculture. The array comprised short oligonucleotide probes (30 mer complementary to the polymorphic regions of 16S rRNA genes for the target pathogens. Targets annealed to the microarray probes were reacted with streptavidin-conjugated alkaline phosphatase and nitro blue tetrazolium/5-bromo-4-chloro-3′-indolylphosphate, p-toluidine salt (NBT/BCIP, resulting in blue spots that are easily visualized by the naked eye. Testing was performed against a total of 168 bacterial strains, i.e., 26 representative collection strains, 81 isolates of target fish pathogens, and 61 ecologically or phylogenetically related strains. The results showed that each probe consistently identified its corresponding target strain with 100% specificity. The detection limit of the microarray was estimated to be in the range of 1 pg for genomic DNA and 103 CFU/mL for pure pathogen cultures. These high specificity and sensitivity results demonstrate the feasibility of using DNA microarrays in the diagnostic detection of fish pathogens.

  7. ROS Accumulation and TTC Reduction in Growing Embryo of Crithmum maritimum L. Isolated from Water or Salt Imbibed Seeds

    Directory of Open Access Journals (Sweden)

    Abdallah ATIA

    2011-05-01

    Full Text Available The salinity induced inhibition of seeds germination remains not clear at physiological levels. The aims of this study is to investigate the effect of salt on germination, embryo growth, superoxide anion radical (O2·- and the respiratory activity (TTC reduction in Crithmum maritimum L. seeds. Thus the embryo growth, in situ localization of respiratory activity and superoxide anion radical (O2·- localization, were investigated. Chlorure 2, 3, 5-triphényltétrazolium (TTC reduction test and superoxide anion radical (O2·- localization with Nitroblue Tetrazolium Chloride (NBT were performed in embryo isolated from seeds of the halophyte Crithmum maritimum L either sown in distilled water or in 200 mM NaCl. The key results show that germination was maximal (90 % in distilled water, but was fully inhibited following seed exposure to NaCl. The completion of the embryo growth (ca. 2 mm length leading to the radicle emergence took 6 d in H2O, but was markedly delayed by salt. NaCl reduced the elongation zone in the embryo axis, hence indicating that the cell division and/or cell elongation were disturbed by salinity. The respiratory activity (TTC reduction and O2·- production in the cotyledon were significantly lowered by salinity.

  8. In vitro activity of antiamoebic drugs against clinical isolates of Entamoeba histolytica and Entamoeba dispar

    Directory of Open Access Journals (Sweden)

    Mahajan Ramesh

    2004-12-01

    Full Text Available Abstract Background Amoebiasis is a major public health problem in tropical and subtropical countries. Although a number of antiamoebic agents are used for its treatment, yet the susceptibility data on clinical isolates of Entamoeba histolytica and Entamoeba dispar are not available. Therefore, the present study was aimed to assess the in vitro susceptibility of clinical isolates of E. histolytica and E. dispar to metronidazole, chloroquine, emetine and tinidazole. Methods A total of 45 clinical isolates (15 E. histolytica and 30 E. dispar were maintained in polyxenic cultures followed by monoxenic cultures. In vitro drug sensitivity (IC50 of clinical isolates and standard reference strain of E. histolytica (HM1: IMSS was assessed by nitro blue tetrazolium (NBT reduction assay after exposure to various concentrations of each drug. Results The results showed that all clinical isolates had a higher IC50 compared to reference strain to all the four drugs. E. histolytica isolates appeared to be more susceptible [IC50 (μm 13.2,26.3,31.2 and 12.4] compared to E. dispar isolates [IC50(μm 15.6,28.9,32.8 and 13.2] and the reference strain of E. histolytica [IC50 (��m 9.5, 15.5, 29.9 and 10.2] to the metronidazole, chloroquine, emetine and tinidazole respectively. Conclusions The results indicate that till date, Entamoeba isolates in India do not seem to be resistant to the commonly used antiamoebic drugs.

  9. Sickle cell anaemia and the NBT test

    Science.gov (United States)

    Walters, Thomas R.; Reddy, B. Narasimha

    1974-01-01

    Patients with sickle cell anaemia have an increased susceptibility to bacterial infections Previous reports of false-negative nitro blue tetrazolium (NBT) tests in the presence of bacteria infection and of a faulty phagocytic response following stimulation in vitro have suggested the possibility of polymorphonuclear dysfunction in certain patients with sickle cell anaemia. In the present study an unstimulated, histochemical NBT technique was used to evaluate the test in patients with sickle cell anaemia. There was a significant difference between the results in the group of patients with infection (mean NBT-positive cells 42·7%) compared to those without infection (mean 9·4%). There was no significant correlation between the total white blood cell count, absolute number of polymorphonuclear cells, and infectious complications. These findings indicate an appropriate polymorphonuclear cell response, as evaluated by the NBT test, in patients with sickle cell anaemia and bacterial infection. The NBT test may be used as an additional parameter in the differentiation of those patients with sickle cell anaemia with bacterial infection. PMID:4426971

  10. The effects of polyanions on NBT Reductions hexose monophosphate shunt activity, and ultrastructure of polymorphonuclear leukocytes.

    Science.gov (United States)

    Czarnetzki, B M; Cowan, D H; Belcher, R W

    1975-07-01

    Heparin causes enhanced nitroblue tetrazolium (NBT) reduction by polymorphonuclear leukocytes (PMN's). To determine the mechanism of this stimulation, samples of 1 to 3 x 10(7) PMN's were incubated with various concentrations of heparin, chondroitin sulfate A (CSA), and chondroitin sulfate B (CSB), with and without NBT. The effect of the polyanions (PA) on PMN hexose monophosphate shunt (HMPS) activity was determined by the production of 14CO2 from glucose-1-14C by the leukocytes. NBT reduction was evaluated histochemically and spectrophotometrically at 515 mmu. Samples of PMN's in heparin and heparin-NBT mixtures were examined by electron microscopy after various incubation periods. Increased NBT reductions by PMN's was found when leukocytes were incubated with heparin, CSA, and CSB, but these compounds had no effect on the HMPS activity of PMN's unless NBT was added. Electron microscopy of samples that contained heparin-NBT revealed an insoluble complex that was phagocytosed by the leukocytes. The stimulation of PMN oxidative metabolism and NBT reduction that follows incubation with PA-NBT appears to be directly related to ingestion of this particulate complex by the leukocytes.

  11. NBT reactivity correlates to the distribution of PMNs between rat pulmonary and systemic circulation.

    Science.gov (United States)

    Wikström, T; Braide, M; Bagge, U; Risberg, B

    1995-10-01

    The rat systemic and pulmonary vascular transit times of radiolabeled polymorphonuclear granulocytes (experimental population referred to as "Polys") and mononuclear leukocytes (experimental population referred to as "Monos") (111In) in relation to those of erythrocytes (51Cr) were measured under physiological conditions. Results were also expressed as "circulating" pool and "marginated" pool of the two leukocyte fractions, and it was investigated whether the degree of spontaneous granulocyte activation, measured with the nitro blue tetrazolium (NBT) reduction test, was of importance for the retention of Polys in the lungs. The measured mean vascular transit was similar for Monos and Polys and several times slower than that of the erythrocytes in the pulmonary (17.4 times) and the systemic (4.1 times) vascular beds. The percentage of NBT-positive cells was NBT-positive cells correlated with a redistribution of granulocytes from the systemic marginated cell pool to the pulmonary marginated cell pool. Microscopic evaluation of sections from embedded lung tissue biopsies, obtained after intravital staining of the leukocytes, confirmed the isotope data on pulmonary transit.

  12. Comparative study of NBT reduction method for estimation of glycated protein (serum fructoseamine) with glycated HbA1c estimated on DCA 2000+Analyzer (immunoagglutination inhibition).

    Science.gov (United States)

    Sahu, Ashok; Sarkar, Purnima Dey

    2008-01-01

    Glycated protein estimation is a diagnostic tool, used for the long term and short term monitoring of the glycemic status of diabetic patients. The present study is designed to compare and correlate modified NBT reduction method for the estimation of Glycated protein (serum fructosamine) with HbAlc estimated on DCA+2000 Analyzer. Glycated protein (serum fructosamine) reduces Nitro Blue Tetrazolium (NBT) reagent in alkaline medium to tetrazinolyl radical NBT+ which forms a highly colored monoformazen compound, absorbance of which is directly proportional to the concentration of glycated protein (serum fructosamine) present in the sample and is recorded as delta A/min. The results of modified NBT were then compared with HbA1c estimated by immunoagglutination inhibition method. Correlation coefficient between HbAlc with serum fructosamine was found to be r = 0.739 using Schimadzu CL-750 spectrophotometer and r = 0.731 using colorimeter. Results of this study were found to be statistically significant P < 0.001. Hence this method could be used for routine monitoring of blood glucose control in diabetics as HbA1c estimation.

  13. Re-evaluation of superoxide scavenging capacity of xanthohumol.

    Science.gov (United States)

    Schempp, Harald; Vogel, Susanne; Hückelhoven, Ralph; Heilmann, Jörg

    2010-12-01

    The chemopreventive chalcone xanthohumol (Xh) has been reported to decrease xanthine oxidase (XOD) catalysed formation of formazan from nitroblue tetrazolium (NBT) and is discussed as a potent scavenger of superoxide. Re-evaluation of the scavenging capacity indicated that Xh disturbed detection of superoxide with NBT, in case of an insufficient NBT/Xh ratio. Xh lacked superoxide scavenging activity in contrast to the Xh-derivative 3'-hydroxy-Xh with catechol substructure, used as positive control. This was shown by the use of sufficient concentration of NBT and other detectors such as hydroxylamine, XTT, cytochrome c and hydroethidine. HPLC analysis of reaction products in a xanthine/XOD/peroxidase system demonstrated beside enhanced inhibition of NBT-formazan by Xh that NBT even prevented oxidation of Xh. p-coumaric acid or ferulic acid could replace Xh in that system, indicating that superoxide detection using NBT is likely jeopardized by interference of phenoxyl-radicals. Furthermore, this study provides evidence that Xh can moderately generate superoxide via auto-oxidation.

  14. Reactive Oxygen Species Play a Role in the Infection of the Necrotrophic Fungi, Rhizoctonia solani in Wheat.

    Directory of Open Access Journals (Sweden)

    Rhonda C Foley

    Full Text Available Rhizoctonia solani is a nectrotrophic fungal pathogen that causes billions of dollars of damage to agriculture worldwide and infects a broad host range including wheat, rice, potato and legumes. In this study we identify wheat genes that are differentially expressed in response to the R. solani isolate, AG8, using microarray technology. A significant number of wheat genes identified in this screen were involved in reactive oxygen species (ROS production and redox regulation. Levels of ROS species were increased in wheat root tissue following R. solani infection as determined by Nitro Blue Tetrazolium (NBT, 3,3'-diaminobenzidine (DAB and titanium sulphate measurements. Pathogen/ROS related genes from R. solani were also tested for expression patterns upon wheat infection. TmpL, a R. solani gene homologous to a gene associated with ROS regulation in Alternaria brassicicola, and OAH, a R. solani gene homologous to oxaloacetate acetylhydrolase which has been shown to produce oxalic acid in Sclerotinia sclerotiorum, were highly induced in R. solani when infecting wheat. We speculate that the interplay between the wheat and R. solani ROS generating proteins may be important for determining the outcome of the wheat/R. solani interaction.

  15. Oral intake of zirconia nanoparticle alters neuronal development and behaviour of Drosophila melanogaster

    Science.gov (United States)

    Mishra, Monalisa; Sabat, Debabrat; Ekka, Basanti; Sahu, Swetapadma; P, Unnikannan; Dash, Priyabrat

    2017-08-01

    Zirconia nanoparticles (ZrO2 NPs) have been extensively used in teeth and bone implants and thus get a chance to interact with the physiological system. The current study investigated the oral administration of various concentrations of ZrO2 NPs synthesized by the hydrothermal method (0.25 to 5.0 mg L-1) on Drosophila physiology and behaviour. The size of the currently studied nanoparticle varies from 10 to 12 nm. ZrO2 NPs accumulated within the gut in a concentration-dependent manner and generate reactive oxygen species (ROS) only at 2.5 and 5.0 mg L-1 concentrations. ROS was detected by nitroblue tetrazolium (NBT) assay and 2',7'-dichlorofluorescein http://www.ncbi.nlm.nih.gov/pubmed/20370560 (H2DCF) staining. The ROS toxicity alters the larval gut structure as revealed by DAPI staining. The NP stress of larvae affects the Drosophila development by distressing pupa count and varying the phenotypic changes in sensory organs (eye, thorax bristle, wings). Besides phenotypic changes, flawed climbing behaviour against gravity was seen in ZrO2 NP-treated flies. All together, for the first time, we have reported that a ROS-mediated ZrO2 NP toxicity alters neuronal development and functioning using Drosophila as a model organism. [Figure not available: see fulltext.

  16. Novel radio-chromic solution dosimeter for radiotherapy treatment planning.

    Science.gov (United States)

    Rabaeh, Khalid A; Moussa, Akram A; Basfar, Ahmed A; Msalam, Rashed I

    2013-06-01

    Nitro blue tetrazolium (NBT) solution dosimeters were prepared and investigated based on radiation-induced reduction of NBT(2+). NBT solution dosimeters containing different concentrations of NBT dye from 1 to 5 mM were prepared in a solution of ethanol. The dosimeters were irradiated with 6 MV X-ray beam at doses up to 30 Gy. The dose sensitivity of NBT solution increases strongly with increase of concentrations of NBT dye. The dose response of NBT dosimeters increases remarkably by addition of various concentrations of sodium formate (0.5, 2.5 and 5 mM). It becomes more remarkable with increasing pH value of NBT-sodium formate dosimeters. The sensitivity of the solution increased fairly with increase of irradiation temperature, therefore, the response of the solutions has to be corrected under actual processing conditions. The stability of solution dosimeters after irradiation was very high up to 30 days. Copyright © 2012 Associazione Italiana di Fisica Medica. Published by Elsevier Ltd. All rights reserved.

  17. The Critical Role of Redox Homeostasis in Shikonin-Induced HL-60 Cell Differentiation via Unique Modulation of the Nrf2/ARE Pathway

    Directory of Open Access Journals (Sweden)

    Bo Zhang

    2012-01-01

    Full Text Available Among various cancer cell lines, the leukemia cell line HL-60 was most sensitive to Shikonin, with evidence showing both the prooxidative activities and proapoptotic effects of micromolar concentrations of Shikonin. However, the mechanism involved in the cytotoxicity of Shikonin in the submicromolar range has not been fully characterized. Using biochemical and free radical biological experiments in vitro, we identified the prodifferentiated profiles of Shikonin and evaluated the redox homeostasis during HL-60 differentiation. The data showed a strong dose-response relationship between Shikonin exposure and the characteristics of HL-60 differentiation in terms of morphology changes, nitroblue tetrazolium (NBT reductive activity, and the expression level of surface antigens CD11b/CD14. During drug exposure, intercellular redox homeostasis changes towards oxidation are necessary to support Shikonin-induced differentiation, which was proven by additional enzymatic and non-enzymatic redox modulators. A statistically significant and dose-dependent increase (P<0.05 was recorded with regard to the unique expression levels of the Nrf2/ARE downstream target genes in HL-60 cells undergoing late differentiation, which were restored with further antioxidants employed with the Shikonin treatment. Our research demonstrated that Shikonin is a differentiation-inducing agent, and its mechanisms involve the Nrf2/ARE pathway to modulate the intercellular redox homeostasis, thus facilitating differentiation.

  18. The effect of heparin and EDTA on the NBT test.

    Science.gov (United States)

    Rothwell, D J; Doumas, B T

    1975-06-01

    Heparin precipitates nitroblue tetrazolium (NBT) in the absence of protein (plasma). This NBT-heparin precipitate, when presented to neutrophils, results in NBT scores higher than those obtained with NBT in solution. It is postulated that the high NBT scores obtained with increasing heparin concentrations are due to increased NBT precipitation and hence increased phagocytosis. It is proposed that the variability of NBT scores obtained when heparin is used as the anticoagulant is related to the amount of precipitate formed and to inhibition of phagocytosis by excess heparin. EDTA, in the presence of plasma and NBT, forms a precipitate consisting of protein and little, if any, NBT. In the presence of EDTA, NBT scores are lower than those obtained with heparin. EDTA, at concentrations used to prevent coagulation, inhibits phagocytosis and this could explain the low NBT scores. These observations provide a mechanism for entry of NBT dye into neutrophils and may help explain the inconsistent results found in both normal and disease states.

  19. Quantitative determination of superoxide in plant leaves using a modified NBT staining method.

    Science.gov (United States)

    Bournonville, Carlos F Grellet; Díaz-Ricci, Juan Carlos

    2011-01-01

    In plants, the ROS (reactive oxygen species) level is tightly regulated because their accumulation produces irreversible damage leading to cell death. However, ROS accumulation plays a key role in plant signaling under biotic or abiotic stress. Although various methods were reported to evaluate ROS accumulation, they are restricted to model plants or provide only qualitative information. Develop a simple method to quantify superoxide radicals produced in plant tissues, based on the selective extraction of the formazan produced after nitroblue tetrazolium (NBT) reduction in histochemical staining. Plant leaves were stained with a standard NBT method and the formazan precipitated in tissues was selectively extracted using chloroform. The organic phase was dried and formazan residue dissolved in dimethylsulfoxide-potassium hydroxide and quantified by spectrophotometry. The method was tested in strawberry plant leaves under different stressing conditions. Formazan extracted from leaves subjected to stress conditions showed similar absorption spectra to those obtained from standard solutions using pure formazan. Calibration curves showed a linear relationship between absorbance and formazan amounts, within the range 0.5-8 µg. Outcomes suggested that formazan was retained in the solid residue of leaf tissues. This protocol allowed us to quantify superoxide radicals produced under different stress conditions. Chloroform allowed a selective formazan extraction and removal of potential endogenous, exogenous or procedural artefacts that may interfere with the quantitative determination. This protocol can be used to quantify the superoxide produced in plant tissues using any traditional qualitative NBT histochemical staining method. Copyright © 2011 John Wiley & Sons, Ltd.

  20. Biocompatibility and Corrosion Protection Behaviour of Hydroxyapatite Sol-Gel-Derived Coatings on Ti6Al4V Alloy

    Directory of Open Access Journals (Sweden)

    Amir A. El Hadad

    2017-01-01

    Full Text Available The aim of this work was to prepare hydroxyapatite coatings (HAp by a sol-gel method on Ti6Al4V alloy and to study the bioactivity, biocompatibility and corrosion protection behaviour of these coatings in presence of simulated body fluids (SBFs. Thermogravimetric/Differential Thermal Analyses (TG/DTA and X-ray Diffraction (XRD have been applied to obtain information about the phase transformations, mass loss, identification of the phases developed, crystallite size and degree of crystallinity of the obtained HAp powders. Fourier Transformer Infrared Spectroscopy (FTIR has been utilized for studying the functional groups of the prepared structures. The surface morphology of the resulting HAp coatings was studied by Scanning Electron Microscopy (SEM. The bioactivity was evaluated by soaking the HAp-coatings/Ti6Al4V system in Kokubo’s Simulated Body Fluid (SBF applying Inductively Coupled Plasma (ICP spectrometry. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT and Alamar blue cell viability assays were used to study the biocompatibility. Finally, the corrosion behaviour of HAp-coatings/Ti6Al4V system was researched by means of Electrochemical Impedance Spectroscopy (EIS. The obtained results showed that the prepared powders were nanocrystalline HAp with little deviations from that present in the human bone. All the prepared HAp coatings deposited on Ti6Al4V showed well-behaved biocompatibility, good bioactivity and corrosion protection properties.

  1. Meloxicam in the treatment of in vitro and in vivo models of urinary bladder cancer.

    Science.gov (United States)

    Arantes-Rodrigues, Regina; Pinto-Leite, Rosário; Ferreira, Rita; Neuparth, Maria João; Pires, Maria João; Gaivão, Isabel; Palmeira, Carlos; Santos, Lúcio; Colaço, Aura; Oliveira, Paula

    2013-05-01

    To assess the efficacy of meloxicam, a non-steroidal anti-inflammatory drug (NSAID), on three human urinary bladder-cancer cell lines (HT1376, T24 and 5637) and on mice urinary bladder cancer chemically induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). The in vitro effects of meloxicam were assessed by optical microscopy, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method, flow cytometry and comet assay. In vivo, Hsd:ICR male mice were exposed to BBN in drinking water, over the course of 12 weeks. Subsequently, animals were treated with meloxicam by intraperitoneal route, for 6 consecutively weeks. Tumour development was evaluated by haematoxylin and eosin staining. Renal and hepatic functions, interleucin-6 (IL-6), C-reactive protein (CRP) and tumour necrosis factor (TNFα) were also evaluated. In vitro, meloxicam induced a significant (Pmeloxicam-treated cells. In vivo, the incidence of pre-neoplastic lesions induced by BBN was not affected by meloxicam treatment. However, although not statistically significant, the development of neoplastic lesions was inhibited by meloxicam treatment without significant alterations of renal or hepatic parameters. Meloxicam is effective on in vitro and in vivo models of urinary bladder cancer. These findings support that meloxicam deserves more attention on urinary bladder cancer study. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  2. Fermented Chinese Formula Shuan-Tong-Ling Protects Brain Microvascular Endothelial Cells against Oxidative Stress Injury

    Directory of Open Access Journals (Sweden)

    Lingjing Tan

    2016-01-01

    Full Text Available Fermented Chinese formula Shuan-Tong-Ling (STL, composed of fourteen medicinal herbs, was an experiential formula by Dr. Zhigang Mei for treating vascular encephalopathy, but the underlying mechanisms remained unknown. In this study, we aimed to investigate the protective effects of fermented STL on hydrogen peroxide- (H2O2- induced injury in rat brain microvascular endothelial cells (BMECs and the possible mechanisms. Cultured BMECs were treated with H2O2, STL, or nicotinamide (NAM, a SIRT1 inhibitor. Then, 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl-2H-tetrazolium bromide (MTT assay was employed to detect cell proliferation and senescence-associated beta-galactosidase (SA-β-gal was used to examine cell senescence. Cell nuclei were observed by 4′,6-diamidino-2-phenylindole. Additionally, changes in reactive oxygen species (ROS, superoxide dismutase (SOD, and glutathione (GSH levels were measured. Expression of SIRT1, p21, and PGC-1α was determined by western blot. Cell proliferation significantly increased with STL treatment in a dose-dependent manner. H2O2 treatment could intensify cell senescence and nuclei splitting or pyknosis. With STL treatment, the reduced ROS level was accompanied by increased SOD and GSH activity. Further assays showed upregulation of SIRT1 and PGC-1α and downregulation of p21 after STL treatment. The results revealed that STL could protect BMECs against oxidative stress injury at least partially through the SIRT1 pathway.

  3. Exploring Bioactive Properties of Marine Cyanobacteria Isolated from the Portuguese Coast: High Potential as a Source of Anticancer Compounds

    Directory of Open Access Journals (Sweden)

    Margarida Costa

    2013-12-01

    Full Text Available The oceans remain a major source of natural compounds with potential in pharmacology. In particular, during the last few decades, marine cyanobacteria have been in focus as producers of interesting bioactive compounds, especially for the treatment of cancer. In this study, the anticancer potential of extracts from twenty eight marine cyanobacteria strains, belonging to the underexplored picoplanktonic genera, Cyanobium, Synechocystis and Synechococcus, and the filamentous genera, Nodosilinea, Leptolyngbya, Pseudanabaena and Romeria, were assessed in eight human tumor cell lines. First, a crude extract was obtained by dichloromethane:methanol extraction, and from it, three fractions were separated in a Si column chromatography. The crude extract and fractions were tested in eight human cancer cell lines for cell viability/toxicity, accessed with the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT and lactic dehydrogenase release (LDH assays. Eight point nine percent of the strains revealed strong cytotoxicity; 17.8% showed moderate cytotoxicity, and 14.3% assays showed low toxicity. The results obtained revealed that the studied genera of marine cyanobacteria are a promising source of novel compounds with potential anticancer activity and highlight the interest in also exploring the smaller filamentous and picoplanktonic genera of cyanobacteria.

  4. [Change of autophagy in endplate chondrocytes of rats during aging process].

    Science.gov (United States)

    Yu, Yun-fei; Xu, Hong-guang; Wang, Hong; Zhang, Wei; Xiong, Shou-liang; Zhang, Min

    2013-12-03

    To explore the expression and significance of autophagy in endplate cartilage of rats during aging process. The end-plate chondrocytes were isolated from 3, 6 and 12-month SD rats respectively. And the natural culture and rapamycin groups were assigned. Alizarin red staining was used to observe the morphological changes of cells. And RT-PCR was employed to detect the expressions of type II collagen, proteoglycan, SOX-9 and matrix metalloproteinase (MMP-13). The expressions of Beclin-I and LC3 were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The rate of autophagy was observed by monodansylcadaverine (MDC) staining and methyl thiazolyl tetrazolium (MTT) for cell survival rate. Alizarin red staining showed that cells might reflect the process of intervertebral disc degeneration. The expressions of polysaccharide, Sox-9, type II collagen, Beclin-1 and LC3 in endplate chondrocytes significantly decreased with advancing age (P aging process, the expressions of autophagy related-gene LC3 and Beclin-1 significantly decrease with the reduced activity of end-plate chondrocyte. And autophagy activity may be correlated with the development and degeneration of intervertebral disc.

  5. Microstructure, microhardness, and biocompatibility characteristics of yttrium hydroxyapatite doped with fluoride.

    Science.gov (United States)

    Toker, S M; Tezcaner, A; Evis, Z

    2011-02-01

    The current study focused on doping of hydroxyapatite (HA) with constant yttrium (Y(3+) ) and varying fluoride (F(-) ) compositions to investigate its microstructure, microhardness, and biocompatibility. HA was synthesized by precipitation method and sintered at 1100°C for 1 h. Y(3+) and F(-) ion dopings resulted in changes in densities. In x-ray diffraction analysis, no secondary phase formation was observed. Lattice parameters decreased upon ion substitutions. Scanning electron microscopy (SEM) results showed that ion addition resulted in smaller grains. In Fourier transform infrared spectroscopy analysis, F(-) ion substitution was confirmed. HA doped with 2.5% Y(3+) and 1% F(-) exhibited the highest microhardness. Y(3+) and F(-) ions improved Saos-2 cell proliferation on discs in Methylthiazolyldiphenyl-tetrazolium (MTT) assay. In SEM analysis, cells attached and proliferated on all disc surfaces. Alkaline phosphatase (ALP) assay showed that cell differentiation on the discs was improved by doping HA with an optimum F(-) amount. Dissolution tests revealed that structural stability of HA was improved with F(-) ion incorporation. The dissolution behavior of fluoridated samples exhibited a parallel pattern with the cell proliferation and differentiation behavior on these samples. Overall, this work shows that fluoride and yttrium cosubstitution into HA HA2.5Y1F was the most promising material for biomedical applications. Copyright © 2010 Wiley Periodicals, Inc.

  6. Properties and in vitro biological evaluation of nano-hydroxyapatite/chitosan membranes for bone guided regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Cheng Xianmiao [Research Center for Nano-Biomaterials, Analytical and Testing Center, Sichuan University, 610064 Chengdu (China); Department of Chemistry, Sichuan University, 610064 Chengdu (China); Li Yubao [Research Center for Nano-Biomaterials, Analytical and Testing Center, Sichuan University, 610064 Chengdu (China)], E-mail: nic7504@scu.edu.cn; Zuo Yi; Zhang Li [Research Center for Nano-Biomaterials, Analytical and Testing Center, Sichuan University, 610064 Chengdu (China); Li Jidong [Research Center for Nano-Biomaterials, Analytical and Testing Center, Sichuan University, 610064 Chengdu (China); Department of Chemistry, Sichuan University, 610064 Chengdu (China); Wang Huanan [Research Center for Nano-Biomaterials, Analytical and Testing Center, Sichuan University, 610064 Chengdu (China)

    2009-01-01

    Nano-hydroxyapatite(n-HA)/chitosan(CS) composite membranes were prepared by solvent casting and evaporation methods for the function of guided bone regeneration (GBR). The effect of n-HA content and solvent evaporation temperature on the properties of the composite membranes was studied. The n-HA/CS membranes were analyzed by scanning electron microscopy, Fourier transformed infrared spectroscopy, X-ray diffraction, swelling measurement, mechanical test, cell culture and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenylte-2H-tetrazolium bromide) assay. The results show that the surface roughness and micropores of the composite membranes increase with the rise of n-HA content, suitable for adhesion, crawl and growth of cells. The hydroxyapatite holds nano size and distributes uniformly in the composite membranes. Chemical bond interaction exists between Ca ions and -OH groups of n-HA and -NH{sub 2} or -OH groups of CS. The n-HA content and solvent evaporation temperature have obvious influence on the swelling ratio, tensile strength and elongation rate of the composite membranes. Cell culture and MTT assays show that n-HA and its content can affect the proliferation of cells. The n-HA/CS composite membranes have no negative effect on the cell morphology, viability and proliferation and possess good biocompatibility. This study makes the n-HA/CS composite membrane be a prospective biodegradable GBR membrane for future applications.

  7. Aquaporin 9 in rat brain after severe traumatic brain injury

    Directory of Open Access Journals (Sweden)

    Hui Liu

    2012-03-01

    Full Text Available OBJECTIVE: To reveal the expression and possible roles of aquaporin 9 (AQP9 in rat brain, after severe traumatic brain injury (TBI. METHODS: Brain water content (BWC, tetrazolium chloride staining, Evans blue staining, immunohistochemistry (IHC, immunofluorescence (IF, western blot, and real-time polymerase chain reaction were used. RESULTS: The BWC reached the first and second (highest peaks at 6 and 72 hours, and the blood brain barrier (BBB was severely destroyed at six hours after the TBI. The worst brain ischemia occurred at 72 hours after TBI. Widespread AQP9-positive astrocytes and neurons in the hypothalamus were detected by means of IHC and IF after TBI. The abundance of AQP9 and its mRNA increased after TBI and reached two peaks at 6 and 72 hours, respectively, after TBI. CONCLUSIONS: Increased AQP9 might contribute to clearance of excess water and lactate in the early stage of TBI. Widespread AQP9-positive astrocytes might help lactate move into neurons and result in cellular brain edema in the later stage of TBI. AQP9-positive neurons suggest that AQP9 plays a role in energy balance after TBI.

  8. Effect of noble gases on oxygen and glucose deprived injury in human tubular kidney cells.

    Science.gov (United States)

    Rizvi, Maleeha; Jawad, Noorulhuda; Li, Yuantao; Vizcaychipi, Marcela P; Maze, Mervyn; Ma, Daqing

    2010-07-01

    The noble gas xenon has been shown to be protective in preconditioning settings against renal ischemic injury. The aims of this study were to determine the protective effects of the other noble gases, helium, neon, argon, krypton and xenon, on human tubular kidney HK2 cells in vitro. Cultured human renal tubular cells (HK2) were exposed to noble gas preconditioning (75% noble gas; 20% O(2); 5% CO(2)) for three hours or mock preconditioning. Twenty-four hours after gas exposure, cell injury was provoked with oxygen-glucose deprived (OGD) culture medium for three hours. Cell viability was assessed 24 h post-OGD by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Other cohorts of cultured cells were incubated in the absence of OGD in 75% noble gas, 20% O(2) and 5% CO(2) and cellular signals phospho-Akt (p-Akt), hypoxia-inducible factor-1alpha (HIF-1alpha) and Bcl-2 were assessed by Western blotting. OGD caused a reduction in cell viability to 0.382 +/- 0.1 from 1.0 +/- 0.15 at control (P 0.05). Helium by comparison significantly enhanced cell injury (0.191 +/- 0.05; P noble gases did not modify protein expression. These results suggest that unlike other noble gases, preconditioning with the anesthetic noble gas xenon may have a role in protection against renal ischemic injury.

  9. Green tea polyphenols protect against okadaic acid-induced acute learning and memory impairments in rats.

    Science.gov (United States)

    Li, Hongyu; Wu, Xiukui; Wu, Qiong; Gong, Dezheng; Shi, Meijun; Guan, Lili; Zhang, Jun; Liu, Jing; Yuan, Bo; Han, Guozhu; Zou, Yuan

    2014-03-01

    Green tea polyphenols (GTPs) are now being considered possible protective agents in neurodegenerative diseases such as Alzheimer's disease (AD). Previous studies suggested that GTPs could inhibit amyloid fibril formation and protect neurons from toxicity induced by β-amyloid. However, whether GTPs can ameliorate learning and memory impairments and also reduce tau hyperphosphorylation induced by okadaic acid (OA) in rats remains unclear. The aim of this study was to determine if GTPs have neuroprotection against OA-induced neurotoxicity. In this work, rats were pretreated with GTPs by intragastric administration for 4 wk. Then OA was microinjected into the right dorsal hippocampus. Morris water maze tests were used to test the ethologic changes in all groups, and tau protein hyperphosphorylation was detected both in vivo and in vitro. The ethologic test indicated that the staying time and swimming distance in the target quadrant were significantly decreased after OA treatment, whereas rats pretreated with GTPs stayed longer in the target quadrant. Methyl thiazolyl tetrazolium assay and lactate dehydrogenase leakage showed that GTPs greatly ameliorated primary hippocampal neurons damage induced by OA. Furthermore, reduced hyperphosphorylated tau protein was detected with GTPs pretreatment. Taken together, our results suggest that GTPs have neuroprotection against OA-induced neurotoxicity. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Effect of 940 nm low-level laser therapy on osteogenesis in vitro

    Science.gov (United States)

    Jawad, Mohammed Mahmood; Husein, Adam; Azlina, Ahmad; Alam, Mohammad Khursheed; Hassan, Rozita; Shaari, Rumaizi

    2013-12-01

    Bone regeneration is essential in medical treatment, such as in surgical bone healing and orthodontics. The aim of this study is to examine the effect of different powers of 940 nm diode low-level laser treatment (LLLT) on osteoblast cells during their proliferation and differentiation stages. A human fetal osteoblast cell line was cultured and treated with LLLT. The cells were divided into experimental groups according to the power delivered and periods of exposure per day for each laser power. The (3-(4,5-dimethylthiazol-2yl)-2,5 diphenyl tetrazolium bromide) (MTT) assay was used to determine cell proliferation. Both alkaline phosphatase and osteocalcin activity assays were assessed for cell differentiation. All treatment groups showed a significant increase in cell proliferation and differentiation compared to the control group. Regarding the exposure time, the subgroups treated with the LLLT for 6 min showed higher proliferation and differentiation rates for the powers delivered, the 300-mW LLLT group significantly increased the amount of cell proliferation. By contrast, the 100 and 200 mW groups showed significantly greater amounts of cell differentiation. These results suggest that the use of LLLT may play an important role in stimulating osteoblast cells for improved bone formation.

  11. Rapid in situ assessment of physiological activities in bacterial biofilms using fluorescent probes

    Science.gov (United States)

    Yu, F. P.; McFeters, G. A.

    1994-01-01

    Two rapid in situ enumeration methods using fluorescent probes were used to assess the physiological activities of Klebsiella pneumoniae biofilms on stainless steel. Fluorescent dyes, 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and rhodamine 123 (Rh 123), were chosen to perform this study. CTC is a soluble redox indicator which can be reduced by respiring bacteria to fluorescent CTC-formazan crystals. Rh 123 is incorporated into bacteria with respect to cellular proton motive force. The intracellular accumulation of these fluorescent dyes can be determined using epifluorescence microscopy. The results obtained with these two fluorescent probes in situ were compared to the plate count (PC) and in situ direct viable count (DVC) methods. Viable cell densities within biofilms determined by the three in situ methods were comparable and always showed approximately 2-fold higher values than those obtained with the PC method. As an additional advantage, the results were observed after 2 h, which was shorter than the 4 h incubation time required for the DVC method and 24 h for colony formation. The results indicate that staining with CTC and Rh 123 provides rapid information regarding cell numbers and physiological activities of bacteria within biofilms.

  12. Synthesis and properties of novel water-soluble fullerene-glycine derivatives as new materials for cancer therapy.

    Science.gov (United States)

    Jiang, Guichang; Yin, Fen; Duan, Jihua; Li, Guangtao

    2015-01-01

    Novel water-soluble fullerene-glycine derivatives were synthesized by means of simple organic chemistry. They are completely soluble in water, yielding a clear brown solution. The products were characterized by fourier transform infrared (FTIR), ultraviolet-visible spectroscopy (UV-Vis), (1)H NMR, (13)C NMR, thermogravimetric analyses (TGA), and scanning electron microscopy (SEM). The assembly behavior of water-soluble fullerene-glycine derivatives was investigated by SEM. The results show that the fullerene-glycine derivatives create morphology that is sphere-like. The cytotoxicity to cancer cell lines of the fullerene-glycine derivatives was evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and flow cytometry. The results show that fullerene-glycine derivatives exhibit mortality and apoptosis of the cells which increased with the increase of fullerene-glycine derivative concentration. The cytotoxicity mechanism of fullerene-glycine derivatives was investigated for the first time. Novel water-soluble fullerene-glycine derivatives were synthesized by means of simple organic chemistry. The products were characterized by FTIR, UV-Vis, (1)H NMR, (13)C NMR, TGA, and SEM. The bioactivities of fullerene-glycine derivative materials have been tested, and the results show that compared with the fullerene complex, the fullerene-glycine derivative materials exhibit mortality and apoptosis of the cells which increased with the increase of fullerene-glycine derivative concentration. SEM images showed the macrostructure of fullerene-glycine derivative materials was spheres.

  13. Internal seed dispersal by parrots: an overview of a neglected mutualism

    Science.gov (United States)

    Bravo, Carolina; Pacifico, Erica C.; Chamorro, Daniel; Speziale, Karina L.; Lambertucci, Sergio A.; Hiraldo, Fernando; Tella, José L.

    2016-01-01

    Despite the fact that parrots (Psitacifformes) are generalist apex frugivores, they have largely been considered plant antagonists and thus neglected as seed dispersers of their food plants. Internal dispersal was investigated by searching for seeds in faeces opportunistically collected at communal roosts, foraging sites and nests of eleven parrot species in different habitats and biomes in the Neotropics. Multiple intact seeds of seven plant species of five families were found in a variable proportion of faeces from four parrot species. The mean number of seeds of each plant species per dropping ranged between one and about sixty, with a maximum of almost five hundred seeds from the cacti Pilosocereus pachycladus in a single dropping of Lear’s Macaw (Anodorhynchus leari). All seeds retrieved were small (parrot species were viable according to the tetrazolium test and germination experiments. The conservative results of our exploratory sampling and a literature review clearly indicate that the importance of parrots as endozoochorous dispersers has been largely under-appreciated due to the lack of research systematically searching for seeds in their faeces. We encourage the evaluation of seed dispersal and other mutualistic interactions mediated by parrots before their generalized population declines contribute to the collapse of key ecosystem processes. PMID:26925322

  14. An experimental study on the change of the radiosensitivity of several tumor cell lines and primary cultured gingi cal fibrobrast

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Sam Sun; You Dong Soo [Dept. of Oral and Maxillofacial Radiology, College of Dentistry, Seoul National University, Seoul (Korea, Republic of)

    1997-02-15

    Radiation sensitivity data was generated for two human cancer cell lines (KB, RPMI 2650) and human primary gingival fibroblast was tested three times using a viable cell number counting with a hemocytometer, MTT (3-[4,5-dimethylthiazol 2-yl]-2,5-dipheny tetrazolium bromide) assay, and LDH (Lactate dehydrogenase) assay. Single irradiation of 2, 4, 6, 10, 15, 20 Gy were applied to the tumor cell lines and the primary cultured gingical fibroblast. The two fractions of 4 Gy an d 10 Gy were separated with a 4 hour time interval. The irradiation was done with 241.5 cGy/min dose rate using 137 Cs MK cell irradiator at room temperature. The obtained results were as followed : 1. There was significantly different viable cell numbers as the amount of radiation dose on the tested cells were cell number counted with a hemocytometer, In fractions, there were more viable cells remaining. 2. Phase-contrast microscopically, radiation-induced morphologic changes were pronounced on the tumor cells, however, almost no differences on the gingival fibroblast. 3. There was significantly different absorbance at 2 Gy on RPMI 2650, 4 Gy on KB and GF in MTT assay. In fractions, the absorbance was significantly higher on KB. 4. The level of extracellular LDH activity in the experimental group was significantly higher in the 2-4 Gy than the control group. 5. The total level of extracellular and intracellular LDH activity was decreased as increased amounts of radiation dose was applied.

  15. Histological analysis and 3D reconstruction of winter cereal crowns recovering from freezing: a unique response in oat (Avena sativa L.).

    Science.gov (United States)

    Livingston, David P; Henson, Cynthia A; Tuong, Tan D; Wise, Mitchell L; Tallury, Shyamalrau P; Duke, Stanley H

    2013-01-01

    The crown is the below ground portion of the stem of a grass which contains meristematic cells that give rise to new shoots and roots following winter. To better understand mechanisms of survival from freezing, a histological analysis was performed on rye, wheat, barley and oat plants that had been frozen, thawed and allowed to resume growth under controlled conditions. Extensive tissue disruption and abnormal cell structure was noticed in the center of the crown of all 4 species with relatively normal cells on the outside edge of the crown. A unique visual response was found in oat in the shape of a ring of cells that stained red with Safranin. A tetrazolium analysis indicated that tissues immediately inside this ring were dead and those outside were alive. Fluorescence microscopy revealed that the barrier fluoresced with excitation between 405 and 445 nm. Three dimensional reconstruction of a cross sectional series of images indicated that the red staining cells took on a somewhat spherical shape with regions of no staining where roots entered the crown. Characterizing changes in plants recovering from freezing will help determine the genetic basis for mechanisms involved in this important aspect of winter hardiness.

  16. Essential Oils from Ugandan Medicinal Plants: In Vitro Cytotoxicity and Effects on IL-1β-Induced Proinflammatory Mediators by Human Gingival Fibroblasts

    Directory of Open Access Journals (Sweden)

    Francis Ocheng

    2016-01-01

    Full Text Available The study investigated cytotoxicity of essential oils from four medicinal plants (Bidens pilosa, Ocimum gratissimum, Cymbopogon nardus, and Zanthoxylum chalybeum on human gingival fibroblasts and their effects on proinflammatory mediators’ secretion. Cytotoxicity of essential oils was investigated using 3-(4, 5-dimethylthiazol-2-yl-2,5-diphenyl-tetrazolium bromide assay. Effects of essential oils at subcytotoxicity concentrations on interleukin- (IL- 6, IL-8, and prostaglandin E2 (PGE2 secretions by gingival fibroblasts treated with IL-1β (300 pg/mL were evaluated by ELISA and EIA. IC50 values of the essential oils ranged from 26 μg/mL to 50 μg/mL. Baseline and IL-1β-induced secretion of PGE2 was inhibited by treatment with essential oil from O. gratissimum. Essential oils from B. pilosa and C. nardus had synergistic effects with IL-1β on PGE2 seceretion. In conclusion, the study suggests that essential oil from O. gratissimum decreases gingival fibroblasts secretion of PGE2, while essential oils from B. pilosa and C. nardus increase PGE2 secretion. Essential oil from Z. chalybeum was the most cytotoxic, while oil from C. nardus was the least cytotoxic. Although the clinical significance of these findings remains to be determined, it may be suggested that essential oil from O. gratissimum, applied at subcytotoxicity concentrations, could reduce the participation of gingival fibroblasts in the gingival inflammation and tissue destruction associated with periodontitis.

  17. Medicinal plants from the Yanesha (Peru): evaluation of the leishmanicidal and antimalarial activity of selected extracts.

    Science.gov (United States)

    Valadeau, Céline; Pabon, Adriana; Deharo, Eric; Albán-Castillo, Joaquina; Estevez, Yannick; Lores, Fransis Augusto; Rojas, Rosario; Gamboa, Dionicia; Sauvain, Michel; Castillo, Denis; Bourdy, Geneviève

    2009-06-25

    Ninety-four ethanolic extracts of plants used medicinally by the Yanesha, an Amazonian Peruvian ethnic group, for affections related to leishmaniasis and malaria were screened in vitro against Leishmania amazonensis amastigotes and against a Plasmodium falciparum chloroquine resistant strain. The viability of Leishmania amazonensis amastigote stages was assessed by the reduction of tetrazolium salt (MTT) while the impact on Plasmodium falciparum was determined by measuring the incorporation of radio-labelled hypoxanthine. Six plant species displayed good activity against Plasmodium falciparum chloroquine resistant strain (IC(50) Siparuna aspera (Ruiz & Pavon), A. DC., two Zingiberaceae, Renealmia thyrsoidea (Ruiz & Pavon) Poepp. & Endl. and Renealmia alpinia (Rottb.), two Piperaceae (Piper aduncum L. and Piper sp.) and the leaves of Jacaranda copaia (Aubl.) D. Don (Bignoniaceae). Eight species displayed interesting leishmanicidal activities (IC50 < 10 microg/ml): Carica papaya L. (Caricaceae), Piper dennisii Trel (Piperaceae), Hedychium coronarium J. König (Zingiberaceae), Cestrum racemosum Ruiz & Pav. (Solanaceae), Renealmia alpinia (Rottb.) Zingiberaceae, Lantana sp. (Verbenaceae), Hyptis lacustris A. St.-Hil. ex Benth. (Lamiaceae) and Calea montana Klat. (Asteraceae). Most of them are used against skin affections by Yanesha people. Results are discussed herein, according to the traditional use of the plants and compared with data obtained from the literature.

  18. Antibacterial properties and cytocompatibility of tantalum oxide coatings with different silver content

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Heng-Li [School of Dentistry, China Medical University, Taichung 404, Taiwan (China); Chang, Yin-Yu, E-mail: yinyu@mail2000.com.tw; Chen, Hung-Jui; Chou, Yu-Kai [Department of Mechanical and Computer-Aided Engineering, National Formosa University, Yunlin 632, Taiwan (China); Lai, Chih-Ho [School of Medicine, China Medical University, Taichung 404, Taiwan (China); Chen, Michael Y. C. [Division of Oral and Maxillofacial Surgery, China Medical University Hospital, Taichung 404, Taiwan (China)

    2014-03-15

    Tantalum (Ta) oxides and their coatings have been proved to increase their applications in the biomedical fields by improving osseointegration and wear resistance. In this study, Ta oxide coatings containing different proportions of Ag are deposited on SS304 materials. A twin-gun magnetron sputtering system is used to deposit the tantalum oxide-Ag coating. In this study, Staphylococcus aureus, which exhibits physiological commensalism on the human skin, nares, and mucosal and oral areas, is chosen as the model for in vitro antibacterial analyses via a fluorescence staining method using Syto9. The cytocompatibility and adhesive morphology of human skin fibroblast cells (CCD-966SK) on the coatings are also determined by using the microculture tetrazolium assay. This study shows that Ta{sub 2}O{sub 5} and Ta{sub 2}O{sub 5}-Ag coatings with 12.5 at. % of Ag exhibit improved antibacterial effects against S. aureus and have good skin fibroblast cell cellular biocompatibility.

  19. Assessment of penetrating thermal tissue damage/spread associated with PhotonBlade™, Valleylab™ Pencil, Valleylab™ EDGE™ Coated Pencil, PlasmaBlade® 3.0S and PlasmaBlade® 4.0 for intraoperative tissue dissection using the fresh extirpated porcine muscle model

    Science.gov (United States)

    Bennett, Haydon E.; Taylor, Scott D.; Fugett, James H.; Shrout, Joshua L.; Davison, Paul O.; Ryan, S. Eric; Coad, James E.

    2017-02-01

    Penetrating thermal tissue damage/spread is an important aspect of many electrosurgical devices and correlates with effective tissue cutting, hemostasis, preservation of adjacent critical structures and tissue healing. This study compared the thermal damage/spread associated with the PhotonBlade, Valleylab Pencil, Valleylab EDGE Coated Pencil, PlasmaBlade 3.0S and PlasmaBlade 4.0, when performing a single pass dynamic tissue cut in fresh extirpated porcine longissimus muscle. These devices were used in a fashion that emulated their use in the clinical setting. Each device's thermal damage/spread, at Minimum, Median and Maximum power input settings, was assessed with nitroblue tetrazolium viability staining in the WVU Pathology Laboratory for Translational Medicine. The thermal damage/spread associated with the PhotonBlade was compared with the other devices tested based on the individual treatment results (n=179 cuts combined). In summary, the PhotonBlade overall demonstrated the least penetrating thermal tissue damage/spread, followed by the PlasmaBlade 4.0, then Valleylab Pencil and PlasmaBlade 3.0S and then Valleylab EDGE Coated Pencil in order of increasing thermal damage/spread depths.

  20. Antioxidant and Anticancer Activities of Walnut (Juglans regia L.) Protein Hydrolysates Using Different Proteases.

    Science.gov (United States)

    Jahanbani, Raheleh; Ghaffari, S Mahmood; Salami, Maryam; Vahdati, Kourosh; Sepehri, Houri; Sarvestani, Nazanin Namazi; Sheibani, Nader; Moosavi-Movahedi, Ali Akbar

    2016-12-01

    Walnut (Juglans regia L.) contains approximately 20-25 % protein with abundant essential amino acids. The enzymatic hydrolysate of Persian walnut (Chandler) seed proteins was prepared by incubation with three different proteases, including pancreatic chymotrypsin and trypsin, and a microbial enzyme proteinase K. The hydrolysates were found to possess excellent antioxidant capacities. The peptide fractions scavenged the 2, 2'-anizo-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) free radicals and inhibited the activity of reactive oxygen species. Walnut protein hydrolysates were also tested, for the first time, against the viability of human breast (MDA-MB231) and colon (HT-29) cancer cell lines. MTT, [3-(4, 5dimethylthiazolyl)-2,5-diphenyl-tetrazolium bromide], assay was used to assess in vitro cancer cell viability upon treatment with the peptide fractions. The peptide fractions showed cell growth inhibition of 63 ± 1.73 % for breast cancer and 51 ± 1.45 % for colon cancer cells. Thus, a direct correlation between antioxidant and anticancer activities of walnut peptide fractions exists and supports their potential therapeutic benefit.

  1. Effective Method of Purification of Betulin from Birch Bark: The Importance of Its Purity for Scientific and Medicinal Use.

    Directory of Open Access Journals (Sweden)

    Pavel Šiman

    Full Text Available A new and relatively simple method for purification of betulin from birch bark extract was developed in this study. Its five purification steps are based on the differential solubility of extract components in various solvents and their crystallization and/or precipitation, on their affinity for Ca(OH2 in ethanol, and on the affinity of some impurities for silica gel in chloroform. In addition, all used solvents can be simply recycled. Betulin of more than 99% purity can be prepared by this method with minimal costs. Various observations including crystallization of betulin, changes in crystals during heating, and attempt of localization of betulin in outer birch bark are also described in this work. The original extract, fraction without betulinic acid and lupeol, amorphous fraction of pure betulin, final crystalline fraction of pure betulin and commercial betulin as a standard were employed to determine the antiproliferative/cytotoxic effect. We used WST-1 tetrazolium-based assays with triple negative breast cancer cell line BT-549. The decrease in cell survival showed clear relationship with the purity of the samples, being most pronounced using our final product of pure crystalline betulin. WST-1 proliferation/cytotoxicity test using triple negative breast cancer cell line BT-549 clearly showed the importance of purity of betulin for biological experiments and, apparently, for its medicinal use.

  2. In vitro study of RRS HA injectable mesotherapy/biorevitalization product on human skin fibroblasts and its clinical utilization.

    Science.gov (United States)

    Deglesne, Pierre-Antoine; Arroyo, Rodrigo; Ranneva, Evgeniya; Deprez, Philippe

    2016-01-01

    Mesotherapy/biorevitalization with hyaluronic acid (HA) is a treatment approach currently used for skin rejuvenation. Various products with a wide range of polycomponent formulations are available on the market. Most of these formulations contain noncross-linked HA in combination with a biorevitalization cocktail, formed by various amounts of vitamins, minerals, amino acids, nucleotides, coenzymes, and antioxidants. Although ingredients are very similar among the different products, in vitro and clinical effects may vary substantially. There is a real need for better characterization of these products in terms of their action on human skin or in vitro skin models. In this study, we analyzed the effect of the RRS(®) (Repairs, Refills, Stimulates) HA injectable medical device on human skin fibroblasts in vitro. Skin fibroblast viability and its capacity to induce the production of key extracellular matrix were evaluated in the presence of different concentrations of RRS HA injectable. Viability was evaluated through colorimetric MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay, and key extracellular matrix genes, type I collagen and elastin, were quantified by quantitative polymerase chain reaction. Results demonstrated that RRS HA injectable could promote human skin fibroblast viability (+15%) and increase fibroblast gene expression of type I collagen and elastin by 9.7-fold and 14-fold in vitro, respectively. These results demonstrate that mesotherapy/biorevitalization products can, at least in vitro, effectively modulate human skin fibroblasts.

  3. [Determination of the healing effect of Piper aduncum (spiked pepper or matico) on human fibroblasts].

    Science.gov (United States)

    Paco, Karen; Ponce-Soto, Luis Alberto; Lopez-Ilasaca, Marco; Aguilar, José L

    2016-01-01

    To evaluate the healing effect of a Piper aduncum ethanol-water extract on an adult human dermal fibroblast cell line (hDFa). After obtaining the extract via solid-liquid extraction, concentration, and lyophilization, extract proteins were purified using reverse phase high-performance liquid chromatography, identified using tandem mass spectrometry of tryptic peptides, and analyzed using MALDI-TOF-TOF on an ABSciex4800 mass spectrometer. Half maximum effective concentration values (EC50), half maximum inhibiting concentration (IC50), and percentages of cell proliferation were determined using tetrazolium salt assays. Cell migration was evaluated using a "scratch assay". Growth factor expression in cells was analyzed via quantitative real-time reverse transcription polymerase chain reaction. Against the hDFa cell line, the extract had an IC50 of 200 μg/mL and EC50 of 103.5 µg/mL. In the proliferation assay, protein K2 (obtained from the extract) exhibited increased proliferative activity relative to other treatments (1 µg/mL); this agent also exhibited increased activity (50 µg/mL) in the fibroblast migration assay.Furthermore, the relative expression of platelet-derived growth factor increased by 8.6-fold in the presence of K2 protein relative to the control. The hydroethanolic extract of Piper aduncum and its component proteins increased the proliferation and migration of hDFa and increased the expression of growth factors involved in the healing process.

  4. Probit analysis of comparative assays on toxicities of lead chloride and lead acetate to in vitro cultured human umbilical cord blood lymphocytes.

    Science.gov (United States)

    Patnaik, Rajashree; Padhy, Rabindra N

    2015-03-01

    This work describes that cytotoxicity of lead chloride and lead acetate to in vitro cultured lymphocytes from human umbilical cord blood, using four monitoring methods namely, trypan blue staining, acridine orange/ethidium bromide staining, 3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyl tetrazolium bromide (MTT) and neutral red uptake assays; lead genotoxicity to lymphocytes was monitored by comet assay. The MIC value in each method was invariably 300 mg/L for PbCl2. Lethal concentration25 (LC25) values were almost in an agreeable range: 691.83 to 831.76 mg/L; LC50 values in each method were almost in the range: 1174.9 to 1348.9 mg/L; LC100 values were in the range: 3000 to 3300 mg/L, for lead chloride. Similarly, The MIC value in each method were invariably 150 mg/L; LC25 values were almost in the range: 295.12 to 371.53 mg/L; LC50 values were in the range: 501.18 to 588.84 mg/L; LC100 value was 1500 mg/L in all assays, for lead acetate. The comet assay also indicated that the LC100 values were 3300 mg/L lead chloride and 1500 mg/L lead acetate. Thus, both cytotoxicity and genotoxicity were recorded at 3300 mg/L lead chloride and 1500 mg/L lead acetate with lymphocytes.

  5. Evaluation of cytotoxicity of Moringa oleifera Lam. callus and leaf extracts on Hela cells

    Directory of Open Access Journals (Sweden)

    Abbas Jafarain

    2014-01-01

    Full Text Available Background: There are considerable attempts worldwide on herbal and traditional compounds to validate their use as anti-cancer drugs. Plants from Moringaceae family including Moringa oleifera possess several activities such as antitumor effect on tumor cell lines. In this study we sought to determine if callus and leaf extracts of M. oleifera possess any cytotoxicity. Materials and Methods: Ethanol-water (70-30 extracts of callus and leaf of M. oleifera were prepared by maceration method. The amount of phenolic compounds of the extracts was determined by Folin Ciocalteu method. The cytotoxicity of the extracts against Hela tumor cells was carried out using MTT assay. Briefly, cells were seeded in microplates and different concentrations of the extract were added. Cells were incubated for 48 h and their viability was evaluated by addition of tetrazolium salt solution. After 3 h medium was aspirated, dimethyl sulfoxide was added and absorbance was determined at 540 nm with an ELISA plate reader. Cytotoxicity was considered when more than 50% reduction on cell survival was observed. Results: Callus and leaf extracts of M. oleifera significantly decreased the viability of Hela cells in a concentration-dependent manner. However, leaf extract of M. oleifera were more potent than that of callus extract. Conclusion: As the content of phenolic compounds of leaf extract was higher than that of callus extract, it can be concluded that phenolic compounds are involved in the cytotoxicity of M. oleifera.

  6. Evaluation of cytotoxicity of Moringa oleifera Lam. callus and leaf extracts on Hela cells.

    Science.gov (United States)

    Jafarain, Abbas; Asghari, Gholamreza; Ghassami, Erfaneh

    2014-01-01

    There are considerable attempts worldwide on herbal and traditional compounds to validate their use as anti-cancer drugs. Plants from Moringaceae family including Moringa oleifera possess several activities such as antitumor effect on tumor cell lines. In this study we sought to determine if callus and leaf extracts of M. oleifera possess any cytotoxicity. Ethanol-water (70-30) extracts of callus and leaf of M. oleifera were prepared by maceration method. The amount of phenolic compounds of the extracts was determined by Folin Ciocalteu method. The cytotoxicity of the extracts against Hela tumor cells was carried out using MTT assay. Briefly, cells were seeded in microplates and different concentrations of the extract were added. Cells were incubated for 48 h and their viability was evaluated by addition of tetrazolium salt solution. After 3 h medium was aspirated, dimethyl sulfoxide was added and absorbance was determined at 540 nm with an ELISA plate reader. Cytotoxicity was considered when more than 50% reduction on cell survival was observed. Callus and leaf extracts of M. oleifera significantly decreased the viability of Hela cells in a concentration-dependent manner. However, leaf extract of M. oleifera were more potent than that of callus extract. As the content of phenolic compounds of leaf extract was higher than that of callus extract, it can be concluded that phenolic compounds are involved in the cytotoxicity of M. oleifera.

  7. Downregulation of ATP-binding cassette subfamily C member 4 increases sensitivity to neoadjuvant radiotherapy for locally advanced rectal carcinoma.

    Science.gov (United States)

    Yu, Zhi-Qi; Zhang, Chang; Wang, Hao; Lao, Xin-Yuan; Chai, Rui; Gao, Xian-Hua; Cao, Guang-Wen; Fu, Chuan-Gang

    2013-05-01

    This study was designed to verify the effect of ATP-binding cassette subfamily C member 4 on radiosensitivity of locally advanced rectal carcinoma. The expression of ATP-binding cassette subfamily C member 4 protein in 121 pretreatment tissue samples from locally advanced rectal carcinoma patients was detected by immunohistochemistry. Pathological response to radiotherapy was evaluated according to tumor regression grading by postoperative histological examinations after they received long-course preoperative neoadjuvant radiotherapy, and the association between clinicopathological data and tumor regression grading was analyzed retrospectively. For further validation, short hairpin RNA was constructed and transfected into colorectal carcinoma cell line HT29. The knockdown efficiency was confirmed at both RNA and protein levels. The altered radiosensitivity was evaluated by methylthiazolyl tetrazolium assay, colony formation assay, flow cytometry, and Hoechst 33258 staining. Univariate analysis revealed that ATP-binding cassette subfamily C member 4 expression (p member 4 expression (p member 4 expression efficiently and persistently. Downregulation of ATP-binding cassette subfamily C member 4 expression significantly enhanced inhibition of cell proliferation, decreased colony formation capacity, and increased cell apoptosis induced by irradiation, as examined by a series of experiments in vitro. In addition, radiobiological parameters calculated according to the single-hit multitarget model were also decreased significantly. Our data indicate that ATP-binding cassette subfamily C member 4 may be a useful molecular marker in predicting radiosensitivity, and a potential target in improving the response to neoadjuvant radiotherapy in locally advanced rectal carcinoma patients.

  8. Biocompatibility of Bletilla striata Microspheres as a Novel Embolic Agent

    Directory of Open Access Journals (Sweden)

    ShiHua Luo

    2015-01-01

    Full Text Available We have prepared Chinese traditional herb Bletilla striata into microspheres as a novel embolic agent for decades. The aim of this study was to evaluate the biocompatibility of Bletilla striata microspheres (BSMs. After a thermal test of BSMs in vitro, the cell biocompatibility of BSMs was investigated in mouse fibroblasts and human umbilical vein endothelial cells using the methyl tetrazolium (MTT assay. In addition, blood biocompatibility was evaluated. In vivo intramuscular implantation and renal artery embolization in rabbits with BSMs were used to examine the inflammatory response. The experimental rabbits did not develop any fever symptoms after injection of BSMs, and BSMs exhibited no cytotoxicity in cultured mouse fibroblasts and human umbilical vein endothelial cells. Additionally, BSMs exhibited high compatibility with red blood cells and no hemolysis activity. Intramuscular implantation with BSMs resulted in a gradually lessened mild inflammatory reaction that disappeared after eight weeks. The occlusion of small renal vessels was associated with a mild perivascular inflammatory reaction without significant renal and liver function damage. In conclusion, we believe that BSMs exhibit high biocompatibility and are a promising embolic agent.

  9. Resveratrol, piperine and apigenin differ in their NADPH-oxidase inhibitory and reactive oxygen species-scavenging properties.

    Science.gov (United States)

    Whitehouse, Scott; Chen, Pei-Lin; Greenshields, Anna L; Nightingale, Mat; Hoskin, David W; Bedard, Karen

    2016-11-15

    Many plant-derived chemicals have been studied for their potential benefits in ailments including inflammation, cancer, neurodegeneration, and cardiovascular disease. The health benefits of phytochemicals are often attributed to the targeting of reactive oxygen species (ROS). However, it is not always clear whether these agents act directly as antioxidants to remove ROS, or whether they act indirectly by blocking ROS production by enzymes such as NADPH oxidase (NOX) enzymes, or by influencing the expression of cellular pro- and anti- oxidants. Here we evaluate the pro- and anti-oxidant and NOX-inhibiting qualities of four phytochemicals: celastrol, resveratrol, apigenin, and piperine. This work was done using the H661 cell line expressing little or no NOX, modified H661 cells expressing NOX1 and its subunits, and an EBV-transformed B-lymphoblastoid cell line expressing endogenous NOX2. ROS were measured using Amplex Red and nitroblue tetrazolium assays. In addition, direct ROS scavenging of hydrogen peroxide or superoxide generated were measured using Amplex Red and methyl cypridina luciferin analog (MCLA). Of the four plant-derived compounds evaluated, only celastrol displayed NOX inhibitory activities, while celastrol and resveratrol both displayed ROS scavenging activity. Very little impact on ROS was observed with apigenin, or piperine. The results of this study reveal the differences that exist between cell-free and intracellular pro-oxidant and antioxidant activities of several plant-derived compounds. Copyright © 2016 Elsevier GmbH. All rights reserved.

  10. Dual tumor-targeted poly(lactic-co-glycolic acid)–polyethylene glycol–folic acid nanoparticles: a novel biodegradable nanocarrier for secure and efficient antitumor drug delivery

    Science.gov (United States)

    Chen, Jia; Wu, Qi; Luo, Li; Wang, Yi; Zhong, Yuan; Dai, Han-Bin; Sun, Da; Luo, Mao-Ling; Wu, Wei; Wang, Gui-Xue

    2017-01-01

    Further specific target-ability development of biodegradable nanocarriers is extremely important to promote their security and efficiency in antitumor drug-delivery applications. In this study, a facilely prepared poly(lactic-co-glycolic acid) (PLGA)–polyethylene glycol (PEG)–folic acid (FA) copolymer was able to self-assemble into nanoparticles with favorable hydrodynamic diameters of around 100 nm and negative surface charge in aqueous solution, which was expected to enhance intracellular antitumor drug delivery by advanced dual tumor-target effects, ie, enhanced permeability and retention induced the passive target, and FA mediated the positive target. Fluorescence-activated cell-sorting and confocal laser-scanning microscopy results confirmed that doxorubicin (model drug) loaded into PLGA-PEG-FA nanoparticles was able to be delivered efficiently into tumor cells and accumulated at nuclei. In addition, all hemolysis, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, and zebrafish-development experiments demonstrated that PLGA-PEG-FA nanoparticles were biocompatible and secure for biomedical applications, even at high polymer concentration (0.1 mg/mL), both in vitro and in vivo. Therefore, PLGA-PEG-FA nanoparticles provide a feasible controlled-release platform for secure and efficient antitumor drug delivery. PMID:28848351

  11. Laser Phototherapy Enhances Mesenchymal Stem Cells Survival in Response to the Dental Adhesives

    Science.gov (United States)

    Marques, Márcia Martins

    2015-01-01

    Background. We investigated the influence of laser phototherapy (LPT) on the survival of human mesenchymal stem cells (MSCs) submitted to substances leached from dental adhesives. Method. MSCs were isolated and characterized. Oral mucosa fibroblasts and osteoblast-like cells were used as comparative controls. Cultured medium conditioned with two adhesive systems was applied to the cultures. Cell monolayers were exposed or not to LPT. Laser irradiations were performed using a red laser (GaAlAs, 780 nm, 0.04 cm2, 40 mW, 1 W/cm2, 0.4 J, 10 seconds, 1 point, 10 J/cm2). After 24 h, cell viability was assessed by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide reduction assay. Data were statistically compared by ANOVA followed by Tukey's test (P adhesives were less cytotoxic to MSCs than to other cell types. Substances leached from Clearfil SE Bond were highly cytotoxic to all cell types tested, except to the MSCs when applied polymerized and in association with LPT. LPT was unable to significantly increase the cell viability of fibroblasts and osteoblast-like cells submitted to the dental adhesives. Conclusion. LPT enhances mesenchymal stem cells survival in response to substances leached from dental adhesives. PMID:25879065

  12. Cytotoxicity and degree of conversion of orthodontic adhesives.

    Science.gov (United States)

    Jagdish, Nithya; Padmanabhan, Sridevi; Chitharanjan, Arun B; Revathi, J; Palani, Gunasekaran; Sambasivam, Mohana; Sheriff, Khaleefathullah; Saravanamurali, K

    2009-11-01

    To test the hypothesis that there is no difference in the cytotoxicity related to the modes of polymerization of five commercially available orthodontic bonding resins, with and without an oxygen-inhibited layer (OIL), and to evaluate the degree of conversion (DC) of these resins and correlate this to cytotoxicity. Five commercially available orthodontic bonding resins were tested for cytotoxicity and DC. Thirty-six disks of standardized dimensions, for each resin, were used for cytotoxicity assessment. Half of them were washed with 99% acetone to remove the OIL (washed resins), and the remaining disks were left intact (intact resins). Glass disks were used as a control. Vero cells were exposed to intact and washed resins on day 1. Cell viability was determined by tetrazolium bromide reduction assay 1, 3, and 6 days after exposure. The DC of the adhesive specimens of each resin, prepared with a procedure identical to the clinical bonding process, was assessed by Fourier transform infrared spectroscopy. Single-cured systems were comparatively less cytotoxic than dual-cured systems. With removal of the OIL, increased cell viability was noted only with two resins on all three days. Resins tested showed differences in DC. A positive correlation was demonstrated by two resins. The hypothesis is rejected. Single-cured systems are superior to dual-cured systems in exhibiting comparatively less toxicity and higher DC. A significant positive correlation was not established between cytotoxicity and DC.

  13. Citotoxicity evaluation of three dental adhesives on vero cells in vitro.

    Science.gov (United States)

    Catunda, Raisa-Queiroz; Vieira, Jeymesson-Raphael-Cardoso; de Oliveira, Erwelly-Barros; da Silva, Eliete-Cavalcanti; Brasil, Veruska-Lima-Moura; Perez, Danyel-Elias-da Cruz

    2017-01-01

    To evaluate, in vitro, the potential cytotoxicity of three different dental adhesives systems (Adper Single Bond 2 -SB, Silorane System Adhesive Bond -SSAB and Single Bond Universal -SBU) on cultivated Vero cells after different contact times. The cells were cultured in a concentration of 2 x 105 cells/mL for 24h and grown to sub-confluent monolayers. VERO cells were exposed to 25µl of conditioned extracts obtained from 24h, 48h and 72h immersion of adhesive samples in culture medium (DMEM), immediately after polymerization. Fresh DMEM was used as negative control. Cell metabolism was evaluated by the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2, 5diphenyl-tetrazolium bromide). The data were analyzed statistically by ANOVA, considering a significance of 5%. The values of cell viability ranged from 94.2% at 72h (SBU) to 109.6% at 48h (SB). The mean percentage of viability after exposure to the extracts of SB, SSAB and SBU were 103.2%, 100.63% and 97.43%, respectively. There was no statistically significant difference (p= 0.342) between the experimental and negative control groups. At all exposure times, all adhesives tested in this study presented no cytotoxicity to Vero cells in vitro. Key words:Biocompatibility, cytotoxicity, dental adhesives, Vero cells.

  14. Experimental self-etching HEMA-free adhesive systems: cytotoxicity and degree of conversion.

    Science.gov (United States)

    Barbosa, Marília Oliveira; de Carvalho, Rodrigo Varella; Demarco, Flávio Fernando; Ogliari, Fabrício Aulo; Zanchi, Cesar Henrique; Piva, Evandro; da Silva, Adriana Fernandes

    2015-01-01

    The aim of this study was to evaluate the effect of replacing 2-hydroxyethyl methacrylate (HEMA) by methacrylate surfactant monomers on the cytotoxicity and degree of conversion of two-step self-etching dentin adhesive systems. Five HEMA-free adhesive systems were tested: Bis-EMA 10, Bis-EMA 30, PEG400, PEG400UDMA, PEG1000, and a HEMA group was used as positive control. The cytotoxicity of the experimental primers, with different monomer concentrations (2 or 20 wt%), and bond resins, containing 25 wt% surfactant, was assessed using murine fibroblast cell line 3T3 and the tetrazolium assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)). The degree of conversion of the bond resins was analyzed using Fourier transform infrared spectroscopy. The data were submitted to statistical analysis using level of significance set at P cytotoxic than HEMA. With regard to the eluate from bond resin, the data showed that the groups BIS-EMA 10, BIS-EMA 30 and PEG400UDMA were less cytotoxic than HEMA. No statistically significant difference was found among degrees of conversion of the experimental groups and HEMA. PEG 1000, BIS-EMA 10 and 30 monomers showed the biological potential for use in new adhesive system formulations since they showed lower cytotoxicity and similar degree of conversion when compared with the HEMA-containing group.

  15. The antioxidant action of Polypodium leucotomos extract and kojic acid: reactions with reactive oxygen species

    Directory of Open Access Journals (Sweden)

    A.J. Gomes

    2001-11-01

    Full Text Available Two natural products Polypodium leucotomos extract (PL and kojic acid (KA were tested for their ability to scavenge reactive oxygen species (·OH, ·O2-, H2O2, ¹O2 in phosphate buffer. Hydroxyl radicals were generated by the Fenton reaction, and the rate constants of scavenging were 1.6 x 10(9 M-1 s-1 for KA and 1.0 x 10(9 M-1 s-1 for PL, similar to that of ethanol (1.4 x 10(9 M-1 s-1. With superoxide anions generated by the xanthine/hypoxanthine system, KA and PL (0.2-1.0 mg/ml inhibited ·O2-dependent reduction of nitroblue tetrazolium by up to 30 and 31%, respectively. In the detection of ¹O2 by rose bengal irradiation, PL at 1.0 mg/ml quenched singlet oxygen by 43% relative to azide and KA by 36%. The present study demonstrates that PL showed an antioxidant effect, scavenging three of four reactive oxygen species tested here. Unlike KA, PL did not significantly scavenge hydrogen peroxide.

  16. Carboxymethyl chitosan nanoparticles coupled with CD59-specific ligand peptide for targeted delivery of C-phycocyanin to HeLa cells.

    Science.gov (United States)

    Yang, Peng; Li, Bing; Yin, Qi-Feng; Wang, Yu-Juan

    2017-03-01

    The combination of nanotechnology and medicine will be the next generation of vehicles for targeted drug delivery. Carboxymethyl chitosan loaded with the anticancer drug C-phycocyanin and the CD59-specific ligand peptide for cancer cell targeting were used to create C-phycocyanin/carboxymethyl chitosan-CD59-specific ligand peptide nanoparticles using the ionic-gelation method. Optimal synthesis conditions, selected by response surface methodology, comprised the ratio carboxymethyl chitosan:C-phycocyanin = 3:1, and carboxymethyl chitosan and CaCl2 concentrations of 2.0 and 1.0 mg/mL, respectively. The resulting nanoparticles were spherical, with diameters of approximately 200 nm; the entrapment efficient was about 65%; and the drug loading was about 20%. The release of C-phycocyanin from C-phycocyanin/carboxymethyl chitosan nanoparticles was pH sensitive and had a sustainable effect in vitro. Guided by the CD59-specific ligand peptide, the nanoparticles efficiently targeted the surface of HeLa cells and had an obvious inhibitory effect on HeLa cell proliferation as determined by methyl thiazolyl tetrazolium assays. The nanoparticles were hemocompatible and induced apoptosis by upregulation of cleaved caspase-3 and cleaved polyADP-ribose polymerase proteins, and downregulation of Bcl-2 proteins. Our study provides a novel approach to the research and development of marine drugs, and support for targeted therapy using anticancer drugs.

  17. The expression of genes involved in the ergosterol biosynthesis pathway in Candida albicans and Candida dubliniensis biofilms exposed to fluconazole.

    LENUS (Irish Health Repository)

    2009-03-01

    The expression of the ERG1, ERG3, ERG7, ERG9, ERG11 and ERG25 genes in response to incubation with fluconazole and biofilm formation was investigated using reverse-transcription PCR and real-time PCR in Candida albicans and Candida dubliniensis clinical isolates. The viability of biofilm was measured using an 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay and confocal scanning laser microscopy (CSLM). Expression of the ERG11 gene was found to be low or moderate and it was regulated by fluconazole addition more so than by biofilm formation. Very low or non-detectable expression of ERG1, ERG7 and ERG25 genes was detected in C. albicans. The expression of the ERG9 increased in the presence of fluconazole in some isolates. Following incubation with fluconazole, formation of biofilm by C. dubliniensis was coupled with up-regulation of the ERG3 and ERG25 genes as have been observed previously in C. albicans. Planktonic cells of both Candida species released from biofilm displayed similar resistance mechanisms to fluconazole like attached cells. The XTT reduction assay and CSLM revealed that although incubation with fluconazole decreased the biofilm thickness, these were still comprised metabolically active cells able to disseminate and produce biofilm. Our data indicate that biofilm represents a highly adapted community reflecting the individuality of clinical isolates.

  18. [Standardization and regulation of the rate of the superoxide-generating adrenaline autoxidation reaction used for evaluation of pro/antioxidant properties of various materials].

    Science.gov (United States)

    Sirota, T V

    2016-11-01

    The superoxide-generating reaction of adrenaline autoxidation is widely used for determination of the activity of superoxide dismutase and pro/antioxidant properties of various materials. There are two variants of the spectrophotometric registration of the products of this reaction. The first is based on registration of adrenochrome, as adrenaline autooxidation product at 347 nm; the second employs nitro blue tetrazolium (NBT) and registration of diformazan, a product of NBT reduction at 560 nm. In the present work, recommendations for the standardization of the reaction rate in both variants have been proposed. The main approach consists in the use of the pharmaceutical form of 0.1% adrenaline hydrochloride solution. Although each of two adrenaline preparations available in the Russian market has some features in kinetic behavior of its autooxidation; they are applicable in the superoxide generating system based on adrenaline autooxidation. Performing measurements at 560 nm, the reaction rate can be regulated by lowering the concentration of added adrenaline, whereas during spectrophotometric registration at 347 nm, this cannot be done. These features of adrenaline autoxidation may be due to the fact that the intrinsic multistage process of the conversion of adrenaline to adrenochrome, which is recorded at 347 nm, is coupled with the transition of electrons from adrenaline and intermediate products of its oxidation to oxygen, carbon dioxide, and carbonate bicarbonate ions, which is detected in the presence of added NBT.

  19. Characterisation and cytotoxic screening of metal oxide nanoparticles putative of interest to oral healthcare formulations in non-keratinised human oral mucosa cells in vitro.

    Science.gov (United States)

    Best, M; Phillips, G; Fowler, C; Rowland, J; Elsom, J

    2015-12-25

    Nanoparticles are increasingly being utilised in the innovation of consumer product formulations to improve their characteristics; however, established links between their properties, dose and cytotoxicity are not well defined. The purpose of this study was to screen four different nanomaterials of interest to oral care product development in the absence of stabilisers, alongside their respective bulk equivalents, within a non-keratinised oral epithelial cell model (H376). Particle morphology and size were characterised using scanning electron microscopy (SEM) and dynamic light scattering (DLS). The H376 model showed that zinc oxide (ZnO) was the most cytotoxic material at concentrations exceeding 0.031% w/v, as assessed using the lactate dehydrogenase (LDH) and dimethylthiazolyl-diphenyl-tetrazolium-bromide (MTT) assays. ZnO cytotoxicity does not appear to be dependent upon size of the particle; a result supported by SEM of cell-particle interactions. Differences in cytotoxicity were observed between the bulk and nanomaterial forms of hydroxyapatite and silica (SiO2); titanium dioxide (TiO2) was well tolerated in both forms at the doses tested. Overall, nano-size effects have some impact on the cytotoxicity of a material; however, these may not be as significant as chemical composition or surface properties. Our data highlights the complexities involved at the nano-scale, in both the characterisation of materials and in relation to cytotoxic properties exerted on oral epithelial cells. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. The effects of erythromycin on the viability and the secretion of TNF-alpha and TGF-beta1 and expression of connexin43 by human pleural mesothelial cells.

    Science.gov (United States)

    Xie, Canmao; Huang, Jian Qiang; Light, Richard W

    2005-11-01

    The mechanism by which erythromycin produces pleurodesis remains unknown. The purpose of this study was to investigate the effects of erythromycin on human pleural mesothelial cell (HPMC) viability, the secretion of tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta(1) (TGF-beta(1)) and the level of expression of connexin43. HPMC were incubated with different concentrations of erythromycin. The inhibitory effects of erythromycin on HPMC growth were measured using a tetrazolium-based colorimetric assay. The levels of TNF-alpha and TGF-beta(1) in supernatants were measured by ELISA and levels of connexin43 were assessed by Western blot. Erythromycin injured HPMC in a dose and time-dependent manner. The secretion of both TNF-alpha and TGF-beta(1) by HMPC increased significantly when they were incubated with 100 mg/L erythromycin for 3 or 5 days. The levels of connexin43 in HPMC decreased after incubation with 100 mg/L erythromycin and no relationship was observed between the levels and incubation time. Erythromycin injures HPMC in a dose- and time-dependent manner and results in the secretion of TNF-alpha and TGF-beta(1). This is one possible mechanism of pleurodesis with erythromycin. Furthermore, erythromycin decreased the levels of connexin43 in HPMC, which could possibly affect the response of HPMC to pleurodesis with erythromycin.

  1. Electrochemical & osteoblast adhesion study of engineered TiO{sub 2} nanotubular surfaces on titanium alloys

    Energy Technology Data Exchange (ETDEWEB)

    Rahman, Zia Ur [School of Engineering and Technology, Central Michigan University, Mt. Pleasant, MI (United States); Haider, Waseem, E-mail: haide1w@cmich.edu [School of Engineering and Technology, Central Michigan University, Mt. Pleasant, MI (United States); Pompa, Luis [Department of Mechanical Engineering, University of Texas–Pan American, Edinburg, TX (United States); Deen, K.M. [Department of Metallurgy & Materials Engineering, CEET, University of the Punjab, 54590 Lahore (Pakistan); Department of Materials Engineering, University of British Columbia, Vancouver, BC V6T 1Z4 (Canada)

    2016-01-01

    TiO{sub 2} nanotubes were grafted on the surface of cpTi, Ti6Al4V and Ti6Al4V-ELI with the aim to provide a new podium for human pre-osteoblast cell (MC3T3) adhesion and proliferation. The surface morphology and chemistry of these alloys were examined with scanning electron microscopy and energy dispersive x-ray spectroscopy. TiO{sub 2} nanotubes were further characterized by cyclic potentiodynamic polarization tests and electrochemical impedance spectroscopy. The vertically aligned nanotubes were subjected to pre-osteoblast cell proliferation in order to better understand cell–material interaction. The study demonstrated that these cells interact differently with nanotubes of different titanium alloys. The significant acceleration in the growth rate of pre-osteoblast cell adhesion and proliferation is also witnessed. Additionally, the cytotoxicity of the leached metal ions was evaluated by using a tetrazolium-based bio-assay, MTS. Each group of data was operated for p < 0.05, concluded one way ANOVA to investigate the significance difference. - Highlights: • TiO{sub 2} nanotubes were grafted on cpTi, Ti6Al4V and Ti6Al4V-ELI via anodization. • MC3T3 cells interact differently with nanotubes of different titanium alloys. • TiO{sub 2} nanotubes have a positive impact on the osteoblast cell viability.

  2. PI3K/Akt Pathway Contributes to Neurovascular Unit Protection of Xiao-Xu-Ming Decoction against Focal Cerebral Ischemia and Reperfusion Injury in Rats

    Directory of Open Access Journals (Sweden)

    Rui Lan

    2013-01-01

    Full Text Available In the present study, we used a focal cerebral ischemia and reperfusion rat model to investigate the protective effects of Xiao-Xu-Ming decoction (XXMD on neurovascular unit and to examine the role of PI3K (phosphatidylinositol 3-kinase/Akt pathway in this protection. The cerebral ischemia was induced by 90 min of middle cerebral artery occlusion. Cerebral infarct area was measured by tetrazolium staining, and neurological function was observed at 24 h after reperfusion. DNA fragmentation assay, combined with immunofluorescence, was performed to evaluate apoptosis of neuron, astrocyte, and vascular endothelial cell which constitute neurovascular unit. The expression levels of proteins involved in PI3K/Akt pathway were detected by Western blot. The results showed that XXMD improved neurological function, decreased cerebral infarct area and neuronal damage, and attenuated cellular apoptosis in neurovascular unit, while these effects were abolished by inhibition of PI3K/Akt with LY294002. We also found that XXMD upregulated p-PDKl, p-Akt, and p-GSK3β expression levels, which were partly reversed by LY294002. In addition, the increases of p-PTEN and p-c-Raf expression levels on which LY294002 had no effect were also observed in response to XXMD treatment. The data indicated the protective effects of XXMD on neurovascular unit partly through the activation of PI3K/Akt pathway.

  3. Cytocompatibility of chitosan and collagen-chitosan scaffolds for tissue engineering

    Directory of Open Access Journals (Sweden)

    Ligia L. Fernandes

    2011-01-01

    Full Text Available In this work, chitosan and collagen-chitosan porous scaffolds were produced by the freeze drying method and characterized as potential skin substitutes. Their beneficial effects on soft tissues justify the choice of both collagen and chitosan. Samples were characterized using scanning electron microscope, Fourier Transform InfraRed Spectroscopy (FTIR and thermogravimetry (TG. The in vitro cytocompatibility of chitosan and collagen-chitosan scaffolds was evaluated with three different assays. Phenol and titanium powder were used as positive and negative controls, respectively. Scanning electron microscopy revealed the highly interconnected porous structure of the scaffolds. The addition of collagen to chitosan increased both pore diameter and porosity of the scaffolds. Results of FTIR and TG analysis indicate that the two polymers interact yielding a miscible blend with intermediate thermal degradation properties. The reduction of XTT ((2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide and the uptake of Neutral Red (NR were not affected by the blend or by the chitosan scaffold extracts, but the blend and the titanium powder presented greater incorporation of Crystal Violet (CV than phenol and chitosan alone. In conclusion, collagen-chitosan scaffolds produced by freeze-drying methods were cytocompatible and presented mixed properties of each component with intermediate thermal degradation properties.

  4. Synergistic effect of methionine encephalin (MENK) combined with pidotimod(PTD) on the maturation of murine dendritic cells (DCs).

    Science.gov (United States)

    Meng, Yiming; Wang, Qiushi; Zhang, Zhenjie; Wang, Enhua; Plotnikoff, Nicollas P; Shan, Fengping

    2013-04-01

    To gain new insight into the functional interaction between dendritic cells and methionine encephalin (MENK) combined with pidotimod (PTD), we have analyzed the effect of MENK plus PTD on the morphology, phenotype and functions of murine bone-marrow derived dendritic cells (BMDCs) in vitro. The maturation of BMDCs cultured in the presence of either MENK or PTD alone, or MENK in combination with PTD, was detected. The cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt/phenazinemethosulphate (MTS/PMS). The changes of BMDCs morphology were confirmed with light microscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The BMDCs treated with MENK combined with PTD displayed a higher expression of typical maturation markers of CD40, CD80, CD83, CD86 and MHC-IIidentified by fluorescence activated cell sorting (FACS), and stronger ability to drive T cells. The decrease of the endocytic ability was assayed by DAB kit, FITC-dextran and cellular immunohistochemistry. Finally upregulation of cytokines production of IL-12 and TNF-α was determined by ELISA. These data indicate that MENK combined with PTD could exert synergistic action on BMDC maturation.

  5. Injectable Polysaccharide Hydrogels as Biocompatible Platforms for Localized and Sustained Delivery of Antibiotics for Preventing Local Infections.

    Science.gov (United States)

    Li, Ziyi; He, Chaoliang; Yuan, Baoming; Dong, Xiaoming; Chen, Xuesi

    2017-04-01

    Biocompatible and antibacterial hydrogels have received increasing attention for preventing local bacterial infections. In this study, a type of polysaccharide hydrogels is prepared via the Schiff-based reaction at physiological conditions. The gelation time and mechanical property of the hydrogels are found to be dependent on the polysaccharide concentration and the polysaccharide weight ratio. 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and live/dead assay indicate that the hydrogels display nontoxicity in vitro. After subcutaneous injection into rats, the hydrogels exhibit an acceptable biocompatibility in vivo. Furthermore, the bacterial inhibition tests by shaking flask method and agar disc-diffusion method demonstrate that the ceftriaxone-sodium-loaded hydrogels have remarkable antibacterial properties in vitro. The in vivo anti-infective tests further display that the antibiotic-loaded hydrogels display excellent anti-infective efficacies in both superficial and deep tissue infection. Consequently, the injectable and biocompatible polysaccharide hydrogels may serve as promising platforms for localized, sustained delivery of antibiotics for preventing local infections. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Mapping myocardial viability using interleaved T1-T2* weighted imaging.

    Science.gov (United States)

    Li, Gang; Dai, Guangping; Xiang, Bo; Mark, John; Tomanek, Boguslaw; Liu, Hongyu; Deslauriers, Roxanne; Tian, Ganghong

    2004-04-01

    The present study was to evaluate the efficacy of our interleaved T1-T2* weighted imaging for assessing myocardial viability. The left anterior descending coronary artery (LAD) of pig hearts (n = 7) were occluded for 2 h, followed by 1 h reperfusion. After removed from animals, the hearts were perfused in a Langendorff apparatus with a mixture of pig blood and crystalloid solution in 1:1 ratio. T1 relaxation times of the myocardium were measured with a TurboFLASH inversion-recovery sequence. Gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA) (0.05 mmol/kg body wt) was then injected as a bolus into the aortic perfusion line. The first pass of the contrast agent through the heart was followed using the interleaved T1-T2* imaging sequence. Once the concentration of the contrast agent was in an equilibrium state, T1 relaxation times were measured again. It was found that the percentage recovery of T2* intensity (PRT2*) at the maximum T1 intensity measured during the first pass of the contrast agent with the interleaved T1-T2* imaging was significantly higher in infarcted myocardium than in normal myocardium. Moreover, the regions showing a high T2* percentage recovery on PRT2* maps matched well with the infarcted myocardium demarcated with triphenyl tetrazolium chloride (TTC) staining. We therefore conclude that infarcted myocardium can be delineated using the interleaved T1-T2* imaging method.

  7. Synthesis, characterization and in vitro anti-cancer evaluation of hesperetin-loaded nanoparticles in human oral carcinoma (KB) cells

    Science.gov (United States)

    Gurushankar, K.; Gohulkumar, M.; Rajendra Prasad, N.; Krishnakumar, N.

    2014-03-01

    Hesperetin (HET), a naturally occurring plant bioflavonoid present in citrus fruits, possesses potential anti-inflammatory and anti-carcinogenic activities but poor aqueous solubility limits its applications. To improve its applicability in cancer therapy, hesperetin was encapsulated in Eudragit® E (EE) 100 nanoparticles in the presence of polyvinyl alcohol (PVA) as a stabilizer and its anticancer efficacy in oral carcinoma (KB) cells was studied. Hesperetin-loaded nanoparticles (HETNPs) were prepared by nanoprecipitation method and characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), Fourier transform infrared (FT-IR) spectroscopy, differential scanning calorimetry (DSC), and x-ray diffraction (XRD). The results thus displayed that the prepared nanoparticles showed a particle size in the range from 55 to 180 nm. The encapsulation efficiency of hesperetin was 83.4% obtained by UV spectroscopy. The in vitro release kinetics of hesperetin under physiological condition show initial rapid release followed by slow and sustained release. 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay revealed higher cytotoxic efficacy of HETNPs than native hesperetin in KB cells. Further, it has been found that reactive oxygen species (ROS) generation, DNA damage and apoptotic indices in HETNPs treated cells are greater than those in native hesperetin treatment. Hence these findings demonstrate that HETNPs could be a potentially useful drug delivery system to produce better hesperetin therapeutics of cancers.

  8. Thin-layer chromatographic (TLC) separations and bioassays of plant extracts to identify antimicrobial compounds.

    Science.gov (United States)

    Kagan, Isabelle A; Flythe, Michael D

    2014-03-27

    A common screen for plant antimicrobial compounds consists of separating plant extracts by paper or thin-layer chromatography (PC or TLC), exposing the chromatograms to microbial suspensions (e.g. fungi or bacteria in broth or agar), allowing time for the microbes to grow in a humid environment, and visualizing zones with no microbial growth. The effectiveness of this screening method, known as bioautography, depends on both the quality of the chromatographic separation and the care taken with microbial culture conditions. This paper describes standard protocols for TLC and contact bioautography with a novel application to amino acid-fermenting bacteria. The extract is separated on flexible (aluminum-backed) silica TLC plates, and bands are visualized under ultraviolet (UV) light. Zones are cut out and incubated face down onto agar inoculated with the test microorganism. Inhibitory bands are visualized by staining the agar plates with tetrazolium red. The method is applied to the separation of red clover (Trifolium pratense cv. Kenland) phenolic compounds and their screening for activity against Clostridium sticklandii, a hyper ammonia-producing bacterium (HAB) that is native to the bovine rumen. The TLC methods apply to many types of plant extracts and other bacterial species (aerobic or anaerobic), as well as fungi, can be used as test organisms if culture conditions are modified to fit the growth requirements of the species.

  9. Isolation and Partial Characterization of Bioactive Fucoxanthin from Himanthalia elongata Brown Seaweed: A TLC-Based Approach.

    Science.gov (United States)

    Rajauria, Gaurav; Abu-Ghannam, Nissreen

    2013-01-01

    Seaweeds are important sources of carotenoids, and numerous studies have shown the beneficial effects of these pigments on human health. In the present study, Himanthalia elongata brown seaweed was extracted with a mixture of low polarity solvents, and the crude extract was separated using analytical thin-layer chromatography (TLC). The separated compounds were tested for their potential antioxidant capacity and antimicrobial activity against Listeria monocytogenes bacteria using TLC bioautography approach. For bio-autography, the coloured band on TLC chromatogram was visualized after spraying with DPPH and triphenyl-tetrazolium chloride reagents which screen antioxidant and antimicrobial compounds, respectively, and only one active compound was screened on the TLC plate. Preliminary identification of this active compound was done by comparing its colour and R f (retention factor) value with the authentic fucoxanthin standard. Further, the active compound was purified using preparative TLC. This purified compound showed a strong antioxidant (EC50: 14.8 ± 1.27 µg/mL) and antimicrobial (inhibition zone: 10.27 mm, 25 µg compound/disc) activities, which were examined by DPPH scavenging and agar disc-diffusion bioassay, respectively. The bioactivity shown by the purified compound was almost similar to the fucoxanthin standard. The characteristic UV-visible and FT-IR spectra of the purified active compound completely matched with the standard. Hence, the main active compound in H. elongata was identified as fucoxanthin.

  10. Rapid synthesis of silver nanoparticles byPseudomonas stutzeriisolated from textile soil under optimised conditions and evaluation of their antimicrobial and cytotoxicity properties.

    Science.gov (United States)

    Rajora, Nishant; Kaushik, Sanket; Jyoti, Anupam; Kothari, Shanker L

    2016-12-01

    Present study utilised textile soil isolated bacterium Pseudomonas stutzeri to synthesise extracellular silver nanoparticles (AgNPs) under optimised conditions. The synthesised AgNPs were characterised using ultraviolet-visible spectroscopy, Fourier transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM). Optimisation showed AgNPs synthesis within 8 h using 2mM Ag nitrate at pH9, temperature 80°C and maximum absorbance toward 400 nm. TEM analysis revealed spherical shape AgNPs and reduction in size upto 8 nm was observed under optimised conditions. FTIR spectra confirmed presence of proteins bound to AgNPs act as reducing agent. AgNPs showed strong antibacterial activity against multi-drug resistant (MDR) Escherichia coli and Klebsiella pneumoniae as demonstrated by disc diffusion and colony forming unit assays. Zone of inhibition increased with increasing concentration of AgNPs with maximum of 19 mm against E. coli and 17 mm against K. pneumoniae at concentration of 2 μg/disc. Furthermore, AgNPs did not show any cytotoxic effects on human epithelial cells as demonstrated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay even at 2 μg/ml concentration of AgNPs. The results of the present study suggest that AgNPs can be synthesised rapidly under optimised conditions and show strong antimicrobial property against MDR pathogens without having toxicity effect on human epithelial cells.

  11. Electromagnetic field-induced converse cell growth during a long-term observation.

    Science.gov (United States)

    Bae, Ji-Eun; Do, Ji-Yeon; Kwon, Soon-Hwan; Lee, Sang-Dae; Jung, Yong Woo; Kim, Soo-Chan; Chae, Kwon-Seok

    2013-12-01

    Professional and public concern about the potential adverse effects of man-made electromagnetic fields (EMF) on the human body has dramatically expanded in recent years. Despite numerous attempts to investigate this issue, the long-standing challenge of reproducibility surrounding alternating EMF effects on human health remains unresolved. Our chief aim was to investigate a plausible mechanism for this phenomenon. Growth of cultured human cancer cells, DU145 and Jurkat, exposed to power frequency magnetic field (MF) (60 Hz, 1 mT) for 3 days, was determined using a 2-(4-Iodophenyl)- 3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) assay and a trypan blue exclusion assay. This experiment was repeated at incubators long-term monitoring period up to 5.3 years. A periodogram analysis was performed to investigate periodic patterns in the MF and sham effects on cell growth. Unlike conventional assumptions, the MF effect on growth in both cell types was promotive or suppressive in a period-dependent manner. The converse cell growth induced by the MF was consistent in incubators, with little variation. Spatiotemporal evidence suggests that the period-dependent converse cell growth by the MF may contribute to the poor reproducibility and explain the adverse effects observed in previous experimental and epidemiological investigations. Additionally, the novel approach of this study may be applied to design features required to experimentally determine the effects of EMF on living organisms in a convincing manner.

  12. Fructus panax ginseng extract promotes hair regeneration in C57BL/6 mice.

    Science.gov (United States)

    Park, Soojin; Shin, Weon-Sun; Ho, Jinnyoung

    2011-11-18

    Radix panax ginseng (Panax ginseng C.A. Meyer, Araliaceae, RPG) has been documented to possess hair growth activity and widely used to treat alopecia, while no report has been issued to date on the effect of Fructus panax ginseng (FPG) on hair regeneration. To investigate the effects of FPG extract on the proliferation of human hair dermal papilla cells (DPCs) and on the promotion of hair regeneration in C57BL6 mice, cell proliferation was evaluated in cultured DPC by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and measured the expressions of Bcl-2 and Bax by immunoblot assay. We also compared the effects of topical FPG extract (1 and 10 mg/ml, 100 μl/d) with the effects of minoxidil as a positive control (5%, 100 μl/d) or vehicle control (30% ethanol) on the depilation-induced hair cycling in 7 week-old-C57BL/6 mice. FPG extract significantly increased the proliferation of DPCs in dose and time dependent manners (Pregeneration activity for the treatment of hair loss. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  13. Oxoglaucine-lanthanide complexes: synthesis, crystal structure and cytotoxicity.

    Science.gov (United States)

    Liu, Yan-Cheng; Chen, Zhen-Feng; Shi, Yan-Fang; Huang, Ke-Bin; Geng, Bo; Liang, Hong

    2014-01-01

    To evaluate the in vitro cytotoxicity of oxoglaucine (OG) complexes: [Sm(OG)2(NO3)3]•H2O (1), [Eu(OG)2(NO3)3]•1.5CH3OH (2) and [Er(OG)2(NO3)3]•H2O (3) through comparison to oxoglaucine and lanthanide salts. The reactions of OG with corresponding lanthanide salts gave rise to complexes 1-3. The crystal structures of complexes 1-3 were determined by single-crystal X-ray diffraction analysis. The in vitro cytotoxicity of oxoglaucine and complexes 1-3 against five human cancer cell lines were evaluated by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium Bromide (MTT) method. Complexes 1-3 have similar mononuclear structures. The 50% inhibitory concentration (IC50) of complex 1 against SGC7901 cells was 32.1 μM; that of complex 2 against MCF-7 cells was 3.2 μM; those of complex 3 on HeLa and MCF-7 cells were 8.3 and 1.4 μM, respectively. The three OG-lanthanide complexes exhibited significantly enhanced cytotoxicity vs. OG and corresponding lanthanide salts.

  14. Tumor-specific RNA interference targeting Pokemon suppresses tumor growth and induces apoptosis in prostate cancer.

    Science.gov (United States)

    Li, Yining; Xu, Shuxiong; Wang, Xiangwei; Shi, Hua; Sun, Zhaolin; Yang, Zhao

    2013-02-01

    To explore the exact mechanism of Pokemon in prostate cancer. Pokemon is a member of the POK family of transcriptional repressors. Its main function is suppression of the p14ARF (alternate reading frame) tumor suppressor gene. Although Pokemon expression has been found to be increased in various types of lymphoma, the exact mechanism of the gene in prostate cancer is not clear. In the present study, prostate cancer cells were transfected with the specific short hairpin ribonucleic acid (RNA) expression vector targeting Pokemon. The expression of Pokemon messenger RNA and its protein was detected by semiquantitative reverse transcriptase-polymerase chain reaction and Western blotting, respectively. The cell growth and cell apoptosis were also examined using the methyl thiazolyl tetrazolium assay and flow cytometry. The results demonstrated that specific RNA interference (RNAi) could decrease the expression levels of Pokemon gene messenger RNA and protein in prostate cancer cells. In addition, that specific RNAi significantly inhibited the cell proliferation and increased the apoptotic rate. In vivo experiments showed that specific RNAi inhibited the tumorigenicity of prostate cancer cells and significantly suppressed tumor growth. Therefore, an RNAi-targeted Pokemon gene strategy could be a potential approach to prostate cancer therapy. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Synthesis, Characterization, and Cytotoxic Activities of a Schiff Base Ligand and Its Binuclear Copper(II and Manganese(III Complexes

    Directory of Open Access Journals (Sweden)

    Zafer Uyar

    2017-09-01

    Full Text Available A novel symmetrical N2O2 type Schiff base (1 and its copper (II (2 and manganese (III (3 complexes were synthesized and characterized by spectroscopic, analytical, and magnetic susceptibility studies. Spectroscopic and magnetic susceptibility studies suggested that copper and manganese ions are in 2+ and 3+ states and their complexes have a binuclear double stranded helical structure in the form of 2:2 (metal to ligand stoichiometry. Cytotoxic effects of the ligand and its metal complexes against MCF-7 (human breast cancer cell line, DLD-1 (human colorectal cancer cell line, ECC-1 (human endometrium cancer cell line, DU-145 (human prostate cancer line, MDA-MB231 (human breast cancer cell line, PC-3 (human prostate cancer line and HEK293 (normal cells were evaluated by determining their cellular viability using the colorimetric 3-(4,5- dimethylthiazole-2-yl-2,5-biphenyl tetrazolium bromide (MTT assay. It has been found that cytotoxicity of the ligand was significantly enhanced towards cancer cells and declined towards normal HEK293 cells by metal chelation. Copper complex yielded better results in comparison with manganese complex. Particularly, copper complex showed a selective cytotoxicity, harming the cancerous cell lines while not impairing the normal cells, which is considered as the key to the future of cytotoxic therapy.

  16. Tetrandrine, a Compound Common in Chinese Traditional Medicine, Preferentially Kills Breast Cancer Tumor Initiating Cells (TICs) In Vitro

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Wei; Debeb, Bisrat G.; Lacerda, Lara; Li, Jessica; Woodward, Wendy A., E-mail: wwoodward@mdanderson.org [Division of Radiation Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030 (United States)

    2011-05-04

    Tetrandrine is a bisbenzylisoquinoline alkaloid found in Stephania tetrandra, a Chinese medicine commonly used as an anti-inflammatory. It has extensive pharmacological activity, including positive ion channel blockade and inhibition of multiple drug resistance proteins. These activities are very similar to that of salinomycin, a known drug targeting breast cancer initiation cells (TICs). Herein, we tested tetrandrine targeting of breast cancer TICs. SUM-149, an inflammatory breast cancer cell line and SUM-159, a non-inflammatory metaplastic breast cancer cell line were used in these studies. In proliferation assays using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl) -2H-tetrazolium (MTS), we found that the IC{sub 50} for inhibition of proliferation is 15.3 ± 4.1 μM for SUM-149 and 24.3 ± 2.1 μM for SUM-159 cells. Tetrandrine also inhibited mammosphere formation, a surrogate for breast cancer TICs growth in vitro with IC{sub 50} around 1 μM for SUM-149 and around 2 μM for SUM-159 cells. Tetrandrine has similar effects on the mammosphere formation from cells isolated from fresh patient sample. Moreover, tetrandrine decreases the aldehyde dehydrogenase (ALDH) positive population in SUM-159 by 45% ± 5.45% P = 0.005. In summary, tetrandrine demonstrates significant efficacy against in vitro surrogates for inflammatory and aggressive breast cancer TICs.

  17. Necrotic and apoptotic cell death of human malignant melanoma cells following photodynamic therapy using an amphiphilic photosensitizer, ATX-S10(Na).

    Science.gov (United States)

    Nagata, Satoshi; Obana, Akira; Gohto, Yuko; Nakajima, Susumu

    2003-01-01

    To investigate the phototoxic effect on and cell death modes of human malignant melanoma cells following photodynamic therapy (PDT) using ATX-S10(Na), an amphiphilic photosensitizer. Cultured human malignant melanoma cells were incubated in a medium containing various concentrations of ATX-S10(Na) and irradiated with a 670 nm wavelength diode laser. Phototoxicity was analyzed by a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay, and cell death modes were investigated by fluorescence microscopy using a Hoechst 33342-propidium iodide double-staining method as well as by static gel electrophoresis. The subcellular localization of ATX-S10(Na) and mitochondrial destabilization following PDT were observed by fluorescence microscopy. Higher phototoxicity was obtained with higher dye and/or laser doses. Most of the dead cells appeared apoptotic with dye and irradiation doses that induced less than 70% cytotoxicity. In contrast, most of them appeared necrotic with doses that induced 99% cytotoxicity. Cells receiving PDT showed disturbances of mitochondrial trans-membrane potential, although the primary site of ATX-S10(Na) accumulation was in lysosomes. ATX-S10(Na) has a phototoxic effect on malignant melanoma cells and, therefore, potential as a photosensitizing agent for PDT designed to kill these cells. Apoptotic pathways may be activated via mitochondrial destabilization following the damage of lysosomes by PDT. Further study, including investigation of therapeutic efficacy in vivo, is warranted. Copyright 2003 Wiley-Liss, Inc.

  18. Acetoxyroyleanone exhibits selective anti-cancer effects and induces apoptosis in human colon carcinoma cells through the mediation of NF-κB and caspase-3 signalling pathways

    Directory of Open Access Journals (Sweden)

    Xiao-Jun Zhong

    2016-03-01

    Full Text Available The objective of the current study was to evaluate the antiproliferative and apoptotic activities of acetoxyroyleanone against various cancer cells along with studying its effect on chromatin condensation, NF-κB and caspase-3 expressions and cell migration. 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide assay was used to evaluate cell viability while as fluorescence microscopy revealed the effects of acetoxyroyleanone on cellular morphology of Colo-205 cells. Western blotting revealed effects on NF-κB and caspase-3 expressions. The results revealed that acetoxyroyleanone induced potent and dose-dependent antiproliferative effects against a range of cancer cell lines with Colo-205 being the most susceptible cell line. However, it required six to eight times higher concentration of acetoxyroyleanone to induce 50% cell death in normal epithelial (fR-2 cell line. Further, following acetoxyroyleanone treatment to Colo-205, it was observed that acetoxy-royleanone induced substantial down-regulation of NF-κB and up-regulation of caspase-3 expressions. In addition, acetoxyroyleanone impairs cell migration, chromatin condensation, cell shrinkage and membrane blebbing.

  19. Facile Formation of Redox-Active Totally Organic Nanoparticles in Water by In Situ Reduction of Organic Precursors Stabilized through Aromatic-Aromatic Interactions by Aromatic Polyelectrolytes.

    Science.gov (United States)

    Flores, Mario E; Garcés-Jerez, Pablo; Fernández, Daniel; Aros-Perez, Gustavo; González-Cabrera, Diego; Álvarez, Eduardo; Cañas, Ignacio; Oyarzun-Ampuero, Felipe; Moreno-Villoslada, Ignacio

    2016-11-01

    The formation of redox-active, totally organic nanoparticles in water is achieved following a strategy similar to that used to form metal nanoparticles. It is based on two fundamental concepts: i) complexation through aromatic-aromatic interactions of a water-soluble precursor aromatic molecule with polyelectrolytes bearing complementary charged aromatic rings, and ii) reduction of the precursor molecule to achieve stabilized nanoparticles. Thus, formazan nanoparticles are synthesized by reduction of a tetrazolium salt with ascorbic acid using polyelectrolytes bearing benzene sulfonate residues of high linear aromatic density, but cannot be formed in the presence of nonaromatic polyelectrolytes. The red colored nanoparticles are efficiently encapsulated in calcium alginate beads, showing macroscopic homogeneity. Bleaching kinetics with chlorine show linear rates on the order of tenths of milli-meters per minute. A linear behavior of the dependence of the rate of bleaching on the chlorine concentration is found, showing the potential of the nanoparticles for chlorine sensing. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Shed Syndecan-1 is involved in chemotherapy resistance via the EGFR pathway in colorectal cancer.

    Science.gov (United States)

    Wang, X; Zuo, D; Chen, Y; Li, W; Liu, R; He, Y; Ren, L; Zhou, L; Deng, T; Wang, X; Ying, G; Ba, Y

    2014-11-11

    Syndecan-1 (Sdc-1) shedding induced by matrix metalloproteinase-7 (MMP-7) and additional proteases has an important role in cancer development. However, the impact of Sdc-1 shedding on chemotherapeutic resistance has not been reported. We examined Sdc-1 shedding in colorectal cancer by enzyme-linked immunosorbent assay (ELISA), Dot blot, reverse transcription-PCR (RT-PCR), immunohistochemistry and so on, its impact on chemotherapeutic sensitivity by collagen gel droplet embedded culture-drug sensitivity test (CD-DST) and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), and potential mechanisms of action by Dot blot, western blot and immunofluorescence. Sdc-1 shedding was increased in colorectal cancer patients, Sdc-1 serum levels in postoperative patients were lower than in preoperative patients, but still higher than those observed in healthy adults. Patients with high preoperative Sdc-1 serum levels were less responsive to 5-Fluorouracil, Oxaliplatin, Irintecan, Cisplatin or Paclitaxel chemotherapy. Moreover, the disease-free survival of patients with high preoperative Sdc-1 serum levels was significantly poorer. The possible mechanism of chemotherapy resistance in colorectal cancer can be attributed to Sdc-1 shedding, which enhances EGFR phosphorylation and downstream signalling. Shed Sdc-1 is involved in chemotherapy resistance via the EGFR pathway in colorectal cancer, and Sdc-1 serum levels could be a new prognostic marker in colorectal cancer.

  1. Both Creatine and Its Product Phosphocreatine Reduce Oxidative Stress and Afford Neuroprotection in an In Vitro Parkinson’s Model

    Directory of Open Access Journals (Sweden)

    Mauricio Peña Cunha

    2014-10-01

    Full Text Available Creatine is the substrate for creatine kinase in the synthesis of phosphocreatine (PCr. This energetic system is endowed of antioxidant and neuroprotective properties and plays a pivotal role in brain energy homeostasis. The purpose of this study was to investigate the neuroprotective effect of creatine and PCr against 6-hydroxydopamine (6-OHDA-induced mitochondrial dysfunction and cell death in rat striatal slices, used as an in vitro Parkinson’s model. The possible involvement of the signaling pathway mediated by phosphatidylinositol-3 kinase (PI3K, protein kinase B (Akt, and glycogen synthase kinase-3β (GSK3β was also evaluated. Exposure of striatal slices to 6-OHDA caused a significant disruption of the cellular homeostasis measured as 3-(4,5 dimethylthiazol-2-yl-2,5-diphenyl-tetrazolium bromide reduction, lactate dehydrogenase release, and tyrosine hydroxylase levels. 6-OHDA exposure increased the levels of reactive oxygen species and thiobarbituric acid reactive substances production and decreased mitochondrial membrane potential in rat striatal slices. Furthermore, 6-OHDA decreased the phosphorylation of Akt (Serine473 and GSK3β (Serine9. Coincubation with 6-OHDA and creatine or PCr reduced the effects of 6-OHDA toxicity. The protective effect afforded by creatine or PCr against 6-OHDA-induced toxicity was reversed by the PI3K inhibitor LY294002. In conclusion, creatine and PCr minimize oxidative stress in striatum to afford neuroprotection of dopaminergic neurons.

  2. Antiproliferative and cytotoxic effects of purple pitanga (Eugenia uniflora L.) extract on activated hepatic stellate cells.

    Science.gov (United States)

    Denardin, Cristiane C; Parisi, Mariana M; Martins, Leo A M; Terra, Silvia R; Borojevic, Radovan; Vizzotto, Márcia; Perry, Marcos L S; Emanuelli, Tatiana; Guma, Fátima T C R

    2014-01-01

    The presence of phenolic compounds in fruit- and vegetable-rich diets has attracted researchers' attention due to their health-promoting effects. The objective of this study was to evaluate the effects of purple pitanga (Eugenia uniflora L.) extract on cell proliferation, viability, mitochondrial membrane potential, cell death and cell cycle in murine activated hepatic stellate cells (GRX). Cell viability by 3-(4,5-dimethylthiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was significantly decreased on cells treated with 50 and 100 µg ml(-1) of purple pitanga extract for 48 and 72 h, and the percentage of dead cell stained with 7-amino-actinomycin D was significantly higher in treated cells. The reduction of cell proliferation was dose dependent, and we also observed alterations on cell cycle progression. At all times studied, GRX cells treated with 50 and 100 µg ml(-1) of purple pitanga showed a significant reduction in cellular mitochondrial content as well as a decrease in mitochondrial membrane potential. Furthermore, our results indicated that purple pitanga extract induces early and late apoptosis/necrosis and necrotic death in GRX cells. This is the first report describing the antiproliferative, cytotoxic and apoptotic activity for E. uniflora fruits in hepatic stellate cells. The present study provides a foundation for the prevention and treatment of liver fibrosis, and more studies will be carried to elucidate this effect. Copyright © 2013 John Wiley & Sons, Ltd.

  3. CONSERVATION OF THE VIABILITY AND VIGOR OF Araucaria angustifolia (Bert. O. Kuntze SEEDS DURING THE STORAGE

    Directory of Open Access Journals (Sweden)

    Cristhyane Garcia

    2014-12-01

    Full Text Available The conservation of Araucaria seeds is widely compromised in function of their recalcitrant feature, which hampers the planning of recovery actions of the degraded populations. Therefore, the objective of this study was to monitor the physiological changes in Araucaria seeds under controlled storage conditions, in order to get insights as to the viability and vigor conservation. The physiological quality of freshly harvested seeds was evaluated and every 60 days throughout the 180 days-storage period in laboratory ambient without thermal control, refrigerator (5 ° C, and freezer (-18 ° C until the final period of 180 days. After each sampling period, the seed viability (germination and tetrazolium tests and vigor (artificial aging, germination speed index – IVG and electrical conductivity were assessed. A reduction in the normal seedlings percentage was noticed over the period of storage of Araucaria seeds. The conservation in freezer and the lack of thermal control caused a complete loss of the seed viability at 60 and 180 days of storage, respectively. However, the refrigerator storage promoted the conservation of seed viability, with 64% germination after 180 days of storage, an event associated with the reduction of the metabolic activity of seeds. Based on the viability and vigor tests, it was concluded that storage in refrigerator provided longer storage periods to Araucaria seeds in comparison to the other storage conditions herein studied.

  4. CONSERVAÇÃO DA VIABILIDADE E VIGOR DE SEMENTES DE Araucaria angustifolia (Bert. O. Kuntze DURANTE O ARMAZENAMENTO

    Directory of Open Access Journals (Sweden)

    Cristhyane Garcia

    2014-01-01

    Full Text Available The conservation of Araucaria seeds is widely compromised in function of their recalcitrant feature, which hampers the planning of recovery actions of the degraded populations. Therefore, the objective of this study was to monitor the physiological changes in Araucaria seeds under controlled storage conditions, in order to get insights as to the viability and vigor conservation. The physiological quality of freshly harvested seeds was evaluated and every 60 days throughout the 180 days-storage period in laboratory ambient without thermal control, refrigerator (5 ° C, and freezer (-18 ° C until the final period of 180 days. After each sampling period, the seed viability (germination and tetrazolium tests and vigor (artificial aging, germination speed index – IVG and electrical conductivity were assessed. A reduction in the normal seedlings percentage was noticed over the period of storage of Araucaria seeds. The conservation in freezer and the lack of thermal control caused a complete loss of the seed viability at 60 and 180 days of storage, respectively. However, the refrigerator storage promoted the conservation of seed viability, with 64% germination after 180 days of storage, an event associated with the reduction of the metabolic activity of seeds. Based on the viability and vigor tests, it was concluded that storage in refrigerator provided longer storage periods to Araucaria seeds in comparison to the other storage conditions herein studied.

  5. Ni ion release, osteoblast-material interactions, and hemocompatibility of hafnium-implanted NiTi alloy.

    Science.gov (United States)

    Zhao, Tingting; Li, Yan; Zhao, Xinqing; Chen, Hong; Zhang, Tao

    2012-04-01

    Hafnium ion implantation was applied to NiTi alloy to suppress Ni ion release and enhance osteoblast-material interactions and hemocompatibility. The auger electron spectroscopy, x-ray photoelectron spectroscopy, and atomic force microscope results showed that a composite TiO(2)/HfO(2) nanofilm with increased surface roughness was formed on the surface of NiTi, and Ni concentration was reduced in the superficial surface layer. Potentiodynamic polarization tests displayed that 4 mA NiTi sample possessed the highest E(br) - E(corr), 470 mV higher than that of untreated NiTi, suggesting a significant improvement on pitting corrosion resistance. Inductively coupled plasma mass spectrometry tests during 60 days immersion demonstrated that Ni ion release rate was remarkably decreased, for example, a reduction of 67% in the first day. The water contact angle increased and surface energy decreased after Hf implantation. Cell culture and methyl-thiazol-tetrazolium indicated that Hf-implanted NiTi expressed enhanced osteoblasts adhesion and proliferation, especially after 7 days culture. Hf implantation decreased fibrinogen adsorption, but had almost no effect on albumin adsorption. Platelets adhesion and activation were suppressed significantly (97% for 4 mA NiTi) and hemolysis rate was decreased by at least 57% after Hf implantation. Modified surface composition and morphology and decreased surface energy should be responsible for the improvement of cytocompatibility and hemocompatibility. Copyright © 2011 Wiley Periodicals, Inc.

  6. Reactive oxygen intermediates from eosinophils in mice infected with Hymenolepis nana.

    Science.gov (United States)

    Niwa, A; Miyazato, T

    1996-06-01

    A large number of eosinophils were recruited to the intestinal villi after infection with Hymenolepis nana. Eosinophil numbers were increased more rapidly in challenged mice than in primary infected mice. Local intestinal eosinophils from challenged mice showed more extracellular oxygen radical release, as assessed by histochemical methods using nitro blue tetrazolium, accompanied with tissue injury and larval degradation. Intestinal eosinophils isolated from the lamina propria induced specific oxygen radical generation in response to H. nana oncosphere extract as measured by luminol-dependent chemiluminescence. This response was stronger in challenged mice than in primary infected mice. Radical generation from uninfected mice was negligible. Lipid peroxidation in the small intestine, as measured by formation of malondialdehyde, was increased during H. nana challenge infection, the peak activity coinciding with the elimination of challenge larvae. Continuous administration of a NADPH oxidase inhibitor to sensitized mice interfered with the degeneration of challenge larvae. These results suggest that intestinal eosinophils may be the major contributor to oxygen radical production in response to H. nana and that reactive oxygen species may play a part of effector molecule in the resistance to reinfection with H. nana.

  7. Onset of Phloem Export from Senescent Petals of Daylily.

    Science.gov (United States)

    Bieleski, R. L.

    1995-10-01

    During senescence, petals of attached daylily (Hemerocallis hybrid cv Cradle Song) flowers lost 95% sugar and 65% dry weight over the first 24 h, with 30% of dry weight loss coming from nonsugar components. Detaching flowers did not delay senescence, but halted loss of carbohydrate and amino acid, suggesting that loss in the intact state was due to phloem export. Petal autolysis occurred mainly in the interveinal parenchyma, causing vascular strands to begin separating from the petal mass. Such vascular strands still stained with tetrazolium and accumulated sucrose, indicating a retained viability. Their sucrose accumulation rates were high in comparison with those of other plant tissues, and the accumulated product was mainly sucrose. Sucrose synthesis took place in the senescent petal, and sucrose was the principal sugar in phloem exudate, whereas hydroxyproline and glutamine were the main transport amino acids. [14C]Sucrose applied to attached senescent flowers was rapidly translocated to other parts of the plant, particularly developing flower buds. Thus, onset of phloem export allowed most of the soluble carbohydrate and amino acid in the senescing flower to be retrieved by the plant. Additional salvaged material came from proteins and possibly from structural carbohydrate. Over a 12-h period, the flower switched from acting as a strong carbohydrate sink during expansion to become a strong source during senescence. This rapid reversal offers potential for phloem transport studies.

  8. Apoptosis of rat hepatic stellate cells induced by diallyl trisulfide and proteomics profiling in vitro.

    Science.gov (United States)

    Zhang, Yajie; Zhou, Xiaoming; Xu, Lipeng; Wang, Lulu; Liu, Jinling; Ye, Jing; Qiu, Pengxin; Liu, Qinghua

    2017-05-01

    Diallyl trisulfide (DATS), a major garlic derivative, inhibits cell proliferation and triggers apoptosis in a variety of cancer cell lines. However, the effects of DATS on hepatic stellate cells (HSCs) remain unknown. The aim of this study was to analyze the effects of DATS on cell proliferation and apoptosis, as well as the protein expression profile in rat HSCs. Rat HSCs were treated with or without 12 and 24 μg/mL DATS for various time intervals. Cell proliferation and apoptosis were determined using tetrazolium dye (MTT) colorimetric assay, bromodeoxyuridine (5-bromo-2'-deoxyuridine; BrdU) assay, Hoechst 33342 staining, electroscopy, and flow cytometry. Protein expression patterns in HSCs were systematically studied using 2-dimensional electrophoresis and mass spectrometry. DATS inhibited cell proliferation and induced apoptosis of HSCs in a time-dependent manner. We observed clear morphological changes in apoptotic HSCs and dramatically increased annexin V-positive - propidium iodide negative apoptosis compared with the untreated control group. Twenty-one significant differentially expressed proteins, including 9 downregulated proteins and 12 upregulated proteins, were identified after DATS administration, and most of them were involved in apoptosis. Our results suggest that DATS is an inducer of apoptosis in HSCs, and several key proteins may be involved in the molecular mechanism of apoptosis induced by DATS.

  9. Conjugated Linoleic Acid Stimulates Apoptosis in RH and Tehran Strains of Toxoplasma gondii, in Vitro.

    Directory of Open Access Journals (Sweden)

    Jebreil Shamseddin

    2015-06-01

    Full Text Available The aim of this study was to evaluate the effects of conjugated linoleic acid (CLA on apoptosis of tachyzoites of T. gondii, RH strain (type I and the cyst-forming Tehran strain (type II in vitro.Toxoplasma strains were injected into the peritoneal cavity of BALB/c mice. The Tehran strain forms cysts in the brain of mice. Bradyzoites within the cysts are reactivated to proliferative tachyzoites, by dexamethasone. Tachyzoites were aspirated from the peritoneum of infected mice, and the percentage of viable parasites was estimated with trypan blue staining. Tachyzoites were inoculated into HeLa cells cultivated in DMEM medium. Different concentrations of CLA were evaluated on T. gondii in HeLa cells by the tetrazolium (MTT colorimetric assay. Differentiation between apoptosis and cell death was determined by flow cytometry using Annexin V and propidium iodide (PI double staining. The statistical analysis performed by GraphPad Prism version 6.00.CLA induces apoptosis in virulent (RH and avirulent (Tehran strains of T. gondii. The results of MTT indicated that CLA could decrease the proliferation of tachyzoites of both strains in HeLa cells.Conjugated linoleic acid has anti-toxoplasmacidal activity on tachyzoites of T. gondii. Therefore, we recommended further studies on this component in order to achieve a new drug against the parasite.

  10. Tramadol inhibits proliferation, migration and invasion via α2-adrenoceptor signaling in breast cancer cells.

    Science.gov (United States)

    Xia, M; Tong, J-H; Zhou, Z-Q; Duan, M-L; Xu, J-G; Zeng, H-J; Wang, S-H

    2016-01-01

    The aim of this study was to examine the function of tramadol on cell proliferation, migration and invasion in breast cancer cells in vitro, and to evaluate the effect of tramadol in vivo. Further, we explore the mechanism accounting for the role of tramadol on breast cancer cells. Cell proliferation was detected by the methyl thiazolyl tetrazolium (MTT) assay. Wound healing assay and transwell assay was applied to quantify the migration and invasion ability of MDA-MB-231 cells. The expression of endogenous α2-adrenoceptor and ERK was measured by Western blotting. Tramadol at a clinical dose of up to 2 μM significantly inhibited the proliferation, migration and invasion in a time-dependent manner from day 0 to 28 in vitro. Moreover, tramadol suppressed the growth of xenotransplant tumor in vivo markedly. Furthermore, the protein levels of α2-adrenoceptor and phosphorylated ERK were decreased by tramadol, whereas the expression of total ERK remained unchanged. In addition, downregulation of α2-adrenoceptor by yohimbine could mimic the effect of tramadol treatment. Collectively, we demonstrated that tramadol could inhibit proliferation, migration and invasion of breast cancers via inactivating α2-adrenoceptor signaling pathway. Our data provide the experimental fundamental for further investigation of the anti-cancer effect of tramadol in breast cancer cells.

  11. Effects of hesperidin loaded poly(lactic-co-glycolic acid) scaffolds on growth behavior of costal cartilage cells in vitro and in vivo.

    Science.gov (United States)

    Cho, Sun Ah; Cha, Se Rom; Park, Sang Mi; Kim, Kyoung Hee; Lee, Hyun Gu; Kim, Eun Young; Lee, Dongwon; Khang, Gilson

    2014-01-01

    It has been widely accepted that costal cartilage cells (CCs) have more excellent initial proliferation capacity than articular cartilage cells. Biodegradable synthetic polymer poly(lactic-co-glycolic acid) (PLGA) was approved by Food and Drug Administration. Hesperidin has antifungal, antiviral, antioxidant, anti-inflammatory, and anticarcinogenic properties. Hesperidin loaded (0, 3, 5, and 10 wt.%) PLGA scaffolds were prepared and in vitro and in vivo properties were characterized. Scaffolds were seeded with CCs isolated from rabbit, which were kept in culture to harvest for histological analysis. Hesperidin/PLGA scaffolds were also implanted in nude mice for 7 and 28 days. Assays of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfo-phenyl)-2H-tetrazolium, monosodium salt (WST), and scanning electron microscope were carried out to evaluate attachment and proliferation of CCs in hesperidin/PLGA scaffolds. Glycosaminoglycan assay was performed to confirm the effects of hesperidin on extracellular matrix formation. Reverse-transcriptase polymerase chain reaction was carried out to confirm the expression of the specific genes for CCs. In these results, we demonstrated that cell attachment and proliferation on hesperidin/PLGA scaffolds were more excellent compared with on PLGA scaffold. Specially, 5 wt.% hesperidin/PLGA scaffold represented the best results among other scaffolds. Thus, 5 wt.% hesperidin/PLGA scaffold will be applicable to tissue engineering cartilage.

  12. Changes in the expression of pituitary adenylate cyclase-activating polypeptide in the human placenta during pregnancy and its effects on the survival of JAR choriocarcinoma cells.

    Science.gov (United States)

    Brubel, R; Boronkai, A; Reglodi, D; Racz, B; Nemeth, J; Kiss, P; Lubics, A; Toth, G; Horvath, G; Varga, T; Szogyi, D; Fonagy, E; Farkas, J; Barakonyi, A; Bellyei, Sz; Szereday, L; Koppan, M; Tamas, A

    2010-11-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide with survival-promoting actions, has been observed in endocrine organs and is thought to play a role in reproductive functions, including pregnancy. PACAP occurs in two forms, 27 and 38 amino acid residues, with PACAP38 being the predominant form in human tissues. In the present study, we determined the concentrations of PACAP38 and PACAP27 in first-trimester and full-term human placentas using radioimmunoassay. We found high levels of PACAP38 and lower levels of PACAP27 in different parts of the full-term human placenta. PACAP38 content increased in the placenta during pregnancy, both on the maternal side and on the fetal side. The effects of PACAP on the survival of JAR human choriocarcinoma cells were investigated using flow cytometry and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) cell viability assay in cells exposed to the widely used chemotherapeutic agent methotrexate (MTX). It was found that PACAP neither influenced the survival of JAR cytotrophoblast cells nor affected cellular response to the death-inducing effect of the chemotherapeutic agent MTX. The present observations further support the significance of PACAP in the human placenta. The observation that PACAP did not influence the effects of MTX may have future clinical importance, showing that PACAP does not decrease the effects of certain chemotherapeutic agents.

  13. Increasing of antioxidant and superoxide dismutase activity in chicory transgenic plants

    Directory of Open Access Journals (Sweden)

    Matvieieva N. A.

    2013-03-01

    Full Text Available Aim. Determination of the antioxidant activity (AOA and superoxide dismutase (SOD activity in transgenic chicory plants carrying the human interferon α2b target and nptII or bar selective genes. Methods. AOA was measured by a method based on the determination of kinetics of the reduced 2,6-dichlorophenolindophenol oxidation. SOD activity was assayed using the system consisting of ethionine, riboflavin, and nitroblue tetrazolium. Results. Antioxidant activity of transformed plants extracts was more than 1,91–2,59 and 2,04–2,43 times over the activity of control non-transgenic plants (at nptII and bar gene presence respectively. SOD activity was higher in transgenic plants than in the control, and was 2,03 ± 0,46–3,33 ± 0,54 U/g weight (nptII gene and 2,25 ± 0,46–2,68 ± 0,08 U/g weight (bar gene. Conclusions. Transgenic C. intybus plants have higher antioxidant and superoxide dismutase activity compared to non-transgenic plants. The increasing of AOA and SOD activity is a response of plants to transformation stress factor and integration of foreign genes in plant genome.

  14. Calcium Hydroxide-induced Proliferation, Migration, Osteogenic Differentiation, and Mineralization via the Mitogen-activated Protein Kinase Pathway in Human Dental Pulp Stem Cells.

    Science.gov (United States)

    Chen, Luoping; Zheng, Lisha; Jiang, Jingyi; Gui, Jinpeng; Zhang, Lingyu; Huang, Yan; Chen, Xiaofang; Ji, Jing; Fan, Yubo

    2016-09-01

    Calcium hydroxide has been extensively used as the gold standard for direct pulp capping in clinical dentistry. It induces proliferation, migration, and mineralization in dental pulp stem cells (DPSCs), but the underlying mechanisms are still unclear. The aim of this study was to investigate the role of the mitogen-activated protein (MAP) kinase pathway in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. Human DPSCs between passages 3 and 6 were used. DPSCs were preincubated with inhibitors of MAP kinases and cultured with calcium hydroxide. The phosphorylated MAP kinases were detected by Western blot analysis. Cell viability was analyzed via the methylthiazol tetrazolium assay. Cell migration was estimated using the wound healing assay. Alkaline phosphatase (ALP) expression was analyzed using the ALP staining assay. Mineralization was studied by alizarin red staining analysis. Calcium hydroxide significantly promoted the phosphorylation of the c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase. The inhibition of JNK and p38 signaling abolished calcium hydroxide-induced proliferation of DPSCs. The inhibition of JNK, p38, and extracellular signal-regulated kinase signaling suppressed the migration, ALP expression, and mineralization of DPSCs. Our study showed that the MAP kinase pathway was involved in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  15. Biocompatibility of Accelerated Mineral Trioxide Aggregate on Stem Cells Derived from Human Dental Pulp.

    Science.gov (United States)

    Kulan, Pinar; Karabiyik, Ozge; Kose, Gamze T; Kargul, Betul

    2016-02-01

    The aim of this study was to evaluate the effects of several additives on the setting time and cytotoxicity of accelerated-set mineral trioxide aggregate (MTA) on stem cells of human dental pulp. ProRoot white MTA (WMTA) (Dentsply Tulsa Dental, Johnson City, TN) was mixed with various additives including distilled water, 2.5% disodium hydrogen phosphate (Na2HPO4) (Merck, Darmstadt, Germany), K-Y Jelly (Johnson & Johnson, Markham, ON, Canada), and 5% and 10% calcium chloride (CaCl2) (Merck). The setting times were evaluated using a Vicat apparatus (Alsa Lab, Istanbul, Turkey). Human dental pulp stem cells were isolated and seeded into 48-well plates at 2 × 10(3) cells per well and incubated with MTA samples for 24 hours, 3 days, and 7 days. Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. MTA mixed with 10% CaCl2 showed the lowest setting time (P cell viability at all time points (P cell viability of MTA mixed with distilled water, 5% CaCl2, 10% CaCl2, and Na2HPO4 increased significantly through time (P dental pulp stem cells in terms of cell viability. Further in vitro and in vivo investigations are required to prove the clinical applications of MTA mixed with various additives. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  16. Antitumor and Antiviral Activity of Colombian Medicinal Plant Extracts

    Directory of Open Access Journals (Sweden)

    Betancur-Galvis LA

    1999-01-01

    Full Text Available Extracts of nine species of plants traditionally used in Colombia for the treatment of a variety of diseases were tested in vitro for their potential antitumor (cytotoxicity and antiherpetic activity. MTT (Tetrazolium blue and Neutral Red colorimetric assays were used to evaluate the reduction of viability of cell cultures in presence and absence of the extracts. MTT was also used to evaluate the effects of the extracts on the lytic activity of herpes simplex virus type 2 (HSV-2. The 50% cytotoxic concentration (CC50 and the 50% inhibitory concentration of the viral effect (EC50 for each extract were calculated by linear regression analysis. Extracts from Annona muricata, A. cherimolia and Rollinia membranacea, known for their cytotoxicity were used as positive controls. Likewise, acyclovir and heparin were used as positive controls of antiherpetic activity. Methanolic extract from Annona sp. on HEp-2 cells presented a CC50 value at 72 hr of 49.6x103mg/ml. Neither of the other extracts examined showed a significant cytotoxicity. The aqueous extract from Beta vulgaris, the ethanol extract from Callisia grasilis and the methanol extract Annona sp. showed some antiherpetic activity with acceptable therapeutic indexes (the ratio of CC50 to EC50. These species are good candidates for further activity-monitored fractionation to identify active principles.

  17. Electrospun polyurethane membranes for Tissue Engineering applications.

    Science.gov (United States)

    Gabriel, Laís P; Rodrigues, Ana Amélia; Macedo, Milton; Jardini, André L; Maciel Filho, Rubens

    2017-03-01

    Tissue Engineering proposes, among other things, tissue regeneration using scaffolds integrated with biological molecules, growth factors or cells for such regeneration. In this research, polyurethane membranes were prepared using the electrospinning technique in order to obtain membranes to be applied in Tissue Engineering, such as epithelial, drug delivery or cardiac applications. The influence of fibers on the structure and morphology of the membranes was studied using scanning electron microscopy (SEM), the structure was evaluated by Fourier transform infrared spectroscopy (FT-IR), and the thermal stability was analyzed by thermogravimetry analysis (TGA). In vitro cells attachment and proliferation was investigated by SEM, and in vitro cell viability was studied by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays and Live/Dead® assays. It was found that the membranes present an homogeneous morphology, high porosity, high surface area/volume ratio, it was also observed a random fiber network. The thermal analysis showed that the membrane degradation started at 254°C. In vitro evaluation of fibroblasts cells showed that fibroblasts spread over the membrane surface after 24, 48 and 72h of culture. This study supports the investigation of electrospun polyurethane membranes as biocompatible scaffolds for Tissue Engineering applications and provides some guidelines for improved biomaterials with desired properties. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Immunization with a novel chimeric peptide representing B and T cell epitopes from HER2 extracellular domain (HER2 ECD) for breast cancer.

    Science.gov (United States)

    Mahdavi, Manijeh; Keyhanfar, Mehrnaz; Jafarian, Abbas; Mohabatkar, Hassan; Rabbani, Mohammad

    2014-12-01

    Because of direct stimulating immune system against disease, vaccination or active immunotherapy is preferable compared to passive immunotherapy. For this purpose, a newly designed chimeric peptide containing epitopes for both B and T cells from HER2 ECD subdomain III was proposed. To evaluate the effects of the active immunization, a discontinuous B cell epitope peptide was selected based on average antigenicity by bioinformatics analysis. The selected peptide was collinearly synthesized as a chimera with a T helper epitope from the protein sequence of measles virus fusion (208-302) using the GPSL linker. Three mice were immunized with the chimeric peptide. Reactive antibodies with HER2 protein in ELISA and immunofluorescence assays with no cross-reactivity were generated. The 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay indicated that the anti-peptide sera had inhibitory effects on proliferation of SK-BR-3 cells. Hence, the newly designed, discontinuous chimeric peptide representing B and T cell epitopes from subdomain III of HER2-ECD can form the basis for future vaccines design, where these data can be applied for monoclonal antibody production targeting the distinct epitope of HER2 receptor compared to the two broadly used anti-HER2 monoclonal antibodies, Herceptin and pertuzumab.

  19. In Vitro Biological Evaluation of 3-D Hydroxyapatite/Collagen (50/50 wt. (% Scaffolds

    Directory of Open Access Journals (Sweden)

    Doris Moura Campos

    2012-02-01

    Full Text Available Hydroxyapatite-collagen (HA/Col composites are potential scaffolds for bone tissue engineering. In this work, three-dimensional (3-D HA/Col (50/50 wt. (% scaffolds were synthesized using a self-assembly method and cross-linked with a 0.125% glutaraldehyde solution. Scaffolds were evaluated in vitro by cytotoxicity testing using MC3T3 cells; proliferation and differentiation were studied using STRO-1A human stromal cells for up to 21 days. Morphological and histological examinations showed a fibrous structure with a good distribution and homogeneous HA particles distribution. By thermogravimetric analysis, a ratio of 1.2 between inorganic and organic phase was found. The scaffolds presented no cytotoxicity when evaluated using three different parameters of cell survival and integrity: 2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl] -2H-tetrazolium-5-carboxanilide (XTT, Neutral Red (NR and Crystal Violet Dye Elution (CVDE. STRO-1A cells were found to adhere, proliferate and differentiate on the 3-D scaffold, but limited cell penetration was observed.

  20. Aluminum Templates of Different Sizes with Micro-, Nano- and Micro/Nano-Structures for Cell Culture

    Directory of Open Access Journals (Sweden)

    Ming-Liang Yen

    2017-10-01

    Full Text Available This study investigates the results of cell cultures on aluminum (Al templates with flat-structures, micro-structures, nano-structures and micro/nano-structures. An Al template with flat-structure was obtained by electrolytic polishing; an Al template with micro-structure was obtained by micro-powder blasting; an Al template with nano-structure was obtained by aluminum anodization; and an Al template with micro/nano-structure was obtained by micro-powder blasting and then anodization. Osteoblast-like cells were cultured on aluminum templates with various structures. The microculture tetrazolium test assay was utilized to assess the adhesion, elongation, and proliferation behaviors of cultured osteoblast-like cells on aluminum templates with flat-structures, micro-structures, nano-structures, and micro/nano-structures. The results showed that the surface characterization of micro/nano-structure of aluminum templates had superhydrophilic property, and these also revealed that an aluminum template with micro/nano-structure could provide the most suitable growth situation for cell culture.

  1. Investigation of photobiomodulation potentiality by 635 and 809 nm lasers on human osteoblasts.

    Science.gov (United States)

    Bölükbaşı Ateş, Gamze; Ak Can, Ayşe; Gülsoy, Murat

    2017-04-01

    Photobiomodulation (PBM) describes light-induced photochemical reactions achieved by the application of red or near infrared lasers/LED light with low energy densities. This noninvasive and painless method has been used in some clinical areas but controversial outcomes demand a skeptical look for its promising and potential effects. In this detailed in vitro study, the osteoblast cells were irradiated with 635 and 809 nm diode lasers at energy densities of 0.5, 1, and 2 J/cm2. Cell viability, proliferation, bone formation, and osteoblast differentiation were evaluated by methylthiazole tetrazolium (MTT) assay, Alamar Blue assay, acridine orange/propidium iodide staining, alkaline phosphatase (ALP) activity, Alizarin red staining, and reverse-transcription polymerase chain reaction (RT-PCR) to test the expression of collagen type I, ALPL, and osteocalcin. The results indicate that studied energy doses have a transient effect (48 h after laser irradiation) on the osteoblast viability and proliferation. Similarly, laser irradiation did not appear to have any effect on ALP activity. These results were confirmed by RT-PCR analysis of osteoblast markers. This study suggests that several irradiation parameters and variations in the methods should be clearly established in the laboratory before laser treatment becomes a postulated application for bone tissue regeneration in clinical level.

  2. Rapid prototyping: porous titanium alloy scaffolds produced by selective laser melting for bone tissue engineering.

    Science.gov (United States)

    Warnke, Patrick H; Douglas, Timothy; Wollny, Patrick; Sherry, Eugene; Steiner, Martin; Galonska, Sebastian; Becker, Stephan T; Springer, Ingo N; Wiltfang, Jörg; Sivananthan, Sureshan

    2009-06-01

    Selective laser melting (SLM), a method used in the nuclear, space, and racing industries, allows the creation of customized titanium alloy scaffolds with highly defined external shape and internal structure using rapid prototyping as supporting external structures within which bone tissue can grow. Human osteoblasts were cultured on SLM-produced Ti6Al4V mesh scaffolds to demonstrate biocompatibility using scanning electron microscopy (SEM), fluorescence microscopy after cell vitality staining, and common biocompatibility tests (lactate dihydrogenase (LDH), 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), 5-bromo-2-deoxyuridine (BrdU), and water soluble tetrazolium (WST)). Cell occlusion of pores of different widths (0.45-1.2 mm) was evaluated. Scaffolds were tested for resistance to compressive force. SEM investigations showed osteoblasts with well-spread morphology and multiple contact points. Cell vitality staining and biocompatibility tests confirmed osteoblast vitality and proliferation on the scaffolds. Pore overgrowth increased during 6 weeks' culture at pore widths of 0.45 and 0.5 mm, and in the course of 3 weeks for pore widths of 0.55, 0.6, and 0.7 mm. No pore occlusion was observed on pores of width 0.9-1.2 mm. Porosity and maximum compressive load at failure increased and decreased with increasing pore width, respectively. In summary, the scaffolds are biocompatible, and pore width influences pore overgrowth, resistance to compressive force, and porosity.

  3. Laser Phototherapy Enhances Mesenchymal Stem Cells Survival in Response to the Dental Adhesives

    Directory of Open Access Journals (Sweden)

    Ivana Márcia Alves Diniz

    2015-01-01

    Full Text Available Background. We investigated the influence of laser phototherapy (LPT on the survival of human mesenchymal stem cells (MSCs submitted to substances leached from dental adhesives. Method. MSCs were isolated and characterized. Oral mucosa fibroblasts and osteoblast-like cells were used as comparative controls. Cultured medium conditioned with two adhesive systems was applied to the cultures. Cell monolayers were exposed or not to LPT. Laser irradiations were performed using a red laser (GaAlAs, 780 nm, 0.04 cm2, 40 mW, 1 W/cm2, 0.4 J, 10 seconds, 1 point, 10 J/cm2. After 24 h, cell viability was assessed by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide reduction assay. Data were statistically compared by ANOVA followed by Tukey’s test (P<0.05. Results. Different cell types showed different viabilities in response to the same materials. Substances leached from adhesives were less cytotoxic to MSCs than to other cell types. Substances leached from Clearfil SE Bond were highly cytotoxic to all cell types tested, except to the MSCs when applied polymerized and in association with LPT. LPT was unable to significantly increase the cell viability of fibroblasts and osteoblast-like cells submitted to the dental adhesives. Conclusion. LPT enhances mesenchymal stem cells survival in response to substances leached from dental adhesives.

  4. In vitro proliferation and osteogenic differentiation of endometrial stem cells and dental pulp stem cells.

    Science.gov (United States)

    Tabatabaei, Fahimeh Sadat; Torshabi, Maryam

    2017-06-01

    Stem-cell-based therapies were introduced aiming to overcome the limitations of the existing procedures for regeneration of mineralized tissues. Stem cells isolated from the endometrial tissue and dental pulp have the capacity to differentiate into various functional cells including osteoblasts. However, studies comparing their ability to regenerate mineralized tissue are lacking. The purpose of this study was to compare the proliferation and osteogenic differentiation potential of endometrial stem cells (EnSCs) and dental pulp stem cells (DPSCs) using in vitro cell culture technique. The DPSCs and EnSCs were isolated from human dental pulp and endometrium, respectively. Their proliferation and osteogenic potential were compared in the same osteogenic medium (OM) after 3, 5, 7 and 10 days using the methyl thiazol tetrazolium assay, alizarin red staining, and real-time quantitative reverse transcription polymerase chain reaction (Real-Time qRT-PCR). The EnSCs showed higher proliferation rate compared to DPSCs. Regarding osteogenesis, alizarin red-positive colonies appeared earlier and in greater amounts in DPSCs group. The real-time qRT-PCR demonstrated significantly greater osteogenic potential of DPSCs compared to EnSCs. Our findings revealed significant differences in stem cell properties based on the tissue source. The EnSCs had greater proliferation rate than DPSCs, while DPSCs showed greater osteogenic potential compared to EnSCs in the same OM.

  5. Biofilm formation by Fusarium oxysporum f. sp. cucumerinum and susceptibility to environmental stress.

    Science.gov (United States)

    Peiqian, Li; Xiaoming, Pu; Huifang, Shen; Jingxin, Zhang; Ning, Huang; Birun, Lin

    2014-01-01

    To the authors' knowledge, most studies on biofilm formation have focused on bacteria and yeasts. So far, biofilm formation by fungal plant pathogen has not been reported. In this study, the biofilm-forming capacity of Fusarium oxysporum f. sp. cucumerinum was evaluated. For biofilm quantification, a colorimetric 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium-hydroxide (XTT) reduction assay was used to observe metabolic activity. Fluorescence and confocal scanning laser microscopy revealed that the biofilms have a highly heterogeneous architecture composed of robust hyphae and extracellular polysaccharide materials. Additionally, the influence of physical factors on F. oxysporum biofilm formation and the susceptibility of biofilms to environmental stress was investigated. Biofilms were less susceptible to heat, cold, UV light and three fungicides than were their planktonic counterparts. Our findings may provide a novel perspective on the pathogenic mechanism associated with biofilms of F. oxysporum f. sp. cucumerinum. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  6. Development of a high-throughput colorimetric Zika virus infection assay.

    Science.gov (United States)

    Müller, Janis A; Harms, Mirja; Schubert, Axel; Mayer, Benjamin; Jansen, Stephanie; Herbeuval, Jean-Philippe; Michel, Detlef; Mertens, Thomas; Vapalahti, Olli; Schmidt-Chanasit, Jonas; Münch, Jan

    2017-04-01

    Zika virus (ZIKV) is an emerging pathogen that causes congenital infections which may result in birth defects, such as microcephaly. Currently, no approved treatment or vaccination is available. ZIKV can be readily detected in cell culture where virally infected cells are normally stained by specific antibodies. As ZIKV regularly causes a cytopathic effect, we were wondering whether this viral property can be used to quantitatively determine viral infectivity. We here describe the use of an 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide-(MTT)-based cell viability assay that allows to determine ZIKV-induced cell death. We show that this colorimetric assay quantifies ZIKV infection over a broad range of viral dilutions in both monkey and human cells. It allows to determine inhibitory activities of antivirals that block ZIKV or to define the neutralizing antibody titers of ZIKV antisera. This MTT-based ZIKV detection assay can be evaluated by naked eye or computational tools, has a broad linear range, does not require large equipment or costly reagents, and thus represents a promising alternative to antibody-based assays, in particular in resource-poor settings. We propose to use this simple, fast, and cheap method for quantification of ZIKV neutralizing antibodies and testing of antiviral compounds.

  7. Antibacterial activity of Thymoquinone, an active principle of Nigella sativa and its potency to prevent bacterial biofilm formation

    Directory of Open Access Journals (Sweden)

    Bakhrouf Amina

    2011-04-01

    Full Text Available Abstract Background Thymoquinone is an active principle of Nigella sativa seed known as "Habbah Al-Sauda" in Arabic countries and "Sinouj" in Tunisia. Bacterial biofilms tend to exhibit significant tolerance to antimicrobials drugs during infections. Methods The antibacterial activity of Thymoquinone (TQ and its biofilm inhibition potencies were investigated on 11 human pathogenic bacteria. The growth and development of the biofilm were assessed using the crystal violet (CV and the 2, 3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT reduction assay. Results TQ exhibited a significant bactericidal activity against the majority of the tested bacteria (MICs values ranged from 8 to 32 μg/ml especially Gram positive cocci (Staphylococcus aureus ATCC 25923 and Staphylococcus epidermidis CIP 106510. Crystal violet assay demonstrated that the minimum biofilm inhibition concentration (BIC50 was reached with 22 and 60 μg/ml for Staphylococcus aureus ATCC 25923 and Staphylococcus epidermidis CIP 106510 respectively. In addition our data revealed that cells oxidative activity was influenced by TQ supplementation. In the same way, TQ prevented cell adhesion to glass slides surface. Conclusion The ability of TQ to prevent biofilm formation warrants further investigation to explore its use as bioactive substances with antibiofilm potential.

  8. Cytotoxicity of different extracts of arial parts of Ziziphus spina-christi on Hela and MDA-MB-468 tumor cells

    Science.gov (United States)

    Jafarian, Abbas; Zolfaghari, Behzad; Shirani, Kobra

    2014-01-01

    Background: It has been shown that plants from the family Rhamnaceae possess anticancer activity. In this study, we sought to determine if Ziziphus spina-christi, a species from this family, has cytotoxic effect on cancer cell lines. Materials and Methods: Using maceration method, different extracts of leaves of Z. spina-christi were prepared. Hexane, chloroform, chloroform-methanol (9:1), methanol-water (7:1) methanol, butanol and water were used for extraction, after preliminary phytochemical analyses were done. The cytotoxic activity of the extracts against Hela and MDA-MB-468 tumor cells was evaluated by MTT assay. Briefly, cells were seeded in microplates and different concentrations of extracts were added. After incubation of cells for 72 h, their viability was evaluated by addition of tetrazolium salt solution. After 3 h medium was aspirated, dimethyl sulfoxide was added and absorbance was determined at 540 nm with an ELISA plate reader. Extracts were considered cytotoxic when more than 50% reduction on cell survival was observed. Results: Hexane, chloroform, chloroform-methanol, butanol, methanol-water and aqueous extracts of Z. spina-christi significantly and concentration-dependently reduced viability of Hela and MAD-MB-468 cells. In the both cell lines, chloroform-methanol extract of Z. spina-christi was more potent than the other extracts. Results: From the finding of this study it can be concluded that Z. spina-christi is a good candidate for further study for new cytotoxic agents. PMID:24627846

  9. Synthesis and Anticancer Activities of 4-[(Halophenyldiazenyl]phenol and 4-[(Halophenyldiazenyl]phenyl Aspirinate Derivatives against Nasopharyngeal Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Boon Kui Ho

    2017-01-01

    Full Text Available Aspirin and azo derivatives have been widely studied and have drawn considerable attention due to diverse biological activities. In this study, a series of 4-[(halophenyldiazenyl]phenyl aspirinate derivatives were synthesized from the reaction of aspirin with 4-[(halophenyldiazenyl]phenol via esterification, in the presence of DCC/DMAP in DCM with overall yield of 45–54%. 4-[(Halophenyldiazenyl]phenol was prepared prior to esterification from coupling reaction of aniline derivatives and phenol in basic solution. All compounds were characterized using elemental analysis, FTIR, and 1H and 13C NMR spectroscopies. All compounds were screened for their anticancer activities against nasopharyngeal cancer (NPC HK-1 cell lines and the viability of cultured cells was determined by MTS [3-(4,5-dimethylthiazol-2-yl-5-(3-carboxylmethoxylphenyl-2-(4-sulfophenyl-2H-tetrazolium]-based colorimetric assay. 4-[(E-(Fluorophenyldiazenyl]phenol showed the highest anticancer activity against NPC HK-1 cell lines compared to other synthesized compounds. 4-[(Halophenyldiazenyl]phenyl aspirinate showed low cytotoxicity against NPC HK-1 cell lines compared to 4-[(halophenyldiazenyl]phenol but better anticancer activity than aspirin alone.

  10. Reduction of Oxidative Stress Attenuates Lipoapoptosis Exacerbated by Hypoxia in Human Hepatocytes

    Directory of Open Access Journals (Sweden)

    Sang Youn Hwang

    2015-02-01

    Full Text Available Chronic intermittent hypoxia, a characteristic of obstructive sleep apnea (OSA, is associated with the progression of simple hepatic steatosis to necroinflammatory hepatitis. We determined whether inhibition of a hypoxia-induced signaling pathway could attenuate hypoxia-exacerbated lipoapoptosis in human hepatocytes. The human hepatocellular carcinoma cell line (HepG2 was used in this study. Palmitic acid (PA-treated groups were used for two environmental conditions: Hypoxia (1% O2 and normoxia (20% O2. Following the treatment, the cell viability was determined by the 3,4-(5-dimethylthiazol-2-yl-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl-2H-tetrazolium salt (MTS assay, and the mechanism of lipoapoptosis was evaluated by Western blotting. Hypoxia exacerbated the suppression of hepatocyte growth induced by palmitic acid via activation of mitochondrial apoptotic pathways as a result of endoplasmic reticulum (ER and oxidative stresses. Ammonium pyrrolidine dithiocarbamate, a scavenger of reactive oxygen species, attenuated the hypoxia-exacerbated lipoapoptosis in hepatocytes, whereas glycerol, which reduces ER stress, did not. This may have been because inhibition of oxidative stress decreases the expression of pro-apoptotic proteins, such as caspase 9 and cytochrome c. These results suggested that modulation of apoptotic signaling pathways activated by oxidative stress can aid in identifying novel therapeutic strategies for the treatment of nonalcoholic steatohepatitis (NASH with OSA. Further in vivo studies are necessary to understand the pathophysiologic mechanism of NASH with OSA and to prove the therapeutic effect of the modulation of the signaling pathways.

  11. Biocompatibility of two novel root repair materials.

    Science.gov (United States)

    Ma, Jingzhi; Shen, Ya; Stojicic, Sonja; Haapasalo, Markus

    2011-06-01

    The purpose of the present study was to evaluate the biocompatibility of 2 root-end filling materials, Endosequence Root Repair Material Putty (ERRM Putty) and Paste (ERRM Paste) and compare them with gray mineral trioxide aggregate (MTA). ERRM Putty, ERRM Paste, MTA, intermediate restorative material (IRM), and Cavit G were tested. For cytotoxicity assay, human gingival fibroblasts were incubated for 1, 3, and 7 days with extracts of varying concentrations from materials set for 2 days or 7 days. Cell viability was evaluated by methyl-thiazol-tetrazolium (MTT) assay. For cell adhesion assay, materials set for 7 days were examined under scanning electron microscope directly after setting, after incubation in cell culture medium for 7 days, and after incubation in gingival fibroblast suspension at a density of 5 × 10(4) cells/well for 2 and 7 days. The constituents of crystals formed on surface of materials were determined by energy dispersive analysis by x-ray. Cell viability was significantly correlated with the type of material, setting time, and incubation time (P Cavit G were significantly lower than with the other 3 materials (P < .001). Similar surface crystallographic features and cell adhesion were observed on ERRM Paste, ERRM Putty, and MTA. ERRM Putty and ERRM Paste displayed similar in vitro biocompatibility to MTA. Copyright © 2011 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  12. Candida species from oral cavity of HIV-infected children exhibit reduced virulence factors in the HAART era.

    Science.gov (United States)

    Portela, Maristela Barbosa; Lima de Amorim, Elaine; Santos, Adrielle Mangabeira; Alexandre da Rocha Curvelo, José; de Oliveira Martins, Karol; Capillé, Cauli Lima; Maria de Araújo Soares, Rosangela; Barbosa de Araújo Castro, Gloria Fernanda

    2017-01-01

    This study aimed to assess, in vitro, the biofilm viability and the phospholipase and protease production of Candida spp. from the saliva of HIV infected children and healthy controls, and to correlate the results with the use of medical data. A total of 79 isolates were analyzed: 48 Candida albicans isolates (33/15) and 20 Candida parapsilosis sensu lato complex isolates (12/8) (from HIV/control patients, respectively), and 8 Candida krusei, 1 Candida tropicalis, 1 Candida dubliniensis and 1 Candida guilliermondii from HIV patients. The XTT (2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-Carboxanilide) reduction assay analyzed the biofilm viability. Phospholipase and protease assays were performed using the egg yolk and Bovine Serum Albumin agar plate methods, respectively. All isolates were able to form biofilm with cell viability. Quantitatively, Candida isolates from both groups presented a similar ability to form biofilm (p > 0.05). The biofilm viability activity was higher in C. albicans isolates than in non-albicans Candida isolates (p Candida spp. isolates from HIV-positive children presented higher phospholipase production, in vitro they exhibited reduced virulence factors compared to isolates from healthy individuals. This finding may enlighten the role played by immunosuppression in the modulation of Candida virulence attributes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Development of a novel bioactive glass suitable for osteosarcoma-related bone grafts.

    Science.gov (United States)

    Rahimnejad Yazdi, Alireza; Torkan, Lawrence; Waldman, Stephen D; Towler, Mark R

    2017-05-31

    In this study, zinc borate-based glasses with increasing gallium content (0, 2.5, 5, 10, and 15 wt % Ga) were synthesized and their effect on the viability and proliferation of preosteoblasts and osteosarcoma cancer cells were investigated. Methyl thiazolyl tetrazolium (MTT) cell viability assays using glass degradation extracts revealed that the extracts from glasses with lower Ga contents could enhance the viability of preosteoblasts, while extracts from the glass composition with 15 wt % Ga caused statistically significant reduction of their viability. MTT cell viability assays using the extracts and osteosarcoma cells showed that only extracts from the glass composition with 5 wt % Ga (G3) did not cause a statistically significant increase in the viability of cancer cells for all degradation periods (1 day, 7 days, and 28 days). G3 was selected as the most suitable composition for the osteosarcoma-related graft operations as it could improve the viability of preosteoblasts without increasing the viability of cancer cells. The viability of preosteoblasts and osteosarcoma cells in contact with the glass powders were also investigated using MTT assays. The results showed that the G3 powders could enhance the viability of preosteoblasts while decreasing the viability of osteosarcoma cells. Finally, live/dead assays revealed that suppression of proliferation appeared to be the mechanism causing the observed reductions in the viability of osteosarcoma cells exposed to G3 powders. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2017. © 2017 Wiley Periodicals, Inc.

  14. In vitro cytotoxic and antibacterial potentials of extracts from three marine isolates of Actinomycetes isolated from coastal ecosystems of Tanur, Kerala, India

    Directory of Open Access Journals (Sweden)

    Prashanthi Kuruvalli

    2015-03-01

    Full Text Available Three Actinomyctes with potential bioactivity are successfully isolated from the marine water samples and identified as Prauserella marina, Streptomyces sindenensis and S. spiroverticillatus. The ethyl acetate extracts from the three Actinomycetes are found to have significant bioactivity. The highest anti-bacterial activity was given by the extract from P. marina on B. cereus showing 28 mm of zone of inhibition. Cytotoxicity screening of the crude extracts using 3-(4, 5-dimethylthiazol-2yl-2, 5-diphenyl tetrazolium bromide (MTT cell viability assay revealed that extract from P. marina noticeably effected the viability of the human cervical cancer cell grown in vitro. Thin layer chromatography of the crude extract with methanol and chloroform (8:2 as solvent system yielded three distinct fractions, of which fraction with Rf value 0.8 resulted in 77, 68, 54 and 40% growth inhibition of HeLa cells at 15, 10, 5, 2.5 µg/mL, respectively with the IC50 value as 3.3 µg/mL. HPLC analysis of the fraction resulted in single major peak at 3.7 min.

  15. Anticancer Activity of a Hexapeptide from Skate (Raja porosa) Cartilage Protein Hydrolysate in HeLa Cells.

    Science.gov (United States)

    Pan, Xin; Zhao, Yu-Qin; Hu, Fa-Yuan; Chi, Chang-Feng; Wang, Bin

    2016-08-16

    In this study, the hexapeptide Phe-Ile-Met-Gly-Pro-Tyr (FIMGPY), which has a molecular weight of 726.9 Da, was separated from skate (Raja porosa) cartilage protein hydrolysate using ultrafiltration and chromatographic methods, and its anticancer activity was evaluated in HeLa cells. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay indicated that FIMGPY exhibited high, dose-dependent anti-proliferation activities in HeLa cells with an IC50 of 4.81 mg/mL. Acridine orange/ethidium bromide (AO/EB) fluorescence staining and flow cytometry methods confirmed that FIMGPY could inhibit HeLa cell proliferation by inducing apoptosis. Western blot assay revealed that the Bax/Bcl-2 ratio and relative intensity of caspase-3 in HeLa cells treated with 7-mg/mL FIMGPY were 2.63 and 1.83, respectively, significantly higher than those of the blank control (p skate cartilage protein hydrolysate may have applications as functional foods and nutraceuticals for the treatment and prevention of cancer.

  16. Anticancer Activity of a Hexapeptide from Skate (Raja porosa Cartilage Protein Hydrolysate in HeLa Cells

    Directory of Open Access Journals (Sweden)

    Xin Pan

    2016-08-01

    Full Text Available In this study, the hexapeptide Phe-Ile-Met-Gly-Pro-Tyr (FIMGPY, which has a molecular weight of 726.9 Da, was separated from skate (Raja porosa cartilage protein hydrolysate using ultrafiltration and chromatographic methods, and its anticancer activity was evaluated in HeLa cells. Methylthiazolyldiphenyl-tetrazolium bromide (MTT assay indicated that FIMGPY exhibited high, dose-dependent anti-proliferation activities in HeLa cells with an IC50 of 4.81 mg/mL. Acridine orange/ethidium bromide (AO/EB fluorescence staining and flow cytometry methods confirmed that FIMGPY could inhibit HeLa cell proliferation by inducing apoptosis. Western blot assay revealed that the Bax/Bcl-2 ratio and relative intensity of caspase-3 in HeLa cells treated with 7-mg/mL FIMGPY were 2.63 and 1.83, respectively, significantly higher than those of the blank control (p < 0.01. Thus, FIMGPY could induce apoptosis by upregulating the Bax/Bcl-2 ratio and caspase-3 activation. Using a DNA ladder method further confirmed that the anti-proliferation activity of FIMGPY was attributable to its role in inducing apoptosis. These results suggest that FIMGPY from skate cartilage protein hydrolysate may have applications as functional foods and nutraceuticals for the treatment and prevention of cancer.

  17. Preparation, in vitro mineralization and osteoblast cell response of electrospun 13-93 bioactive glass nanofibers.

    Science.gov (United States)

    Deliormanlı, Aylin M

    2015-08-01

    In this study, silicate based 13-93 bioactive glass fibers were prepared through sol-gel processing and electrospinning technique. A precursor solution containing poly (vinyl alcohol) and bioactive glass sol was used to produce fibers. The mixture was electrospun at a voltage of 20 kV by maintaining tip to a collector distance of 10 cm. The amorphous glass fibers with an average diameter of 464±95 nm were successfully obtained after calcination at 625 °C. Hydroxyapatite formation on calcined 13-93 fibers was investigated in simulated body fluid (SBF) using two different fiber concentrations (0.5 and 1 mg/ml) at 37 °C. When immersed in SBF, conversion to a calcium phosphate material showed a strong dependence on the fiber concentration. At 1mg/ml, the surface of the fibers converted to the hydroxyapatite-like material in SBF only after 30 days. At lower solid concentrations (0.5 mg/ml), an amorphous calcium phosphate layer formation was observed followed by the conversion to hydroxyapatite phase after 7 days of immersion. The XTT (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) assay was conducted to evaluate the osteoblast cell response to the bioactive glass fibers. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Cytotoxicity of ZnO Nanowire Arrays on Excitable Cells

    Directory of Open Access Journals (Sweden)

    Yongchen Wang

    2017-04-01

    Full Text Available Zinc oxide (ZnO nanowires have been widely studied for their applications in electronics, optics, and catalysts. Their semiconducting, piezoelectric, fluorescent, and antibacterial properties have also attracted broad interest in their biomedical applications. Thus, it is imperative to evaluate the biosafety of ZnO nanowires and their biological effects. In this study, the cellular level biological effects of ZnO nanowire arrays are specifically tested on three types of excitable cells, including NG108-15 neuronal cell line, HL-1 cardiac muscle cell line, and neonatal rat cardiomyocytes. Vertically aligned and densely packed ZnO nanowire arrays are synthesized using a solution-based method and used as a substrate for cell culture. The metabolism levels of all three types of cells cultured on ZnO nanowire arrays are studied using the 3-(4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2H-tetrazolium bromide (MTT assays of a full factorial design. Under the studied settings, the results show statistically significant inhibitory effects of ZnO nanowire arrays on the metabolism of NG108-15 and HL-1 cells in comparison to gold, glass, and polystyrene substrates, and on the metabolism of cardiomyocytes in comparison to gold substrate.

  19. Interdependence of antioxidants and micronutrients in oral cancer and potentially malignant oral disorders: a serum and saliva study.

    Directory of Open Access Journals (Sweden)

    Shishir Ram Shetty

    2014-12-01

    Full Text Available In our previous studies we have evaluated the role of antioxidants and trace elements in potentially malignant disorders and cancers of the oral cavity, taking into consideration the importance of antioxidants as biomarkers in cancer detection. We felt that other than evaluation, the correlation and interdependence that existed among antioxidants and trace elements require further evaluation in order to develop a better understanding.Serum and salivary zinc, glutathione, and superoxide dismutase levels were evaluated in 65 healthy controls, 115 subjects with potentially malignant oral disorders, and 50 subjects with oral squamous cell carcinoma, using the atom absorption photometry, [5, 5-Dithiobis (2 nitrobenzoic acid], and nitroblue tetrazolium methods, respectively.Serum zinc and serum glutathione showed significant positive correlation (r=0.76, P=0.01. Similarly, salivary glutathione and salivary zinc levels had a positive correlation (r=0.68, P=0.01. Serum superoxide dismutase showed a strong positive correlation with serum zinc (r=0.64, P=0.01. Similarly, there was a moderate positive correlation between salivary superoxide dismutase and salivary zinc (r=0.67, P=0.01.Our findings showed that trace elements and antioxidants exhibited interdependence in serum, as well as in saliva, in both physiologic and pathologic states such as oral cancer.

  20. In vitro investigating of anticancer activity of focuxanthin from marine brown seaweed species

    Directory of Open Access Journals (Sweden)

    M. Karkhane Yousefi

    2018-01-01

    Full Text Available Breast cancer is the most common cancer type among women all over the world. Chemotherapy is the use of anticancer medicines for treating cancer but it has many side effects and cells may become resistant to these chemical medicines. Therefore, finding new compounds of natural origin could be a promising solution to this problem. The aim of the current study was to evaluate anticancer activity of fucoxanthin which is the most important carotenoid found in the marine brown seaweeds and diatoms. fucoxanthin has many properties (antioxidant, antibacterial, anticancer, antiobesity, anti-inflammatory and etc. due to its unique structure. Samples with different concentrations (10, 25 and 50 µg/ml and at various incubation times were collected (6, 24 and 48 hours from four different species (Padina tenuis, Colpomenia sinuosa, Iyengaria stellate and Dictyota indica of brown seaweeds from Qeshm Island, Persian Gulf. Moreover, the anticancer activity of fucoxanthin-containing extracts on breast cancer cells line and normal human skin fibroblast cells line was assessed by MTT [3-(4,5-dimethylthiazolyl-2,5-diphenyl-tetrazolium bromide] assay to specify the cytotoxic effects. The results showed that fucoxanthin extract from Dictyota. indica at 24-hour treatment and 50 µg/ml concentration has the most effective anticancer activity on the breast cancer cells line, without toxic effects to the normal cells. According to the obtained results, it seems that Dictyota. Indica is a good candidate for further analysis and can be introduced to the food and pharmaceutical industries.

  1. Polypropylene Biocomposites with Boron Nitride and Nanohydroxyapatite Reinforcements

    Directory of Open Access Journals (Sweden)

    Kai Wang Chan

    2015-03-01

    Full Text Available In this study, we develop binary polypropylene (PP composites with hexagonal boron nitride (hBN nanoplatelets and ternary hybrids reinforced with hBN and nanohydroxyapatite (nHA. Filler hybridization is a sound approach to make novel nanocomposites with useful biological and mechanical properties. Tensile test, osteoblastic cell culture and dimethyl thiazolyl diphenyl tetrazolium (MTT assay were employed to investigate the mechanical performance, bioactivity and biocompatibility of binary PP/hBN and ternary PP/hBN-nHA composites. The purpose is to prepare biocomposite nanomaterials with good mechanical properties and biocompatibility for replacing conventional polymer composites reinforced with large hydroxyapatite microparticles at a high loading of 40 vol%. Tensile test reveals that the elastic modulus of PP composites increases, while tensile elongation decreases with increasing hBN content. Hybridization of hBN with nHA further enhances elastic modulus of PP. The cell culture and MTT assay show that osteoblastic cells attach and proliferate on binary PP/hBN and ternary PP/hBN-20%nHA nanocomposites.

  2. Flavonoid C-glucosides Derived from Flax Straw Extracts Reduce Human Breast Cancer Cell Growth In vitro and Induce Apoptosis.

    Science.gov (United States)

    Czemplik, Magdalena; Mierziak, Justyna; Szopa, Jan; Kulma, Anna

    2016-01-01

    Flax straw of flax varieties that are grown for oil production is a by product which represents a considerable biomass source. Therefore, its potential application for human use is of high interest. Our research has revealed that flax straw is rich in flavonoid C-glucosides, including vitexin, orientin, and isoorientin. The objective of this study was to evaluate the cytotoxicity and possible proapoptotic effect of flax straw derived C-glucosides of flavonoids in the human breast adenocarcinoma cell line (MCF-7). The effects of flax straw derived flavonoid C-glucosides on cell proliferation of MCF-7 cells were evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) and sulforhodamine B assays. The expression of apoptosis-related genes was assessed by real-time PCR. Our data revealed that flax C-glucosides as well as pure compounds are cytotoxic toward MCF-7 cells and inhibit their proliferation. Moreover, the induction of apoptosis was correlated with the changes in the mRNA level of pro-apoptotic genes. Increased expression of bax and caspase-7, -8, and -9 and decreased mRNA expression of bcl-2 was observed, whereas the mRNA levels of p53 and mdm2 were not altered. These results clearly demonstrated that flax straw metabolites effectively induced growth inhibition and apoptosis in human breast adenocarcinoma cells.

  3. Flavonoid C-glucosides derived from flax straw extracts reduce human breast cancer cell growth in vitro and induce apoptosis.

    Directory of Open Access Journals (Sweden)

    Magdalena Czemplik

    2016-08-01

    Full Text Available Flax straw of flax varieties that are grown for oil production is a byproduct which represents a considerable biomass source. Therefore its potential application for human use is of high interest. Our research has revealed that flax straw is rich in flavonoid C-glucosides, including vitexin, orientin and isoorientin. The objective of this study was to evaluate the cytotoxicity and possible proapoptotic effect of flax straw derived C-glucosides of flavonoids in the human breast adenocarcinoma cell line (MCF-7. The effects of flax straw derived flavonoid C-glucosides on cell proliferation of MCF-7 cells were evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT and sulforhodamine B (SRB assays. The expression of apoptosis-related genes was assessed by real-time PCR. Our data revealed that flax C-glucosides as well as pure compounds are cytotoxic towards MCF-7 cells and inhibit their proliferation. Moreover, the induction of apoptosis was correlated with the changes in the mRNA level of pro-apoptotic genes. Increased expression of bax and caspase-7, -8, and -9 and decreased mRNA expression of bcl-2 was observed, whereas the mRNA levels of p53 and mdm2 were not altered. These results clearly demonstrated that flax straw metabolites effectively induced growth inhibition and apoptosis in human breast adenocarcinoma cells.

  4. KAEMPFEROL, A FLAVONOID COMPOUND FROM GYNURA MEDICA INDUCED APOPTOSIS AND GROWTH INHIBITION IN MCF-7 BREAST CANCER CELL.

    Science.gov (United States)

    Yi, Xiaofang; Zuo, Jiangcheng; Tan, Chao; Xian, Sheng; Luo, Chunhua; Chen, Sai; Yu, Liangfang; Luo, Yucheng

    2016-01-01

    Kaempferol, a natural flavonoid, has been shown to induce cancer cell apoptosis and cell growth inhibition in several tumors. Previously we have conducted a full investigation on the chemical constituents of Gynura medica, kaempferol and its glycosides are the major constituents of G. medica. Here we investigated the growth inhibition and apoptosis induction effect of kaempferol extracted from G. medica. The inhibition effects of kaempferol were evaluated by MTS assay and soft agar colony formation assay. Fluorescence staining and western blotting were be used to study the apoptosis. The structure was identified by (1)H- NMR), (13)C-NMR and ESI-MS analyses. Our results showed that kaempferol's inhibition of MCF-7 breast cancer cell growth may through inducing apoptosis and downregulation of Bcl2 expression. Kaempferol is a promising cancer preventive and therapeutic agent for breast cancer. List of non-standard abbreviations: MTS: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, HPLC: High-performance liquid chromatography, NMR: Nuclear Magnetic Resonance, ESI-MS Electrospray Ionization Mass Spectral, PARP: Poly ADP-ribose polymerase.

  5. Biosynthesis and characterization of copper oxide nanoparticles and its anticancer activity on human colon cancer cell lines (HCT-116).

    Science.gov (United States)

    Gnanavel, V; Palanichamy, V; Roopan, Selvaraj Mohana

    2017-06-01

    The eco-friendly synthesis of nanoparticles through green route from plant extracts have renowned a wide range of application in the field of modern science, due to increased drug efficacy and less toxicity in the nanosized mediated drug delivery model. In the present study, our research groups have biosynthesized the stable and cost effective copper oxide nanoparticles (CuO NPs) from the leaves of (Ormocarpum cochinchinense) O. cochinchinense. The synthesis of crystalline CuO NPs from the leaf extract of O. cochinchinense were confirmed by various analytical techniques like UV-Visible Spectroscopy (UV-Vis), Fourier-Transform Infrared Spectroscopy (FT-IR), X-Ray Diffractometer (XRD), Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM) and Selected Area Electron Diffraction (SAED) pattern. Further the synthesized CuO NPs were screened for anticancer activity on human colon cancer cell lines (HCT-116) by MTT (3-(4,5-dimethyl-2-tiazolyl)-2,5-diphenyl-2-tetrazolium bromide) assay. The obtained result inferred that the synthesized CuO NPs demonstrated high anticancer cytotoxicity on human colon cancer cell lines (HCT-116) with IC50 value of 40μgmL(-1) were discussed briefly in this manuscript. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Cytotoxicity induced by ochratoxin A, zearalenone, and α-zearalenol: effects of individual and combined treatment.

    Science.gov (United States)

    Wang, H W; Wang, J Q; Zheng, B Q; Li, S L; Zhang, Y D; Li, F D; Zheng, N

    2014-09-01

    This study investigated the cytotoxicity of combined mycotoxins of ochratoxin A (OTA), zearalenone (ZEA), and/or α-zearalenol (α-ZOL). The cytotoxicity of two mycotoxin combinations (two two-toxin combinations and one three-toxin combination) on human Hep G2 cells was evaluated using a tetrazolium salt (MTT) assay and isobologram analysis. Our results demonstrated significant cytotoxic effects of the two-toxin combination and the three-toxin combination on Hep G2 cells in a time- and concentration-dependent manner. The combination indexes (CI) were 2.73-7.67 for the OTA+ZEA combination and 1.23-17.82 for the OTA+α-ZOL combination after 24 h, 48 h, and 72 h of exposure at all inhibit concentration (IC) levels (IC10-IC90), indicating an antagonism. The CIs of the ZEA+α-ZOL combination were 1.29-2.55 after 24 h and 72 h of exposure (IC10-IC90), indicating an antagonism. The CIs of the ZEA+α-ZOL combination were 0.74-1.68 after 48 h of exposure, indicating synergism (IC80-IC90), additive effects (IC50-IC70), or antagonism (IC10-IC40). For the OTA+ZEA+α-ZOL combination, the CIs were 1.41-14.65 after 24 h, 48 h, and 72 h of exposure (IC10-IC90), indicating an antagonism. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Synthesis, Characterization, and Cytotoxicity of a Novel Gold(III Complex with O,O′-Diethyl Ester of Ethylenediamine-N,N′-Di-2-(4-MethylPentanoic Acid

    Directory of Open Access Journals (Sweden)

    Nebojša Pantelić

    2016-09-01

    Full Text Available A novel gold(III complex, [AuCl2{(S,S-Et2eddl}]PF6, ((S,S-Et2eddl = O,O′-diethyl ester of ethylenediamine-N,N′-di-2-(4-methylpentanoic acid was synthesized and characterized by IR, 1D (1H and 13C, and 2D (H,H-COSY and H,H-NOESY NMR spectroscopy, mass spectrometry, and elemental analysis. Density functional theory calculations confirmed that (R,R-N,N′ diastereoisomer was energetically the most stable isomer. In vitro antitumor action of ligand precursor [(S,S-H2Et2eddl]Cl2 and corresponding gold(III complex was determined against tumor cell lines: human adenocarcinoma (HeLa, human colon carcinoma (LS174, human breast cancer (MCF7, non-small cell lung carcinoma cell line (A549, and non-cancerous cell line human embryonic lung fibroblast (MRC-5 using microculture tetrazolium test (MTT assay. The results indicate that both ligand precursor and gold(III complex have showed very good to moderate cytotoxic activity against all tested malignant cell lines. The highest activity was expressed by [AuCl2{(S,S-Et2eddl}]PF6 against the LS174 cells, with IC50 value of 7.4 ± 1.2 µM.

  8. TIME REDUCTION FOR SURINAM GRASS SEED GERMINATION TEST

    Directory of Open Access Journals (Sweden)

    Camila de Aquino Tomaz

    2015-10-01

    Full Text Available ABSTRACTThe period for the germination test of Surinam grass seeds established by the Rules for Seeds Testing is 28 days, considered too lengthy by producers, venders, and seed analysis laboratories. So, the objective of this research was to evaluate the possibility of reducing the time for the germination test of Surinam grass seeds and to establish a method for dormancy breaking and the ideal temperature. Ten seed lots were submitted to the following treatments to overcome seed dormancy: control; substrate moistening with 0.2% KNO3; and scarification with sulfuric acid (98% 36 N for 15 minutes. After the treatments, the lots were submitted to seed water content, germination and tetrazolium tests. During the germination test, conducted with four replicates of 100 seeds per treatment for 28 days, two conditions of alternating temperatures (20-35 °C and 15-35 °C with 8 hours of light were tested. Attempting to determine the test end date, daily counts of the number of normal seedlings were made and for each lot, treatment, and temperature, a growth curve for the evaluation of germination was adjusted. The segmented regression model parameter estimations were calculated for each treatment. The germination test of Braquiaria decumbensseeds may be evaluated in 12 days after sowing using alternating temperatures of 20-35 °C and without any treatment to overcome dormancy.

  9. Anti-tumor studies with extracts of Calotropis procera (Ait.) R.Br. root employing Hep2 cells and their possible mechanism of action.

    Science.gov (United States)

    Mathur, Rajani; Gupta, Suresh K; Mathur, Sandeep R; Velpandian, Thirumurthy

    2009-05-01

    Anti-tumor potential of root extracts of Calotropis procera: methanolic extract (CM), hexane extract (CH), aqueous extract (CW) and ethylacetate extract (CE) and its possible mechanism against Hep2 cancer cells has been investigated. Cellular proliferation activities were assayed by tetrazolium bromide (MTT) colorimetry. Morphological changes of cancer cells were observed under inverted microscope and cell cycle parameters were determined by flow cytometry following propidium iodide staining. Treatment with the extracts at various doses of 1, 5, 10 and 25 microg/ml revealed that CM, CH and CE possessed cytotoxicity, whereas CW did not have cytotoxic effect. CE (10 microg/ml) showed strongest cytotoxic effect (96.3%) on Hep2 at 48 hr following treatment, whereas CM and CH showed cytotoxicity of 72.7 and 60.5%, respectively. Extract-treated cells exhibited typical morphological changes of apoptosis. Results of flow cytometric analysis clearly demonstrated that root extracts initiated apoptosis of Hep2 cells through cell cycle arrest at S phase, thus preventing cells from entering G2/M phase. Results of the study indicate that the root extracts of C. procera inhibit the proliferation of Hep2 cells via apoptotic and cell cycle disruption based mechanisms.

  10. Possible activation by the green tea amino acid theanine of mammalian target of rapamycin signaling in undifferentiated neural progenitor cells in vitro

    Directory of Open Access Journals (Sweden)

    Takeshi Takarada

    2016-03-01

    Full Text Available We have shown marked promotion of both proliferation and neuronal differentiation in pluripotent P19 cells exposed to the green tea amino acid theanine, which is a good substrate for SLC38A1 responsible for glutamine transport. In this study, we evaluated the activity of the mammalian target of rapamycin (mTOR kinase pathway, which participates in protein translation, cell growth and autophagy in a manner relevant to intracellular glutamine levels, in murine neural progenitor cells exposed to theanine. Exposure to theanine promoted the phosphorylation of mTOR and downstream proteins in neurospheres from embryonic mouse neocortex. Although stable overexpression of SLC38A1 similarly facilitated phosphorylation of mTOR-relevant proteins in undifferentiated P19 cells, theanine failed to additionally accelerate the increased phosphorylation in these stable transfectants. Theanine accelerated the formation of neurospheres from murine embryonic neocortex and adult hippocampus, along with facilitation of both 5-bromo-2’-deoxyuridine incorporation and 3-(4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2H-tetrazolium bromide reduction in embryonic neurospheres. In embryonic neurospheres previously exposed to theanine, a significant increase was seen in the number of cells immunoreactive for a neuronal marker protein after spontaneous differentiation. These results suggest that theanine activates the mTOR signaling pathway for proliferation together with accelerated neurogenesis in murine undifferentiated neural progenitor cells.

  11. In Vitro Testing of Potential Entamoeba histolytica Pyruvate Phosphate Dikinase Inhibitors.

    Science.gov (United States)

    Saidin, Syazwan; Othman, Nurulhasanah; Noordin, Rahmah

    2017-10-01

    Adverse effects and resistance to metronidazole have motivated the search for new antiamoebic agents against Entamoeba histolytica. Control of amoeba growth may be achieved by inhibiting the function of the glycolytic enzyme and pyruvate phosphate dikinase (PPDK). In this study, we screened 10 compounds using an in vitro PPDK enzyme assay. These compounds were selected from a virtual screening of compounds in the National Cancer Institute database. The antiamoebic activity of the selected compounds was also evaluated by determining minimal inhibitory concentrations (MICs) and IC50 values using the nitro-blue tetrazolium reduction assay. Seven of the 10 compounds showed inhibitory activities against the adenosine triphosphate (ATP)/inorganic phosphate binding site of the ATP-grasp domain. Two compounds, NSC349156 (pancratistatin) and NSC228137 (7-ethoxy-4-[4-methylphenyl] sulfonyl-3-oxido-2, 1, 3-benzoxadiazol-3-ium), exhibited inhibitory effects on the growth of E. histolytica trophozoites with MIC values of 25 and 50 μM, and IC50 values of 14 and 20.7 μM, respectively.

  12. Tetrandrine, a Compound Common in Chinese Traditional Medicine, Preferentially Kills Breast Cancer Tumor Initiating Cells (TICs In Vitro

    Directory of Open Access Journals (Sweden)

    Jessica Li

    2011-05-01

    Full Text Available Tetrandrine is a bisbenzylisoquinoline alkaloid found in Stephania tetrandra, a Chinese medicine commonly used as an anti-inflammatory. It has extensive pharmacological activity, including positive ion channel blockade and inhibition of multiple drug resistance proteins. These activities are very similar to that of salinomycin, a known drug targeting breast cancer initiation cells (TICs. Herein, we tested tetrandrine targeting of breast cancer TICs. SUM-149, an inflammatory breast cancer cell line and SUM-159, a non-inflammatory metaplastic breast cancer cell line were used in these studies. In proliferation assays using 3-(4,5-dimethylthiazol-2-yl-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl-2H-tetrazolium (MTS, we found that the IC50 for inhibition of proliferation is 15.3 ± 4.1 µM for SUM-149 and 24.3 ± 2.1 µM for SUM-159 cells. Tetrandrine also inhibited mammosphere formation, a surrogate for breast cancer TICs growth in vitro with IC50 around 1 µM for SUM-149 and around 2 µM for SUM-159 cells. Tetrandrine has similar effects on the mammosphere formation from cells isolated from fresh patient sample. Moreover, tetrandrine decreases the aldehyde dehydrogenase (ALDH positive population in SUM-159 by 45% ± 5.45% P = 0.005. In summary, tetrandrine demonstrates significant efficacy against in vitro surrogates for inflammatory and aggressive breast cancer TICs.

  13. Compounds used to produce cloned animals are genotoxic and mutagenic in mammalian assays in vitro and in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Oliveira, R.J. [Programa de Pós-Graduação em Biologia Celular e Molecular, Instituto de Biociências de Rio Claro, Universidade Estadual Paulista, Rio Claro, SP (Brazil); Centro de Estudos em Células Tronco, Terapia Celular e Genética Toxicológica, Núcleo de Hospital Universitário, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS (Brazil); Programa de Pós-Graduação em Saúde em Desenvolvimento na Região Centro-Oeste, Faculdade de Medicina “Dr. Hélio Mandetta”, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS (Brazil); Programa de Mestrado em Farmácia, Centro de Ciências Biológicas e da Saúde, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS (Brazil); Mantovani, M.S.; Silva, A.F. da [Departamento de Biologia Geral, Universidade Estadual de Londrina, Londrina, PR (Brazil); Pesarini, J.R. [Centro de Estudos em Células Tronco, Terapia Celular e Genética Toxicológica, Núcleo de Hospital Universitário, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS (Brazil); Programa de Pós-Graduação em Saúde em Desenvolvimento na Região Centro-Oeste, Faculdade de Medicina “Dr. Hélio Mandetta”, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS (Brazil); Mauro, M.O. [Centro de Estudos em Células Tronco, Terapia Celular e Genética Toxicológica, Núcleo de Hospital Universitário, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS (Brazil); Programa de Doutorado em Biotecnologia e Biodiversidade - Rede Pró Centro-Oeste, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS (Brazil); Ribeiro, L.R. [Programa de Pós-Graduação em Biologia Celular e Molecular, Instituto de Biociências de Rio Claro, Universidade Estadual Paulista, Rio Claro, SP (Brazil); Programa de Pós-Graduação em Patologia, Faculdade de Medicina de Botucatu, Universidade Estadual Paulista, Botucatu, SP (Brazil)

    2014-03-28

    The compounds 6-dimethylaminopurine and cycloheximide promote the successful production of cloned mammals and have been used in the development of embryos produced by somatic cell nuclear transfer. This study investigated the effects of 6-dimethylaminopurine and cycloheximide in vitro, using the thiazolyl blue tetrazolium bromide colorimetric assay to assess cytotoxicity, the trypan blue exclusion assay to assess cell viability, the comet assay to assess genotoxicity, and the micronucleus test with cytokinesis block to test mutagenicity. In addition, the comet assay and the micronucleus test were also performed on peripheral blood cells of 54 male Swiss mice, 35 g each, to assess the effects of the compounds in vivo. The results indicated that both 6-dimethylaminopurine and cycloheximide, at the concentrations and doses tested, were cytotoxic in vitro and genotoxic and mutagenic in vitro and in vivo, altered the nuclear division index in vitro, but did not diminish cell viability in vitro. Considering that alterations in DNA play important roles in mutagenesis, carcinogenesis, and morphofunctional teratogenesis and reduce embryonic viability, this study indicated that 6-dimethylaminopurine and cycloheximide utilized in the process of mammalian cloning may be responsible for the low embryo viability commonly seen in nuclear transfer after implantation in utero.

  14. Evaluation of cytotoxic effects of Anbarnesa on fibroblast L929: Can it be used as a mouthwash?

    Directory of Open Access Journals (Sweden)

    Hassan Ali Shafiee

    2014-01-01

    Full Text Available Aims: In Iranian traditional medicine Anbarnesa (derived from smoke from burning female donkey′s stool has been used to treat ulcers and inflammatory conditions like stomatitis and ear infections (otitis. We assess the properties of Anbarnesa as an alternative mouthwash. Materials and Methods: In this experimental study, Anbarnesa smoke was analyzed using aGC-mass device. The smoke collected was dissolved at different densities in propylene glycol and incubated in Dulbecco′s modified Eagle′s medium in direct contact with fibroblast cells. Assessment of cytotoxicity was done at 1, 24 and 72 h. Cell viability was measured by methyl thiazolyl tetrazolium test, and ELISA Reader machine was used to read the results. Data were analyzed using one-way ANOVA test. Results: The findings of this study showed Anbarnesa was nontoxic in 1/64, 1/128 and 1/256 dilutions. In 1/32 dilution, toxicity was seen after 72 h. In dilutions, 1/8 and 1/16 toxicity were seen in the 1 st h. Conclusion: According to the initial results of Anbarnesa may be used as an alternative mouthwash with fewer side-effects for plaque control and prevention of periodontal disease.

  15. The toxicity of silver and silica nanoparticles in comparable human and mouse cell lines

    DEFF Research Database (Denmark)

    Foldbjerg, Rasmus; Beer, Christiane; Sutherland, Duncan S

    the annexin V/PI assay. Death at EC50 in the lung cells was equally due to apoptosis and necrosis after exposure to either NP. However, large discrepancies were found when comparing EC50 values from the WST-8 assay in macrophages to results from the Annexin V/PI assay. The WST-8 assay appeared to overestimate...... to be well dispersed. The primary sizes were 69 nm (Ag) and 27 nm (SiO2) as determined by TEM. Cytotoxicity was tested after 24 h in terms of viability by dehydrogenase activity (WST-8), apoptosis (Annexin V/PI) and the formation of ROS (DCF). Murine cells are more sensitive to NPs than human cells...... cell death caused by Ag NPs in J774A.1 cells and by SiO2 NPs in THP-1 cells, whereas the assay underestimated the EC50 values of SiO2 NPs in J774A.1 cells and Ag NPs in THP-1 cells. These discrepancies suggest that dose-responses based on tetrazolium dyes such as WST-8 should be confirmed by additional...

  16. Effects of Roundabout 5 on adhesion, invasion and potential motility of human tongue carcinoma Tb cells.

    Science.gov (United States)

    Xiao, Rui; Zhao, Yuan; Wang, Li-jing; Li, Wei-ping

    2011-08-01

    Roundabout 5 (R5) is a monoclonal antibody which can neutralize the binding of Roundabout 1 (Robo1) to Slit2. Oral squamous cell carcinoma angiogenesis was significantly inhibited when R5 blocked slit-robo signaling pathway. However, the effect of R5 on the invasion of tongue cancer cells has not been investigated clearly. In this study, we treated human brain metastasis of tongue cancer cell lines (Tb cells) with R5 at different concentrations, and the control Tb cells were treated with 10 mg/ml immunoglobin G 2b (IgG2b). The effect of R5 on the proliferation, adhension, invasion and motility of Tb cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, cell attachment assay on fibronectin (FN), wound assay and chemotaxis assay, respectively. And gelatin-incorporated sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to investigate the activity of matrix metalloproteinase-2 (MMP2) and matrix metalloproteinase-9 (MMP9). R5 had no effect on the proliferation of Tb cells. However, R5 could significantly inhibit the motility, attachment and chemotaxis of Tb cells to FN, and it could also significantly inhibit the activity of MMP2 and MMP9 in Tb cells. R5 can inhibit the adhesion, invasion and motility of human tongue carcinoma Tb cells.

  17. Cytotoxicity and proliferative effects of Iodoform-containing root canal-filling material on RAW 264.7 macrophage and RKO epithelial cell lines.

    Science.gov (United States)

    Petel, Roy; Moskovitz, Moti; Tickotsky, Nili; Halabi, Amal; Goldstein, Judith; Houri-Haddad, Yael

    2013-01-01

    The present study investigated the effect of the Iodoform-containing root canal filling material on the viability of cultured macrophages and epithelial cells, and on cytokine secretion. The effect of Endoflas F.S. on the proliferation of a RAW 264.7 macrophage cell line and on a RKO epithelial cell line, and on the production of tumour necrosis factor alpha (TNFα) from macrophages was examined. Cell vitality was evaluated using a colourimetric XTT (sodium 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium inner salt) assay. The presence of cytokines was determined by two-site enzyme-linked immunosorbent assay (ELISA). Direct exposure of Endoflas F.S. and its media, up to a dilution of 1/8, decreased the viability of macrophages and epithelial cells by ∼70% compared to control media (Pmaterial. Direct and indirect exposure to high concentrations of iodoform-containing root canal filling material showed a cytotoxic effect on macrophages and epithelial cells, while low concentrations induced cell proliferation. Copyright © 2012 Elsevier Ltd. All rights reserved.

  18. Survival Strategy of Erwinia amylovora against Copper: Induction of the Viable-but-Nonculturable State

    Science.gov (United States)

    Ordax, Mónica; Marco-Noales, Ester; López, María M.; Biosca, Elena G.

    2006-01-01

    Copper compounds, widely used to control plant-pathogenic bacteria, have traditionally been employed against fire blight, caused by Erwinia amylovora. However, recent studies have shown that some phytopathogenic bacteria enter into the viable-but-nonculturable (VBNC) state in the presence of copper. To determine whether copper kills E. amylovora or induces the VBNC state, a mineral medium without copper or supplemented with 0.005, 0.01, or 0.05 mM Cu2+ was inoculated with 107 CFU/ml of this bacterium and monitored over 9 months. Total and viable cell counts were determined by epifluorescence microscopy using the LIVE/DEAD kit and by flow cytometry with 5-cyano-2,3-ditolyl tetrazolium chloride and SYTO 13. Culturable cells were counted on King's B nonselective solid medium. Changes in the bacterial morphology in the presence of copper were observed by scanning electron microscopy. E. amylovora entered into the VBNC state at all three copper concentrations assayed, much faster when the copper concentration increased. The addition of different agents which complex copper allowed the resuscitation (restoration of culturability) of copper-induced VBNC cells. Finally, copper-induced VBNC cells were virulent only for the first 5 days, while resuscitated cells always regained their pathogenicity on immature fruits over 9 months. These results have shown, for the first time, the induction of the VBNC state in E. amylovora as a survival strategy against copper. PMID:16672494

  19. Polysaccharides in fungi. XXXVII. Immunomodulating activities of carboxymethylated derivatives of linear (1-->3)-alpha-D-glucans extracted from the fruiting bodies of Agrocybe cylindracea and Amanita muscaria.

    Science.gov (United States)

    Yoshida, I; Kiho, T; Usui, S; Sakushima, M; Ukai, S

    1996-01-01

    Immunomodulating activities of three carboxymethylated derivatives (AG-AL-CMS, AG-AL-CMI, and AM-APP-CM) of linear (1-->3)-alpha-D-glucans from Agrocybe cylindracea and Amanita muscaria were evaluated with murine peritoneal macrophages playing an important role in tumor immunity. The ratio of macrophages in peritoneal exudate cells increased more than 50% after the administration of three carboxymethylated (1-->3)-alpha-D-glucans. These carboxymethylated (1-->3)-alpha-D-glucans exhibited higher potentiating activities for macrophages than carboxymethylated linear (1-->3)-beta-D-glucan (CMPS) in the potency of reduction of nitro blue tetrazolium, products of nitric oxide and the soluble cytotoxic factor, the amount of glucose consumption, and the activation of acid phosphatase. AG-AL-CMS, AG-AL-CMI, and AM-APP-CM were found to induce the tumor regressing factor in mouse serum, although the ability of the induction of this factor was weaker than that of CMPS. The reticuloendothelial system-potentiating activation of three carboxymethylated alpha-D-glucans was similar to that of the carboxymethylated beta-D-glucan. AG-AL-CMS and AG-AL-CMI, but not AM-APP-CM, were suggested to possess a higher-order structure, resulting from the formation of a fluorescent complex with aniline blue.

  20. Effects of starvation on physiological activity and chlorine disinfection resistance in Escherichia coli O157:H7

    Science.gov (United States)

    Lisle, J. T.; Broadaway, S. C.; Prescott, A. M.; Pyle, B. H.; Fricker, C.; McFeters, G. A.

    1998-01-01

    Escherichia coli O157:H7 can persist for days to weeks in microcosms simulating natural conditions. In this study, we used a suite of fluorescent, in situ stains and probes to assess the influence of starvation on physiological activity based on membrane potential (rhodamine 123 assay), membrane integrity (LIVE/DEAD BacLight kit), respiratory activity (5-cyano-2,3-di-4-tolyl-tetrazolium chloride assay), intracellular esterase activity (ScanRDI assay), and 16S rRNA content. Growth-dependent assays were also used to assess substrate responsiveness (direct viable count [DVC] assay), ATP activity (MicroStar assay), and culturability (R2A agar assay). In addition, resistance to chlorine disinfection was assessed. After 14 days of starvation, the DVC values decreased, while the values in all other assays remained relatively constant and equivalent to each other. Chlorine resistance progressively increased through the starvation period. After 29 days of starvation, there was no significant difference in chlorine resistance between control cultures that had not been exposed to the disinfectant and cultures that had been exposed. This study demonstrates that E. coli O157:H7 adapts to starvation conditions by developing a chlorine resistance phenotype.

  1. Enhancement of expression of survivin promoter-driven CD/TK double suicide genes by the nuclear matrix attachment region in transgenic gastric cancer cells.

    Science.gov (United States)

    Niu, Ying; Li, Jian-Sheng; Luo, Xian-Run

    2014-01-25

    This work aimed to study a novel transgenic expression system of the CD/TK double suicide genes enhanced by the nuclear matrix attachment region (MAR) for gene therapy. The recombinant vector pMS-CD/TK containing the MAR-survivin promoter-CD/TK cassette was developed and transfected into human gastric cancer SGC-7901 cells. Expression of the CD/TK genes was detected by quantitative real-time PCR (qPCR) and Western blot. Cell viability and apoptosis were measured using the methyl thiazolyl tetrazolium (MTT) assay and flow cytometry. When the MAR fragment was inserted into the upstream of the survivin promoter, the qPCR result showed that the expression of the CD/TK genes significantly increased 7.7-fold in the transgenic SGC-7901 cells with plasmid pMS-CD/TK compared with that without MAR. MTT and flow cytometry analyses indicated that treatment with the prodrugs (5-FC+GCV) significantly decreased the cellular survival rate and enhanced the cellular apoptosis in the SGC-7901 cells. The expression of the CD/TK double suicide genes driven by the survivin promoter can be enhanced by the MAR fragment in human gastric cancer cells. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Neuroprotective effects of cactus polysaccharide on oxygen and glucose deprivation induced damage in rat brain slices.

    Science.gov (United States)

    Huang, Xianju; Li, Qin; Zhang, Yingpei; Lü, Qing; Guo, Lianjun; Huang, Lin; He, Zhi

    2008-06-01

    1. The neuroprotective effect of cactus polysaccharide (CP) on oxygen and glucose deprivation (OGD) and reoxygenation (REO)-induced damage in the cortical and hippocampal slices of rat brain was investigated. 2. Cell viability was evaluated by using the 2, 3, 5-triphenyl tetrazolium chloride (TTC) method. The fluorescence of propidium iodide (PI) staining was used for quantification of cellular survival, and lactate dehydrogenase (LDH) activity in incubation medium was assessed by LDH assay to evaluate the degree of injury. 3. The OGD ischemic condition significantly decreased cellular viability and increased LDH release in the incubation medium. CP (0.2 mg/l approximately 2 mg/l) protected brain slices from OGD injury in a dosage dependent manner as demonstrated by increased A 490 value of TTC, decreased PI intensity and LDH release. At the above concentration, CP also prevented the increase of nitric oxide (NO) content and inducible nitric oxide synthase (iNOS) activity induced by OGD. 4. CP can protect the brain slices (cortical and hippocampus) against injury induced by OGD. Its neuroprotective effect may be partly mediated by the NO/iNOS system induced by OGD insult.

  3. The Ciprofloxacin Impact on Biofilm Formation by Proteus Mirabilis and P. Vulgaris Strains

    Science.gov (United States)

    Kwiecinska-Pirog, Joanna; Skowron, Krzysztof; Bartczak, Wojciech; Gospodarek-Komkowska, Eugenia

    2016-01-01

    Background Proteus spp. bacilli belong to opportunistic human pathogens, which are primarily responsible for urinary tract and wound infections. An important virulence factor is their ability to form biofilms that greatly reduce the effectiveness of antibiotics in the site of infection. Objectives The aim of this study was to determine the value of the minimum concentration of ciprofloxacin that eradicates a biofilm of Proteus spp. strains. Materials and Methods A biofilm formation of 20 strains of P. mirabilis and 20 strains of P. vulgaris were evaluated by a spectrophotometric method using 0.1% 2, 3, 5-Triphenyl-tetrazolium chloride solution (TTC, AVANTORTM). On the basis of the results of the absorbance of the formazan, a degree of reduction of biofilm and minimum biofilm eradication (MBE) values of MBE50 and MBE90 were determined. Results All tested strains formed a biofilm. A value of 1.0 μg/mL ciprofloxacin is MBE50 for the strains of both tested species. An MBE90 value of ciprofloxacin for isolates of P. vulgaris was 2 μg/mL and for P. mirabilis was 512 μg/mL. Conclusions Minimum biofilm eradication values of ciprofloxacin obtained in the study are close to the values of the minimal inhibition concentration (MIC). PMID:27303616

  4. Purification and neuroprotective effects of polysaccharides from Opuntia Milpa Alta in cultured cortical neurons.

    Science.gov (United States)

    Chen, Yang; Zhao, Bo; Huang, Xiaolan; Zhan, Jinjin; Zhao, Yan; Zhou, Mei; Guo, Lianjun

    2011-11-01

    Opuntia is a traditional plant from China with medicinal applications. In this experiment, polysaccharides from Opuntia Milpa Alta (MAPs) were analyzed using gas chromatograph-mass spectrometer (GC-MS) method and result showed that MAPs contained mannose (6.37%), rhamnose (14.94%), xylose (1.99%), arabinose (24.07%), galactose (38.25%), ribose (2.63%) and glucose (11.48%). The neuroprotective effects of MAPs were evaluated at the mechanistic level in vitro models of cerebral ischemic injury. In vitro oxygen/glucose deprivation (OGD) model, MAPs (0.5 μg/ml, 5 μg/ml, 50 μg/ml) effectively increased cell viability by methyl thiazolyl tetrazolium (MTT) assay, inhibited cell cytotoxicity by lactate dehydrogenase (LDH) assay, reduced neuronal cell death, suppressed the production of intracellular reactive oxygen species (ROS), decreased of intracellular free Ca(2+) concentrations ([Ca(2+)](i)), and reduced extracellular glutamate level. Therefore, MAPs might prevent intracellular calcium overload and decreased glutamate excitotoxicity, both of which can cause neuronal injury and death in vitro models of cerebral ischemic injury. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. Improvement of erythrose reductase activity, deletion of by-products and statistical media optimization for enhanced erythritol production from Yarrowia lipolytica mutant 49.

    Science.gov (United States)

    Ghezelbash, Gholam Reza; Nahvi, Iraj; Emamzadeh, Rahman

    2014-08-01

    The purpose of the present investigation was to produce erythritol by Yarrowia lipolytica mutant without any by-products. Mutants of Y. lipolytica were generated by ultra-violet for enhancing erythrose reductase (ER) activity and erythritol production. The mutants showing the highest ER activity were screened by triphenyl tetrazolium chloride agar plate assay. Productivity of samples was analyzed by thin-layer chromatography and high-performance liquid chromatography equipped with the refractive index detector. One of the mutants named as mutant 49 gave maximum erythritol production without any other by-products (particularly glycerol). Erythritol production and specific ER activity in mutant 49 increased to 1.65 and 1.47 times, respectively, in comparison with wild-type strain. The ER gene of wild and mutant strains was sequenced and analyzed. A general comparison of wild and mutant gene sequences showed the replacement of Asp(270) with Glu(270) in ER protein. In order to enhance erythritol production, we used a three component-three level-one response Box-Behnken of response surface methodology model. The optimum medium composition for erythritol production was found to be (g/l) glucose 279.49, ammonium sulfate 9.28, and pH 5.41 with 39.76 erythritol production.

  6. Upgrading food wastes by means of bromelain and papain to enhance growth and immunity of grass carp (Ctenopharyngodon idella).

    Science.gov (United States)

    Choi, W M; Lam, C L; Mo, W Y; Wong, M H

    2016-04-01

    The fast growing of global aquaculture industry accompanied with increasing pressure on the supply and price of traditional feed materials (e.g., fish meal and soy bean meal). This circumstance has urged the need to search alternative sources of feed stuff. Food waste was used as feed stuff in rearing fish which possess substantial protein and lipid. Grass carp are major species reared in Hong Kong with lower nutritional requirements; it is also an ideal species for investigating the feasibility of using food waste as fish feeds for local aquaculture industry. The growth and immunity, reflected by total protein, total immunologlobulin (IgI), and nitroblue tetrazolium (NBT) activity of grass carp blood, were depressed when feeding with food waste feeds without enzymes. However, the supplementation of bromelain and papain in fish feed enhanced the efficient use of food waste by grass carp, which in turn improved the fish immunity. The present results indicated that the addition of those enzymes could enhance the feed utilization by fish and hematological parameters of grass carp, and the improvement on growth and immunity superior to the control (commercial feed) was observed with the addition of bromelain and papain supplement. Addition of 1 and 2 % mixture of bromelain and papain could significantly enhance the lipid utilization in grass carp.

  7. Evaluation of Inhibitory and Lethal Effects of Aqueous, Ethanolic and Hydroalcoholic Extracts of Aerial Parts of Salvia chorassanica against Some Gram-negative and Gram-positive Bacteria in Vitro

    Directory of Open Access Journals (Sweden)

    Azam Mehraban

    2016-05-01

    Full Text Available Abstract Background and Objectives: Development of bacterial resistance to antibiotics has led to an increased tendency to development of new more effective and non-toxic antimicrobial compounds. In this study, the inhibitory and lethal effects of aqueous, ethanolic, and hydroalcoholic extracts of aerial parts of Salvia chorassanica were evaluated against Listeria monocytogenes, Bacillus cereus, Salmonella typhi, and Escherichia coli O:157. Methods: In this study, Kirby–Bauer disk diffusion method was used to evaluate antimicrobial activity. In this method, bacteria were cultivated as grass culture in Mueller Hinton Agar (MHA media. To determine the minimum inhibitory concentration and minimum bactericidal concentration, micro-dilution method with ELISA and addition of phenyl tetrazolium chloride reagent, were used. Data were analyzed using one-way ANOVA and Duncan’s test at the significance level of p<0.05. Results: The highest diameter of inhibition in agar diffusion method was related to hydroalcoholic extract of aerial parts of Salvia chorassanica against Gram-positive bacteria Bacillus cereus. The amount of calculated MIC of hydro-alcoholic extract for Gram-positive bacteria was 30mg/ml. This amount was the lowest among other measured MIC. Conclusion: Based on the results of this study, Gram-negative bacteria showed more resistance to different extracts of aerial parts of Salvia chorassanica as compared to Gram-positive bacteria, so that Salmonella typhi was found to be the most resistant bacterium among the tested bacteria.

  8. Comparative Efficiency and Impact on the Activity of Blood Neutrophils Isolated by Percoll, Ficoll and Spontaneous Sedimentation Methods.

    Science.gov (United States)

    Mosca, Tainá; Forte, Wilma C N

    2016-01-01

    Studies on the role of cells in physiological and pathological processes generally require isolation of some populations, such as neutrophils. In the literature, several methods used for isolating neutrophils are described; however, there is no consensus on the best technique to be used in cell functional studies. The present study compares the efficiency and impact on the chemotactic and phagocytic activity of neutrophils isolated from blood by three different methods: Percoll and Ficoll density centrifugation gradients and spontaneous sedimentation technique. The neutrophil chemotaxis, stimulated with lipopolysaccharide (LPS), autologous serum or homologous serum, was determined by using Boyden chambers. The phagocytic capacity was assessed by ingestion of zimosan particles, and digestion phase was analyzed by nitroblue tetrazolium test (NBT). The results obtained from neutrophil isolation by Percoll and Ficoll density gradients, as compared to spontaneous sedimentation technique, showed similar degrees of cell yields and higher purity; however, these methods affected neutrophil responsiveness, accompanied by elevated chemotaxis and reduced chemotactic capacity to respond to subsequent stimulation. Neutrophil isolation by spontaneous sedimentation, in contrast, did not affect cellular activity and resulted in cell preparation with high number of neutrophils. Although neutrophil phagocytosis results were similar between the different methods, digestion phase of phagocytosis was significantly enhanced after LPS-stimulation, only in the neutrophils isolated by spontaneous sedimentation technique. In conclusion, the present study shows that isolation of blood neutrophils by the spontaneous sedimentation technique is appropriate for the assessment of cellular activity, since it neither primes or activates the neutrophils nor does it affect their functional responsiveness.

  9. Cytotoxic sesquiterpene lactones from the aerial parts of Inula aucheriana.

    Directory of Open Access Journals (Sweden)

    Ahmad Reza Gohari

    2015-06-01

    Full Text Available Inula aucheriana DC is a member of the family Asteraceae which is known to produce cytotoxic secondary metabolites noted as sesquiterpene lactones. In the present study, sesquiterpene lactones inuchinenolide B, 6-deoxychamissonolide (stevin and 14-acetoxy-1β,5α,7αH-4β-hydroxy-guai-9(10,11(13-dien-12,8α-olide were isolated from I. aucheriana. Inuchinenolide B and 14-acetoxy-1β,5α,7αH-4β-hydroxy-guai-9(10,11(13-dien-12,8α-olide were further evaluated by the MTT (3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide assay to demonstrate cytotoxic activity with IC50 values of (56.6, 19.0, (39.0, 11.8, and (55.7, 15.3 μg/mL against HepG-2, MCF-7 and A-549 cells, respectively. The cytotoxic activity of the two evaluated sesquiterpene lactones partly explains the cytotoxic activity that was previously observed for the extracts of Inula aucheriana. The isolated compounds could be further investigated in cancer research studies.

  10. Effects of Long-Term Starvation on a Host Bivalve (Codakia orbicularis, Lucinidae) and Its Symbiont Population▿

    Science.gov (United States)

    Caro, Audrey; Got, Patrice; Bouvy, Marc; Troussellier, Marc; Gros, Olivier

    2009-01-01

    The bivalve Codakia orbicularis, hosting sulfur-oxidizing gill endosymbionts, was starved (in artificial seawater filtered through a 0.22-μm-pore-size membrane) for a long-term experiment (4 months). The effects of starvation were observed using transmission electron microscopy, fluorescence in situ hybridization and catalyzed reporter deposition (CARD-FISH), and flow cytometry to monitor the anatomical and physiological modifications in the gill organization of the host and in the symbiotic population housed in bacteriocytes. The abundance of the symbiotic population decreased through starvation, with a loss of one-third of the bacterial population each month, as shown by CARD-FISH. At the same time, flow cytometry revealed significant changes in the physiology of symbiotic cells, with a decrease in cell size and modifications to the nucleic acid content, while most of the symbionts maintained a high respiratory activity (measured using the 5-cyano-2,3-ditolyl tetrazolium chloride method). Progressively, the number of symbiont subpopulations was reduced, and the subsequent multigenomic state, characteristic of this symbiont in freshly collected clams, turned into one and five equivalent genome copies for the two remaining subpopulations after 3 months. Concomitant structural modifications appeared in the gill organization. Lysosymes became visible in the bacteriocytes, while large symbionts disappeared, and bacteriocytes were gradually replaced by granule cells throughout the entire lateral zone. Those data suggested that host survival under these starvation conditions was linked to symbiont digestion as the main nutritional source. PMID:19346359

  11. Inhibitory effects of sea buckthorn procyanidins on fatty acid synthase and MDA-MB-231 cells.

    Science.gov (United States)

    Wang, Yi; Nie, Fangyuan; Ouyang, Jian; Wang, Xiaoyan; Ma, Xiaofeng

    2014-10-01

    Fatty acid synthase (FAS) is overexpressed in many human cancers including breast cancer and is considered to be a promising target for therapy. Sea buckthorn has long been used to treat a variety of maladies. Here, we investigated the inhibitory effect of sea buckthorn procyanidins (SBPs) isolated from the seeds of sea buckthorn on FAS and FAS overexpressed human breast cancer MDA-MB-231 cells. The FAS activity and FAS inhibition were measured by a spectrophotometer at 340 nm of nicotinamide adenine dinucleotide phosphate (NADPH) absorption. We found that SBP potently inhibited the activity of FAS with a half-inhibitory concentration (IC50) value of 0.087 μg/ml. 3-4,5-Dimethylthiazol-2-yl-2,3-diphenyl tetrazolium bromide (MTT) assay was used to test the cell viability. SBP reduced MDA-MB-231 cell viability with an IC50 value of 37.5 μg/ml. Hoechst 33258/propidium iodide dual staining and flow cytometric analysis showed that SBP induced MDA-MB-231 cell apoptosis. SBP inhibited intracellular FAS activity with a dose-dependent manner. In addition, sodium palmitate could rescue the cell apoptosis induced by SBP. These results showed that SBP was a promising FAS inhibitor which could induce the apoptosis of MDA-MB-231 cells via inhibiting FAS. These findings suggested that SBP might be useful for preventing or treating breast cancer.

  12. Preparation of electrospun core-sheath yarn with enhanced bioproperties for biomedical materials.

    Science.gov (United States)

    Li, Boyu; Liu, Chengkun; Zhou, Fenglei; Mao, Xue; Sun, Runjun

    2017-11-08

    To create a multifunctional medical material that combines the advantages of both nanofibers and macroyarns. A novel electrospinning-based approach was developed for creating polycaprolactone (PCL) nanofiber covered yarns (PCL-NCYs) in which polyglycolic acid multi-strand filaments (PGA-MFs) were used as the core. BALB/3T3 (mouse embryonic fibroblast cell line) cells were cultured on the PCL-NCYs substrate and cell morphology and proliferation were determined by methylthiazol tetrazolium (MTT) assay. Compared with PGA-MFs, PCL-NCYs had a higher porosity and tensile strength of 88 ± 8% and 348 ± 16 MPa and in particular, the porosity was four times higher. BALB/3T3 cells attached more easily onto the nanofiber structure and proliferated along the direction of nanofibers, indicating that PCL-NCYs can achieve better cell differentiation and proliferation. PCL-NCYs can be created by combining electrospinning covering and textile twisting, and have better mechanical property and higher porosity, and can be used as a novel scaffold in tissue engineering.

  13. Screening for antibacterial and antibiofilm activity in Thai medicinal plant extracts against oral microorganisms.

    Science.gov (United States)

    Teanpaisan, Rawee; Kawsud, Pajaree; Pahumunto, Nuntiya; Puripattanavong, Jindaporn

    2017-04-01

    To evaluate the antibacterial activity of 12 ethanol extracts of Thai traditional herb against oral pathogens. The antibacterial activities were assessed by agar well diffusion, broth microdilution, and time-kill methods. Antibiofilm activity was investigated using a 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium-bromide (MTT) assay. High performance liquid chromatography (HPLC), thin layer chromatography (TLC) fingerprinting, and TLC-bioautography were used to determine the active antibacterial compounds. Piper betle showed the best antibacterial activities against all tested strains in the minimal inhibitory concentration and minimal bactericidal concentration, ranged from 1.04-5.21 mg/mL and 2.08-8.33 mg/mL, respectively. Killing ability depended on time and concentrations of the extract. P. betle extract acts as a potent antibiofilm agent with dual actions, preventing and eradicating the biofilm. The major constituent of P. betle extract was 4-chromanol, which responded for antibacteria and antibiofilm against oral pathogens. It suggests that the ethanol P. betle leaves extract may be used for preventing oral diseases.

  14. Histological Analysis and 3D Reconstruction of Winter Cereal Crowns Recovering from Freezing: A Unique Response in Oat (Avena sativa L.)

    Science.gov (United States)

    Livingston, David P.; Henson, Cynthia A.; Tuong, Tan D.; Wise, Mitchell L.; Tallury, Shyamalrau P.; Duke, Stanley H.

    2013-01-01

    The crown is the below ground portion of the stem of a grass which contains meristematic cells that give rise to new shoots and roots following winter. To better understand mechanisms of survival from freezing, a histological analysis was performed on rye, wheat, barley and oat plants that had been frozen, thawed and allowed to resume growth under controlled conditions. Extensive tissue disruption and abnormal cell structure was noticed in the center of the crown of all 4 species with relatively normal cells on the outside edge of the crown. A unique visual response was found in oat in the shape of a ring of cells that stained red with Safranin. A tetrazolium analysis indicated that tissues immediately inside this ring were dead and those outside were alive. Fluorescence microscopy revealed that the barrier fluoresced with excitation between 405 and 445 nm. Three dimensional reconstruction of a cross sectional series of images indicated that the red staining cells took on a somewhat spherical shape with regions of no staining where roots entered the crown. Characterizing changes in plants recovering from freezing will help determine the genetic basis for mechanisms involved in this important aspect of winter hardiness. PMID:23341944

  15. Antimicrobial activity and cytotoxicity of 3 photosensitizers activated with blue light.

    Science.gov (United States)

    Bulit, Florence; Grad, Iwona; Manoil, Daniel; Simon, Stéphane; Wataha, John C; Filieri, Anna; Feki, Anis; Schrenzel, Jacques; Lange, Norbert; Bouillaguet, Serge

    2014-03-01

    Pulp repair is less likely to occur when dentin or pulpal tissue remains infected after caries excavation. Yet there are currently few options to kill residual bacteria without damaging resident cells. The current study has evaluated the effect of 3 blue light-activated chemicals on the viability of lactobacilli, odontoblast-like cells (MDPC-23), undifferentiated pulp cells (OD21), and human embryonic stem cells (hESC H1). Bacteria were incubated for 15 minutes with curcumin, eosin Y, or rose bengal and then irradiated with blue light (240 seconds). Bacteria were labeled with LIVE/DEAD BacLight Bacterial Viability kit, and viability was assessed by fluorescence-activated cell sorting. Cytotoxicity assays were performed on MDPC-23 cells, OD21, and hESC H1 cells grown in 24-well plates and exposed to the same photosensitizer-light combination. After 24 hours, cellular response was measured by using the methyl-thiazol-diphenyl-tetrazolium assay. Results were statistically analyzed by using one-way analysis of variance and Tukey multiple comparison intervals (α = 0.05). Bacterial viability was significantly reduced after exposure to different combinations of light and photosensitizers; mitochondrial activity of cultured cells remained unaffected when exposed to the same conditions, suggesting a good therapeutic index in vitro. Blue light-mediated disinfection is promising for the development of new treatment strategies designed to promote pulp repair after carious exposure. Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  16. Isolation and activity of two antibacterial biflavonoids from leaf extracts of Garcinia livingstonei (Clusiaceae).

    Science.gov (United States)

    Kaikabo, Adamu A; Samuel, Babatunde B; Eloff, Jacobus N

    2009-10-01

    The antibacterial activity of the acetone extract of Garcinia livingstonei leaves was studied using bioautography and by determining the minimum antibacterial concentration against four nosocomial pathogens. Bioautograms showed that two compounds were mainly responsible for the antibacterial activity. These were isolated by a combination of solvent-solvent fractionation and bioassay-guided chromatographic fractionation and were characterized by 1H NMR, 13C NMR and 2D NMR spectroscopy as amentoflavone and 4"-methoxy amentoflavone. The antibacterial activity of the isolated compounds was determined against Escherichia coli, Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa. Three of the tested organisms were sensitive to both compounds with MIC values ranging from 8-100 microg/mL. P. aeruginosa was resistant with MICs > 100 microg/mL. The safety of the two compounds was assessed with a tetrazolium based colorimetric assay (MTT assay) using Vero monkey kidney cells. The compounds had low toxicity against the cell line with cytotoxic concentrations to 50% of the cells (LD50) of 386 microg/mL and > 600 microg/mL for amentoflavone and 4"-methoxy amentoflavone, respectively. Berberine, the positive control had a CC50 of 170 microg/mL. 4"-Methoxy amentoflavone was more active and much less toxic than amentoflavone.

  17. The neuroprotective effect of immune serum of adeno-associated virus vaccine containing Aβ1-15 gene on amyloid toxicity

    Directory of Open Access Journals (Sweden)

    Ling-Yun Liu

    2013-01-01

    Full Text Available Objective: The aim of this study was to explore the effect of adeno-associated virus (AAV serotype 2 vector vaccine containing amyloid-β peptide (Aβ 1-15 gene fragment (AAV-Aβ15 immunized mice sera on counteracting Aβ1-42 peptide toxicity towards a primary culture cortical neurons. Materials and Methods: BALB/c mice were vaccinated via the intramuscular immunization route with AAV-Aβ15. The anti-Aβ antibody titer of immunized mice sera was quantified by sandwich Enzyme-Linked ImmunoSorbent Assay. The toxicity of Aβ1-42 peptide on neurons was assessed by morphology with an inverse microscopy and cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT assay. Results: AAV-Aβ15 could induce an Aβ-specific immunoglobulin G (IgG humoral immune response in /c mice the anti-Aβ antibodies were detectable at 1 month after immunization, significantly increased at 2 and 4 months after immunization, and the immunized sera could attenuate cytotoxicity of Aβ1-42 peptide on primary culture cortical neurons. Conclusions: The immune serum of AAV-Aβ15 could play a neuroprotective effect against Aβ1-42 peptide toxicity, which would be beneficial for Alzheimer′s disease patients.

  18. Immunomodulatory effects of Aloe vera and its fractions on response of macrophages against Candida albicans.

    Science.gov (United States)

    Farahnejad, Zohreh; Ghazanfari, Tooba; Yaraee, Roya

    2011-12-01

    Natural products are important resources in traditional medicine and have been long used for prevention and treatment of many diseases. Medicinal plants have immunomodulatory properties. Aloe is one of the herbal medicines widely used in natural treatment and alternative therapy for various types of diseases. Aloe vera has been shown to modulate the immune response. Macrophages have been shown to play an essential role as the first line of defense against invading pathogen. Candida albicans is a communal and opportunistic pathogen in humans. In this study, we investigated the effect of A. vera extract and its fractions on infected macrophages with C. albicans. Viability of intraperitoneal macrophages was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test. Cell viability of infected macrophages was increased by the extract and dose of some isolated fractions dependently. The extract as well as R100, R50, R30, and R10 fractions of A. vera significantly increased cell viability of macrophages in most doses. R5 and F5 fractions showed no significant difference in comparison with control group. Further studies in animal models and human are necessary to clarify the modulatory effects of A. vera on macrophage function. Isolation and purification of A. vera components are also needed to find out the effective molecules.

  19. Immunomodulatory activity of methanolic extracts of fruits and bark of Ficus glomerata Roxb. in mice and on human neutrophils.

    Science.gov (United States)

    Heroor, Sanjeev; Beknal, Arun Kumar; Mahurkar, Nitin

    2013-01-01

    To evaluate the immunomodulatory activity of methanolic extracts of fruit and bark of Ficus glomerata Roxb. on cyclophosphamide-induced myelosuppression in mice and the phagocytic effect on human neutrophils. Methanolic extracts of fruits and bark of Ficus glomerata Roxb. at two dose levels of 250 and 500 mg/kg p.o. were administered for 13 days to albino mice and cyclophosphamide (30 mg/kg i.p.) was administered on 11th, 12th, and 13th days, 1 hour after the administration of the respective treatment. On 14th day blood was collected and the hematological parameters were evaluated. The two extracts in the concentration range 100, 50, 25, 12 and 6.25 μg were also tested for phagocytic effect on human neutrophils using the in vitro models-nitroblue tetrazolium (NBT) dye test, phagocytosis of Candida albicans, and chemotaxis assay. Methanolic extracts of fruit and bark of Ficus glomerata Roxb. showed significant counteracting effect (P plant in the concentration range 100, 50, 25, 12, and 6.25 μg also showed significant (P effect on human neutrophils in the parameters studied. Methanolic extracts of fruits and bark of Ficus glomerata Roxb. exhibited immunomodulatory property in both in vivo and in vitro models.

  20. Lidocaine inhibits the proliferation of lung cancer by regulating the expression of GOLT1A.

    Science.gov (United States)

    Zhang, Lei; Hu, Rong; Cheng, Yanyong; Wu, Xiaoyang; Xi, Siwei; Sun, Yu; Jiang, Hong

    2017-10-01

    Lidocaine is the most commonly used local anaesthetic in clinical and can inhibit proliferation, suppress invasion and migration and induce apoptosis in human lung adenocarcinoma (LAD) cells. However, its specific downstream molecular mechanism is unclear. LAD cell lines, A549 and H1299 cells, were treated with lidocaine. The proliferation was evaluated by the methylthiazolyldiphenyl-tetrazolium bromide (MTT) and bromodeoxyuridine (BrdU) assay. The expression level of related proteins was detected by real-time quantitative PCR (qPCR) and Western blot assay. The results indicated that lidocaine dose-dependently suppressed the proliferation of A549 and H1299 cells. In the LAD patients' samples, GOLT1A was upregulated and involved in the poor prognosis and higher grade malignancy. Additionally, GOLT1A mediates the function of lidocaine on repressing proliferation by regulating the cell cycle in A549 cells. Our findings suggest that lidocaine downregulates the GOLT1A expression to repress the proliferation of lung cancer cells. © 2017 John Wiley & Sons Ltd.

  1. Cyclosporine A decreases the fluconazole minimum inhibitory concentration of Candida albicans clinical isolates but not biofilm formation and cell growth.

    Science.gov (United States)

    Wibawa, T; Nurrokhman; Baly, I; Daeli, P R; Kartasasmita, G; Wijayanti, N

    2015-03-01

    Among the genus Candida, Candida albicans is the most abundant species in humans. One of the virulent factors of C. albicans is its ability to develop biofilm. Biofilm forming microbes are characterized by decreasing of its susceptibility to antibiotics and antifungal. The fungicidal effect of fluconazole may be enhanced by cyclosporine A in laboratory engineered C. albicans strains. The aim of this work is to analyze the synergistic effect of cyclosporine A with fluconazole in C. albicans clinical isolates and the effect of cycolsporine A alone in the biofilm formation. Six fluconazole resistant and six sensitive C. albicans clinical isolates were analyzed for its minimum inhibitory concentration (MICs), biofilm formation, and cell growths. A semi-quantitative XTT [2,3-bis(2-methoxy-4-nitro-5- sulfo-phenyl)-2H-tetrazolium-5-carboxanilide] reduction assay was conducted to measure the biofilm formation. Cyclosporine A has synergistic effect with fluconazole that was shown by decreasing MICs of both fluconazole resistant and sensitive C. albicans clinical isolates. However, cyclosporine A alone did not influence the biofilm formation and cell growth of both fluconazole resistant and sensitive C. albicans clinical isolates. These results indicated that cyclosporine A might be a promising candidate of adjuvant therapy for fluconazole against both fluconazole resistant and sensitive C. albicans clinical isolates.

  2. Stimulated Osteogenic Differentiation of Human Mesenchymal Stem Cells by Reduced Graphene Oxide.

    Science.gov (United States)

    Jin, Linhua; Lee, Jong Ho; Jin, Oh Seong; Shin, Yong Cheol; Kim, Min Jeong; Hong, Suck Won; Lee, Mi Hee; Park, Jong-Chul; Han, Dong-Wook

    2015-10-01

    Osteoprogenitor cells play a significant role in the growth or repair of bones, and have great potential as cell sources for regenerative medicine and bone tissue engineering, but control of their specific differentiation into bone cells remains a challenge. Graphene-based nanomaterials are attractive candidates for biomedical applications as substrates for stem cell (SC) differentiation, scaffolds in tissue engineering, and components of implant devices owing to their biocompatible, transferable and implantable properties. This study examined the enhanced osteogenic differentiation of human mesenchymal stem cells (hMSCs) by reduced graphene oxide (rGO) nanoparticles (NPs), and rGO NPs was prepared by reducing graphene oxide (GO) with a hydrazine treatment followed by annealing in argon and hydrogen. The cytotoxicity profile of each particle was examined using a water-soluble tetrazolium-8 (WST-8) assay. At different time-points, a WST-8 assay, alkaline phosphatase (ALP) activity assay and alizarin red S (ARS) staining were used to determine the effects of rGO NPs on proliferation, differentiation and mineralization, respectively. The results suggest that graphene-based materials have potential as a platform for stem cells culture and biomedical applications.

  3. Additive antifungal activity of anidulafungin and human neutrophils against Candida parapsilosis biofilms.

    Science.gov (United States)

    Katragkou, Aspasia; Chatzimoschou, Athanasios; Simitsopoulou, Maria; Georgiadou, Elpiniki; Roilides, Emmanuel

    2011-03-01

    To investigate the activities of two newer triazoles and two echinocandins combined with human phagocytes against Candida parapsilosis biofilms. An in vitro model of C. parapsilosis biofilms was used. Biofilms were grown on silicone elastomer discs in 96-well plates at 37°C for 72 h. Biofilms or planktonic cells were incubated with voriconazole, posaconazole, caspofungin or anidulafungin, at clinically relevant concentrations, and human phagocytes (neutrophils or monocytes) alone or in combination with each of the antifungal agents for a further 22 h. Fungal damage induced by antifungal agents and/or phagocytes was determined by XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)2H-tetrazolium-5-carboxanilide] metabolic assay. Each of the antifungal agents alone and in combination with human phagocytes induced less damage against C. parapsilosis biofilms compared with planktonic cells. No antagonistic interactions between antifungal agents and phagocytes were found. Furthermore, anidulafungin, but not caspofungin, and neutrophils exerted additive activity against C. parapsilosis biofilms. Besides a lack of antagonistic interactions between newer antifungal agents and phagocytes, anidulafungin exerts additive immunopharmacological activity against C. parapsilosis biofilms.

  4. Activities of Triazole-Echinocandin Combinations against Candida Species in Biofilms and as Planktonic Cells▿†

    Science.gov (United States)

    Chatzimoschou, Athanasios; Katragkou, Αspasia; Simitsopoulou, Maria; Antachopoulos, Charalampos; Georgiadou, Elpiniki; Walsh, Thomas J.; Roilides, Emmanuel

    2011-01-01

    Biofilm formation complicates the treatment of various infections caused by Candida species. We investigated the effects of simultaneous or sequential combinations of two triazoles, voriconazole (VRC) and posaconazole (PSC), with two echinocandins, anidulafungin (AND) and caspofungin (CAS), against Candida albicans and Candida parapsilosis biofilms in comparison to their planktonic counterparts. Antifungal activity was assessed by the 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]2H-tetrazolium-5-carboxanilide (XTT) metabolic assay. Antifungal-agent interactions were analyzed by the Bliss independence model in the simultaneous-treatment studies and by analysis of variance (ANOVA) in the sequential-treatment studies. Against C. albicans planktonic cells, the simultaneous combination of PSC (32 to 128 mg/liter) and CAS (0.008 to 0.25 mg/liter) was synergistic; the combinations of PSC (128 to 1,024 mg/liter) with AND (0.03 to 0.5 mg/liter) and VRC (32 to 512 mg/liter) with AND (0.008 to 0.03 mg/liter) were antagonistic. Against C. parapsilosis planktonic cells, the interaction between VRC (32 to 1,024 mg/liter) and CAS (1 to 16 mg/liter) was antagonistic. All simultaneous antifungal combinations demonstrated indifferent interactions against biofilms of both Candida species. Damage to biofilms of both species increased (P Triazole-echinocandin combinations do not appear to produce antagonistic effects against Candida sp. biofilms, while various significant interactions occur with their planktonic counterparts. PMID:21343465

  5. Intra-operative microwave ablation of liver malignancies with tumour permittivity feedback control: a prospective ablate and resect study.

    Science.gov (United States)

    Ratanaprasatporn, Linda; Charpentier, Kevin P; Resnick, Murray; Lu, Shaolei; Dupuy, Damian

    2013-12-01

    Tumour permittivity feedback control is a novel method for microwave ablation (MWA) that theoretically allows for larger, more predictable ablations. This prospective case series evaluates the feasibility and efficacy of MWA of liver malignancies using a device with tumour permittivity feedback control. Ten consecutive patients initially determined to be candidates for surgical resection of a liver malignancy underwent intra-operative MWA with tumour permittivity feedback control followed by a surgical resection. A 14-gauge Medwaves microwave antenna was used to deliver a single treatment according to the manufacturer's recommendations. Tumours were assessed grossly as well as by haematoxylin and eosin (H&E) and tetrazolium chloride staining. The primary end point was per cent tumour necrosis. The median maximum ablation diameter measured was 4.1 cm (range 3.0-6.8). The median ablation volume was 8.7 cm(3) (range 4.84-17.55). Six of the 10 tumours demonstrated a pathological complete response (CR). Six of seven tumours ≤ 3 cm demonstrated a pathological CR. Zero of the three tumours ≥ 3 cm had a pathological CR, but all had ≥ 50% tumour necrosis. All patients survived and there were no ablation-related morbidities. MWA of liver tumours with tumour permittivity feedback control is feasible and appears effective for the treatment of small (< 3 cm) liver tumours. © 2013 International Hepato-Pancreato-Biliary Association.

  6. Effects of Curcuma longa Extract on Telomerase Activity in Lung and Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Nosratollah Zarghami

    2014-10-01

    Full Text Available Background: The purpose of this study is to evaluate the effect of Curcuma longa extract on the telomerase gene expression in QU-DB lung cancer and T47D breast cancer cell lines. Materials and Methods: The present study is an experimental research. Using 3 different phases n-hexane, dichloromethane and methanol, total extract of Curcuma longa in a serial dilution was prepared and three phases was analyzed for determining which phase has more curcuminoids. Then the extract cytotoxicity effect was tested on breast cancer cell line (T47D, and lung cancer cell line (QU-DB by 24, 48 and 72 h MTT (Dimethyl thiazolyl diphenyl tetrazolium assay. Then, the cells were treated with serial concentrations of the extract. Finally, total protein was extracted from the control and test groups, its quantity was determined and telomeric repeat amplification protocol (TRAP assay was performed for measurement of possible inhibition of the telomerase activity. Results: Cell viability and MTT-based cytotoxicity assay show that the total extract of Curcuma longa has cytotoxic effect with different IC50s in breast and lung cancer cell lines. Analysis of TRAP assay also shows a significant reduction in telomerase activity on both cancer cells with different levels. Conclusion: Curcuma longa extract has anti-proliferation and telomerase inhibitory effects on QU-DB lung cancer and T47D breast cancer cells with differences in levels of telomerase inhibition.

  7. Assessment of Cytotoxic Activity of Rosemary (Rosmarinus officinalis L.), Turmeric (Curcuma longa L.), and Ginger (Zingiber officinale R.) Essential Oils in Cervical Cancer Cells (HeLa).

    Science.gov (United States)

    Santos, P A S R; Avanço, G B; Nerilo, S B; Marcelino, R I A; Janeiro, V; Valadares, M C; Machinski, Miguel

    2016-01-01

    The objective of this study was to evaluate the cytotoxic activity of rosemary (REO, Rosmarinus officinalis L.), turmeric (CEO, Curcuma longa L.), and ginger (GEO, Zingiber officinale R.) essential oils in HeLa cells. Cytotoxicity tests were performed in vitro, using tetrazolium (MTT) and neutral red assays for evaluation of antiproliferative activity by different mechanisms, trypan blue assay to assess cell viability and evaluation of cell morphology for Giemsa to observe the cell damage, and Annexin V to evaluate cell death by apoptosis. CEO and GEO exhibited potent cytotoxic activity against HeLa cells. IC50 obtained was 36.6 μg/mL for CEO and 129.9 μg/mL for GEO. The morphology of HeLa cells showed condensation of chromatin, loss of cell membrane integrity with protrusions (blebs), and cell content leakage for cells treated with CEO and GEO, from the lowest concentrations studied, 32.81 μg/mL of CEO and 32.12 μg/mL of GEO. The Annexin V assay revealed a profile of cell death by apoptosis for both CEO and GEO. The results indicate cytotoxic activity in vitro for CEO and GEO, suggesting potential use as anticancer agents for cervical cancer cells.

  8. Viability of various weed seeds in anaerobic conditions (biogas plant)

    Energy Technology Data Exchange (ETDEWEB)

    Hansen, S.; Hansen, J.

    1983-04-01

    Seeds from different weeds, Urtica urens L. (nettle), Solanum nigrum L. (nightshade), Avena fatua L. (wild oat-grass), Brassica napus L. (rape), Chenopodium album L. (goose-foot), were put into small polyester net bags, which were placed in biogas reactors containing cattle manure. These ''biogas reactors'' were placed at different temperatures . Net bags were taken out after 4.5, 10.5, 21.5, 38 and 53 days, and the seeds were tested for their viability by germination tests and the tetrazolium method. Concerning all seeds it was manifested that the viability decreased very steeply at 35degC. Most of the seeds had a T/sub 50/ at 2-5 days; Chenopodium album L seeds had a T/sub 50/ at 16 days. After 4.5 days it was not possible to find living Avena fatua L seeds. The decrease in viability was less steep at 20degC and even less steep at 2degC.

  9. Effect of acetic acid on citric acid fermentation in an integrated citric acid-methane fermentation process.

    Science.gov (United States)

    Xu, Jian; Chen, Yang-Qiu; Zhang, Hong-Jian; Tang, Lei; Wang, Ke; Zhang, Jian-Hua; Chen, Xu-Sheng; Mao, Zhong-Gui

    2014-09-01

    An integrated citric acid-methane fermentation process was proposed to solve the problem of extraction wastewater in citric acid fermentation process. Extraction wastewater was treated by anaerobic digestion and then recycled for the next batch of citric acid fermentation to eliminate wastewater discharge and reduce water resource consumption. Acetic acid as an intermediate product of methane fermentation was present in anaerobic digestion effluent. In this study, the effect of acetic acid on citric acid fermentation was investigated and results showed that lower concentration of acetic acid could promote Aspergillus niger growth and citric acid production. 5-Cyano-2,3-ditolyl tetrazolium chloride (CTC) staining was used to quantify the activity of A. niger cells, and the results suggested that when acetic acid concentration was above 8 mM at initial pH 4.5, the morphology of A. niger became uneven and the part of the cells' activity was significantly reduced, thereby resulting in deceasing of citric acid production. Effects of acetic acid on citric acid fermentation, as influenced by initial pH and cell number in inocula, were also examined. The result indicated that inhibition by acetic acid increased as initial pH declined and was rarely influenced by cell number in inocula.

  10. Optimization of the sulforhodamine B colorimetric assay.

    Science.gov (United States)

    Papazisis, K T; Geromichalos, G D; Dimitriadis, K A; Kortsaris, A H

    1997-10-27

    Sulforhodamine B (SRB) protein staining has been widely used for cell proliferation and chemosensitivity testing, substituting for tetrazolium-based assays. However, the cell fixation step in the original assay is subject to error. We tested whether aspiration of medium with an automatic microplate multiwash device prior to fixation improves the method for adherent cells. A panel of adherent cell lines was used. Signal-to-noise ratios were significantly increased in the new assay. Coefficients of variation (CV) between replicate wells were significantly lower especially at lower cell densities. The linearity of the method improved, with absolute linearity over the whole range of cell densities. The aspiration procedure dislodged only negligible numbers of cells. Cytotoxicity testing using the cytotoxic agent paclitaxel showed no IC50 (50% inhibitory concentration) differences between the new and original methods but a better CV was associated with the optimized protocol. We conclude that aspiration of the growth medium prior to fixing comprises a safe and reliable practice which improves CV, linearity and the signal-to-noise ratio of the SRB assay.

  11. Comparative analysis of two colorimetric assays in dental pulp cell density

    Science.gov (United States)

    Nedel, F.; Soki, F.N.; Conde, M.C.M.; Zeitlin, B.D.; Tarquinio, S.B.C.; Nör, J.E.; Seixas, F.K.; Demarco, F.F.

    2013-01-01

    Aim To compare and contrast two colorimetric assays used for measurement of proliferation using two dental pulp cell types: dental pulp stem cells (DPSC) and human dental pulp fibroblasts (HDPF). Methodology DPSC or HDPF were seeded at 0.25 × 104 cells/well in 96-well plates. Cell proliferation was evaluated after 24-72 hours. At the end of the experimental period, the Sulforhodamine B (SRB) assay or a water-soluble tetrazolium salt (WST-1) assay was performed. Optical densities were determined in a microplate reader (Genius; TECAN). Data were analyzed by Student’s t-test (comparison between cell types), and one-way ANOVA followed by Tukey test (time point intervals). Pearson’ correlation tests were performed to compare the two assays for each cell line. Results Both assays showed that DPSC had higher proliferation rates than HDPF. A positive significant correlation between the two colorimetric assays tested for both cell types DPSC (Pearson’s Correlation Coefficient=0.847; passay is therefore one of practical application. SRB stained plates may be dried and stored so may have utility in laboratories where data may require review or when access to analytical equipment is limited. WST-1 assays have the benefit of both ease and speed and may have utility in laboratories requiring either high throughput or rapid analyses. PMID:20880134

  12. The Analysis of Cu(II)/Zn(II) Cyclopeptide System as Potential Cu,ZnSOD Mimic Center.

    Science.gov (United States)

    Kotynia, Aleksandra; Janek, Tomasz; Czyżnikowska, Żaneta; Bielińska, Sylwia; Kamysz, Wojciech; Brasuń, Justyna

    2017-01-01

    In this paper are presented the features of copper (II) and zinc (II) heteronuclear complexes of the cyclic peptide-c(HKHGPG)2. The coordination properties of ligand were studied by potentiometric, UV-Vis and CD spectroscopic methods. These experiments were carried out in aqueous solutions at 298 K depending on pH. It turned out that in a physiological pH dominates Cu(II)/Zn(II) complex ([CuZnL]4+) which could mimic the active center of superoxide dismutase (Cu,ZnSOD). In next step we performed in vitro research on Cu,ZnSOD activity for [CuZnL]4+ complex existing in 7.4 pH by the method of reduction of nitroblue tetrazolium (NBT). Also mono- and di-nuclear copper (II) complexes of this ligand were examined. The ability of inhibition free radical reaction were compared for all complexes. The results of these studies show that Cu(II) mono-, di-nuclear and Cu(II)/Zn(II) complexes becoming to new promising synthetic superoxide dismutase mimetics, and should be considered for further biological assays.

  13. Cytotoxic Effect of a Novel Synthesized Carbazole Compound on A549 Lung Cancer Cell Line.

    Directory of Open Access Journals (Sweden)

    Refilwe P Molatlhegi

    Full Text Available Increased death rates due to lung cancer have necessitated the search for potential novel anticancer compounds such as carbazole derivatives. Carbazoles are aromatic heterocyclic compounds with anticancer, antibacterial and anti-inflammatory activity. The study investigated the ability of the novel carbazole compound (Z-4-[9-ethyl-9aH-carbazol-3-yl amino] pent-3-en-2-one (ECAP to induce cytotoxicity of lung cancer cells and its mechanism of action. ECAP was synthesized as a yellow powder with melting point of 240-247 °C. The 3-(4,5-dimethythiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT, lipid peroxidation and comet assays were used to assess the cytotoxic effect of the compound on A549 lung cancer cells. Protein expression was determined using western blots, apoptosis was measured by luminometry (caspase-3/7, -8 and -9 assay and flow cytometry was used to measure phosphatidylserine (PS externalisation. ECAP induced a p53 mediated apoptosis of lung cancer cells due to a significant reduction in the expression of antioxidant defence proteins (Nrf2 and SOD, Hsp70 (p < 0.02 and Bcl-2 (p < 0.0006, thereby up-regulating reactive oxygen species (ROS production. This resulted in DNA damage (p < 0.0001, up-regulation of Bax expression and caspase activity and induction of apoptosis in lung cancer cells. The results show the anticancer potential of ECAP on lung cancer.

  14. Establishment and Application of a Visual DNA Microarray for the Detection of Food-borne Pathogens.

    Science.gov (United States)

    Li, Yongjin

    2016-01-01

    The accurate detection and identification of food-borne pathogenic microorganisms is critical for food safety nowadays. In the present work, a visual DNA microarray was established and applied to detect pathogens commonly found in food, including Salmonella enterica, Shigella flexneri, E. coli O157:H7 and Listeria monocytogenes in food samples. Multiplex PCR (mPCR) was employed to simultaneously amplify specific gene fragments, fimY for Salmonella, ipaH for Shigella, iap for L. monocytogenes and ECs2841 for E. coli O157:H7, respectively. Biotinylated PCR amplicons annealed to the microarray probes were then reacted with a streptavidin-alkaline phosphatase conjugate and nitro blue tetrazolium/5-bromo-4-chloro-3'-indolylphosphate, p-toluidine salt (NBT/BCIP); the positive results were easily visualized as blue dots formatted on the microarray surface. The performance of a DNA microarray was tested against 14 representative collection strains and mock-contamination food samples. The combination of mPCR and a visual micro-plate chip specifically and sensitively detected Salmonella enterica, Shigella flexneri, E. coli O157:H7 and Listeria monocytogenes in standard strains and food matrices with a sensitivity of ∼10(2) CFU/mL of bacterial culture. Thus, the developed method is advantageous because of its high throughput, cost-effectiveness and ease of use.

  15. TESTE DE DETERIORAÇÃO CONTROLADA EM SEMENTES DE QUATRO ESPÉCIES DE ORQUÍDEAS PARA AVALIAÇÃO DO COMPORTAMENTO FISIOLÓGICO

    Directory of Open Access Journals (Sweden)

    Jéssica Fontes Filet

    2015-12-01

    Full Text Available An alternative for conservation of this species is seed banking, based in that is possible to make conservation and propagations of the endangered species. But for this the seed quality must be monitored. Vigour tests can offer an option, and one of this is the controlled deterioration, which expose the seeds with high moisture to high temperatures during a determined period of time. The present work aimed to evaluate the controlled deterioration test over four orchid species in different times. Seeds of C. schilleriana, C. tigrina, C. crispata and C. labiata was submitted to deterioration from times ranging from zero to 48h at 43°C. For Cattleya crispata it was not found a effective time for the test. did not suffer any deterioration at any time. It is recommended for C. tigrina, C. labiata and C. schilleriana the use of 27 h at 43°C. However, time and temperature should be better studied for this group of plants. Tetrazolium test was not efficient to evaluate the seed quality after controlled deterioration.

  16. Bonellia albiflora: A Mayan Medicinal Plant That Induces Apoptosis in Cancer Cells.

    Science.gov (United States)

    Moo-Puc, Rosa; Chale-Dzul, Juan; Caamal-Fuentes, Edgar

    2013-01-01

    Few studies have been carried out on the medical flora of Mexico's Yucatan Peninsula in search for new therapeutic agents, in particular against cancer. In this paper, we evaluated the cytotoxic potential of the extract of Bonellia albiflora, a plant utilized in the traditional Mayan medicine for treatment of chronic injuries of the mouth. We carried out the methanolic extracts of different parts of the plant by means of extraction with the Soxhlet equipment. We conducted liquid-liquid fractions on each extract with solvents of increasing polarity. All extracts and fractions were evaluated for cytotoxic activity versus four human cancer cell lines and one normal cell line through a tetrazolium dye reduction (MTT) assay in 96-well cell culture plates. The methanolic root-bark extract possessed much greater cytotoxic activity in the human oropharyngeal cancer cell line (KB); its hexanic fraction concentrated the active metabolites and induced apoptosis with the activation of caspases 3 and 8. The results demonstrate the cytotoxic potential of the B. albiflora hexanic fraction and substantiate the importance of the study of the traditional Mayan medicinal plants.

  17. In vitro assessment of the genotoxic and cytotoxic effects of boiled juice (tucupi) from Manihot esculenta Crantz roots.

    Science.gov (United States)

    Cunha, L A; Mota, T C; Cardoso, P C S; Alcântara, D D F Á; Burbano, R M R; Guimarães, A C; Khayat, A S; Rocha, C A M; Bahia, M O

    2016-10-05

    The population of Pará (a state in Brazil) has a very characteristic food culture, as a majority of the carbohydrates consumed are obtained from cassava (Manihot esculenta Crantz) derivatives. Tucupi is the boiled juice of cassava roots that plays a major role in the culinary footprint of Pará. Before boiling, this juice is known as manipueira and contains linamarin, a toxic glycoside that can decompose to hydrogen cyanide. In this study, the cytotoxic and genotoxic effects of tucupi on cultured human lymphocytes were assessed using the comet assay and detection of apoptosis and necrosis by differential fluorescent staining with acridine orange-ethidium bromide. Tucupi concentrations (v/v) were determined using the methylthiazole tetrazolium biochemical test. Concentrations of tucupi that presented no genotoxic effects (2, 4, 8, and 16%) were used in our experiments. The results showed that under our study conditions, tucupi exerted no genotoxic effects; however, cytotoxic effects were observed with cell death mainly induced by necrosis. These effects may be related to the presence of hydrogen cyanide in the juice.

  18. In vitro cytotoxic and antioxidant activities of phenolic components of Algerian Achillea odorata leaves

    Directory of Open Access Journals (Sweden)

    Hanane Boutennoun

    2017-03-01

    Full Text Available In this study, methanol extract from Achillea odorata was evaluated for its phenolic contents using Folin–Ciocalteu reagent, and antioxidant activity using: 1,1-diphenyl-2-picrylhidrazyl (DPPH radical scavenging activity, reducing activity of H2O2 and ferric reducing power assay. The total phenolic content was determined as gallic acid (GAE equivalent. Flavonoids and flavonols contents were determined as quercetin (QE equivalents. The cytotoxicity of the plant extract was tested against three tumor cell lines: MCF-7, Hep2 and WEHI using 3-(4,5-dimethyl thiazol-2-yl-2,5-diphynyl tetrazolium bromide (MTT assay. Preliminary screening was concluded in the presence of substances with large therapeutic values. The total phenolic content confirmed the presence of total phenolics in the extract and showed strong association with antioxidant activity. An important content of flavonoids and flavonols was also detected. The results of the antioxidant activities obtained indicate that A. odorata recorded a good capacity. For the cytotoxic activity, the results showed the plant extract significantly inhibited tumor cell growth and colony formation at various concentrations.

  19. The association atorvastatin-meloxicam reduces brain damage, attenuating reactive gliosis subsequent to arterial embolism = La asociación atorvastatina-meloxicam reduce el daño cerebral, atenuando la gliosis reactiva consecuente a embolismo arterial

    Directory of Open Access Journals (Sweden)

    Marcela Hernández Torres

    2013-10-01

    Full Text Available The association atorvastatin-meloxicam reduces brain damage, attenuating reactive gliosis subsequent to arterial embolism Introduction: Stroke is the leading cause of disability and the third of death in Colombia and in the world and it is associated with neurodegenerative and mental diseases. Objective: To determine the effects of the atorvastatin- meloxicam association on reactive gliosis in a model of cerebral ischemia produced by arterial embolization. Materials and methods: 56 adult male Wistar rats were used, divided into four ischemic and four control groups, plus 10 additional animals to determine the distribution and extent of infarction by injury in six of them and simulation (sham in the remaining four. The treatments were: placebo, atorvastatin (ATV, meloxicam (MELOX and ATV + MELOX in ischemic and simulated animals. 24 hours post-ischemia mitochondrial enzymatic activity was evaluated with triphenyl- tetrazolium (TTC, and at 120 hours astrocytic reactivity (anti-GFAP was analyzed by conventional immunohistochemistry. Results: The association ATV + MELOX favored the modulation of the response of protoplasmatic and fibrous astrocytes in both the hippocampus and the paraventricular zone by reducing their hypereactivity. Conclusion: Atorvastatin and meloxicam, either individually or associated, reduce cerebral damage by lessening the reactive gliosis produced by arterial embolization; this suggests new mechanisms of neuroprotection against thromboembolic cerebral ischemia, and opens new perspectives in its early treatment.

  20. Glycyrrhetinic Acid Inhibits Cell Growth and Induces Apoptosis in Ovarian Cancer A2780 Cells

    Directory of Open Access Journals (Sweden)

    Venus Haghshenas

    2014-10-01

    Full Text Available Purpose: Accumulating evidence indicates that glycyrrhizin (GZ and its hydrolyzed metabolite 18-β glycyrrhetinic acid (GA exhibit anti-inflammatory and anticancer activities. The objective of this study was to examine the in vitro cytotoxic activity of GA on human ovarian cancer A2780 cells. Methods: A2780 cells were cultured in RPMI1640 containing 10% fetal bovine serum. Cells were treated with different doses of GA and cell viability and proliferation were detected by dye exclusion and 3-bis-(2-methoxy-4-nitro-5-sulfophenyl-2H-tetrazolium-5-carboxanilide (XTT assays. Apoptosis induction and expression of Fas and Fas ligand (FasL were analyzed by flow cytometry. Results: We observed that GA decreases cell viability and suppressed cells proliferation in a dose-dependent manner as detected by dye-exclusion and XTT assayes. In addition, our flow cytometry data show that GA not only induces apoptosis in A2780 cells but also upregulates both Fas and FasL on these cells in a dose-dependent manner. Conclusion: we demonstrate that GA causes cell death in A2780 cells by inducing apoptosis.

  1. Petiveria alliacea extracts uses multiple mechanisms to inhibit growth of human and mouse tumoral cells.

    Science.gov (United States)

    Urueña, Claudia; Cifuentes, Claudia; Castañeda, Diana; Arango, Amparo; Kaur, Punit; Asea, Alexzander; Fiorentino, Susana

    2008-11-18

    There is ethnopharmacological evidence that Petiveria alliacea can have antitumor activity; however, the mechanism of its cytotoxic activity is not well understood. We assessed multiple in vitro biological activities of an ethyl acetate soluble plant fraction over several tumor cell lines. Tumor cell lines were evaluated using the following tests: trypan blue exclusion test, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], flow cytometry, cytoskeleton organization analysis, cell cycle, mitochondria membrane depolarization, clonogenicity test, DNA fragmentation test and differential protein expression by HPLC-Chip/MS analysis. F4 fraction characterization was made by HPLC-MS. Petiveria alliacea fraction characterized by de-replication was found to alter actin cytoskeleton organization, induce G2 cell cycle arrest and cause apoptotic cell death in a mitochondria independent way. In addition, we found down regulation of cytoskeleton, chaperone, signal transduction proteins, and proteins involved in metabolic pathways. Finally up regulation of proteins involved in translation and intracellular degradation was also observed. The results of this study indicate that Petiveria alliacea exerts multiple biological activities in vitro consistent with cytotoxicity. Further studies in animal models are needed but Petiveria alliacea appears to be a good candidate to be used as an antitumor agent.

  2. Petiveria alliacea extracts uses multiple mechanisms to inhibit growth of human and mouse tumoral cells

    Directory of Open Access Journals (Sweden)

    Kaur Punit

    2008-11-01

    Full Text Available Abstract Background There is ethnopharmacological evidence that Petiveria alliacea can have antitumor activity; however, the mechanism of its cytotoxic activity is not well understood. We assessed multiple in vitro biological activities of an ethyl acetate soluble plant fraction over several tumor cell lines. Methods Tumor cell lines were evaluated using the following tests: trypan blue exclusion test, MTT [3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide], flow cytometry, cytoskeleton organization analysis, cell cycle, mitochondria membrane depolarization, clonogenicity test, DNA fragmentation test and differential protein expression by HPLC-Chip/MS analysis. F4 fraction characterization was made by HPLC-MS. Results Petiveria alliacea fraction characterized by de-replication was found to alter actin cytoskeleton organization, induce G2 cell cycle arrest and cause apoptotic cell death in a mitochondria independent way. In addition, we found down regulation of cytoskeleton, chaperone, signal transduction proteins, and proteins involved in metabolic pathways. Finally up regulation of proteins involved in translation and intracellular degradation was also observed. Conclusion The results of this study indicate that Petiveria alliacea exerts multiple biological activities in vitro consistent with cytotoxicity. Further studies in animal models are needed but Petiveria alliacea appears to be a good candidate to be used as an antitumor agent.

  3. Corrosive effects of fluoride on titanium: investigation by X-ray photoelectron spectroscopy, atomic force microscopy, and human epithelial cell culturing.

    Science.gov (United States)

    Stájer, Anette; Ungvári, Krisztina; Pelsoczi, István K; Polyánka, Hilda; Oszkó, Albert; Mihalik, Erzsébet; Rakonczay, Zoltán; Radnai, Márta; Kemény, Lajos; Fazekas, András; Turzó, Kinga

    2008-11-01

    High fluoride (F(-)) concentrations and acidic pH impair the corrosion resistance of titanium (Ti). Effects of F(-)-containing caries-preventive prophylactic rinses, and gels on Ti were investigated by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Human epithelial cell attachment and proliferation were investigated by dimethylthiazol-diphenyl tetrazolium bromide (MTT) and protein content assays. Aqueous 1% NaF solution (3800 ppm F(-), pH 4.5) or high (12,500 ppm) F(-) content gel (pH 4.8) strongly corroded the surface and modified its composition. XPS revealed formation of a strongly bound F(-)-containing complex (Na(2)TiF(6)). AFM indicated an increase in roughness (R(a)) of the surfaces: 10-fold for the NaF solution and smaller for the gel or a mouthwash (250 ppm F(-), pH 4.4). MTT revealed that cell attachment was significantly increased by the gel, but was not disturbed by either the mouthwash or the NaF. Cell proliferation determined by MTT decreased significantly only for the NaF-treated samples; protein content assay experiments showed no such effect. This study indicates that epithelial cell culturing results can depend on the method used, and the adverse effects of a high F(-) concentration and low pH should be considered when prophylactic gels are applied by patients with Ti implants or other dental devices.

  4. Effect of extremely low frequency electromagnetic fields on bacterial membrane.

    Science.gov (United States)

    Oncul, Sule; Cuce, Esra M; Aksu, Burak; Inhan Garip, Ayse

    2016-01-01

    The effect of extremely low frequency electromagnetic fields (ELF-EMF) on bacteria has attracted attention due to its potential for beneficial uses. This research aimed to determine the effect of ELF-EMF on bacterial membrane namely the membrane potential, surface potential, hydrophobicity, respiratory activity and growth. Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli were subjected to ELF-EMF, 50 Hz, 1 mT for 2 h. Membrane potential was determined by fluorescence spectroscopy with or without EDTA (Ethylenediaminetetraacetic acid) with DisC3(5) (3,3-dipropylthiacarbocyanine iodide), zeta potential measurements were performed by electrophoretic mobility, hydrophobicity of the membrane was measured with MATH (Microbial Adhesion to Hydrocarbons) test, respiratory activity was determined with CTC (5-Cyano-2,3-ditolyl tetrazolium chloride), colony forming unit (CFU) and DAPI (4',6-diamidino-2-phenylindole, dihydrochloride) was used for growth determinations. ELF-EMF caused changes in physicochemical properties of both Gram-positive and Gram-negative bacteria. Hyperpolarization was seen in S. aureus and EDTA-treated E. coli. Surface potential showed a positive shift in S. aureus contrariwise to the negative shift seen in EDTA-untreated E. coli. Respiratory activity increased in both bacteria. A slight decrease in growth was observed. These results show that ELF-EMF affects the crucial physicochemical processes in both Gram-positive and Gram-negative bacteria which need further research.

  5. Change of extracellular signal-regulated kinase expression in pulmonary arteries from smokers with and without chronic obstructive pulmonary disease.

    Science.gov (United States)

    Liu, Kui; Liu, Xian-Sheng; Yu, Mu-Qing; Xu, Yong-Jian

    2013-01-01

    Cigarette smoking may contribute to pulmonary hypertension in chronic obstructive pulmonary disease (COPD) by resulting in pulmonary vascular remodeling that involves pulmonary artery smooth muscle cell (PASMC) proliferation. However, the molecular mechanism underlying this process remains poorly understood. The purpose of this study was to investigate the role of extracellular signal-regulated kinase (ERK) in pulmonary arteries from smokers with normal lung function and smokers with mild to moderate COPD. The peripheral lung tissues were obtained from 14 nonsmokers with normal lung function, 18 smokers with normal lung function, and 16 smokers with mild to moderate COPD. The morphological changes of pulmonary arteries were observed by hematoxylin-eosin (HE) staining. Primary cultured human pulmonary artery smooth muscle cells (HPASMCs) were exposed to cigarette smoke extract (CSE). Cell proliferation was determined by cell counting and Methyl thiazolyl tetrazolium assay. Protein expression was analyzed by western blotting. Morphometrical analysis showed that the pulmonary vessel wall thickness in smoker group and COPD group was significantly greater than that in nonsmoker group (P < .01). The protein level of ERK was significantly increased in smoker group and COPD group as compared with nonsmoker group (P < .01). The expression of ERK was significantly increased in HPASMCs at protein levels when HPASMCs were treated with 5% CSE (P < .01), which significantly promoted the proliferation of HPASMCs (P < .01). Increased expression of ERK might be involved in the pathogenesis of abnormal proliferation of PASMCs in smokers with and without COPD.

  6. In Vitro Cytotoxicity and Setting Time Assessment of Calcium-Enriched Mixture Cement, Retro Mineral Trioxide Aggregate and Mineral Trioxide Aggregate.

    Science.gov (United States)

    Pornamazeh, Tahereh; Yadegari, Zahra; Ghasemi, Amir; Sheykh-Al-Eslamian, Seyedeh Mahsa; Shojaeian, Shiva

    2017-01-01

    The present study sought to evaluate and compare biocompatibility and setting time of Retro mineral trioxide aggregate (MTA), calcium-enriched mixture (CEM) and Angelus MTA. CEM cement, Angelus MTA and Retro MTA were assessed in set and fresh states. Extracts transformed to each cavity of three 24-well plates in which 1×104 cell were seeded into each well 24 h earlier. All specimens were incubated in a humidified incubator with 5% CO2 at 37°C. Mosmann's tetrazolium toxicity (MTT) assay was used to determine in vitro cytotoxicity on L929 mouse fibroblast cell line. Cell viability was determined at 1, 24, and 72 h after exposure. The initial setting time was measured by 113.4 g Gilmore needle testing. Then, final setting times were assessed by the 456.5 g Gilmore needle. Data comparisons were performed using the analysis of variance (ANOVA) and Tukey's post hoc test (α=0.05). All groups in both forms indicated higher cell vitality compared to positive control group (PMTA showed better biocompatibility compared to set CEM and set Angelus MTA (PMTA showed significantly lower initial and final setting time compared to CEM and Angelus MTA (PMTA and relatively short period of setting time. It seems a promising alternative material in clinical situations where accelerated setting is required. However, more clinical and in vivo investigations are needed for a clear decision making.

  7. Seed production temperature regulation of primary dormancy occurs through control of seed coat phenylpropanoid metabolism.

    Science.gov (United States)

    MacGregor, Dana R; Kendall, Sarah L; Florance, Hannah; Fedi, Fabio; Moore, Karen; Paszkiewicz, Konrad; Smirnoff, Nicholas; Penfield, Steven

    2015-01-01

    Environmental changes during seed production are important drivers of lot-to-lot variation in seed behaviour and enable wild species to time their life history with seasonal cues. Temperature during seed set is the dominant environmental signal determining the depth of primary dormancy, although the mechanisms though which temperature changes impart changes in dormancy state are still only partly understood. We used molecular, genetic and biochemical techniques to examine the mechanism through which temperature variation affects Arabidopsis thaliana seed dormancy. Here we show that, in Arabidopsis, low temperatures during seed maturation result in an increase in phenylpropanoid gene expression in seeds and that this correlates with higher concentrations of seed coat procyanidins. Lower maturation temperatures cause differences in coat permeability to tetrazolium, and mutants with increased seed coat permeability and/or low procyanidin concentrations are less able to enter strongly dormant states after exposure to low temperatures during seed maturation. Our data show that maternal temperature signalling regulates seed coat properties, and this is an important pathway through which the environmental signals control primary dormancy depth. © 2014 The Authors New Phytologist © 2014 New Phytologist Trust.

  8. Osteogenic differentiation of immature osteoblasts: Interplay of cell culture media and supplements.

    Science.gov (United States)

    Brauer, A; Pohlemann, T; Metzger, W

    2016-01-01

    Differentiation of immature osteoblasts to mature osteoblasts in vitro initially was induced by supplementing the medium with β-gylcerophosphate and dexamethasone. Later, ascorbic acid, vitamin D3, vitamin K3 and TGFβ1 were used in varying concentrations as supplements to generate a mature osteoblast phenotype. We tested the effects of several combinations of cell culture media, seeding protocols and osteogenic supplements on osteogenic differentiation of human primary osteoblasts. Osteogenic differentiation was analyzed by staining alkaline phosphatase (ALP) with 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT) and by von Kossa staining of deposited calcium phosphate. The combinations of culture media and supplements significantly influenced osteogenic differentiation, but the seeding protocol did not. Staining of ALP and calcium phosphate could be achieved only if our own mix of osteogenic supplements was used in combination with Dulbecco's modified Eagle medium or if a commercial mix of osteogenic supplements was used in combination with osteoblast growth medium. Especially for von Kossa, we observed great variations in the staining intensity. Because osteogenic differentiation is a complex process, the origin of the osteoblasts, cell culture media and osteogenic supplements should be established by preliminary experiments to achieve optimal differentiation. Staining of ALP or deposited calcium phosphate should be supplemented with qRT-PCR studies to learn more about the influence of specific supplements on osteogenic markers.

  9. Evaluation of antioxidant potential of Amalakayas Rasayana: A polyherbal Ayurvedic formulation.

    Science.gov (United States)

    Samarakoon, S M S; Chandola, H M; Shukla, V J

    2011-01-01

    Amalakayas Rasayana (AR) is a polyherbal formulation mentioned in Ayurveda to treat aging and age-associated diseases. Being an antiaging drug, AR may have antioxidants and free radical scavenging activity to minimize free radical-induced damage which is a key cause of aging. The methanolic extract of AR was evaluated in vitro for total phenolic and tannin content, free radical scavenging activity, superoxide radical scavenging activity, and reducing power. The total phenolic content was measured using Folin-ciocalteu reagent against gallic acid [relative standard deviation (R(2)) = 0.998]. Total tannin was estimated using the Stephen method and was found to be 2.82% w/w. Free radical scavenging activity was measured by 2,2-diphenyl-1-picryl hydrazyl assay and R(2) was 1. Superoxide radical scavenging activity was done by ethylene diamine tetra acetate and Nitro Blue Tetrazolium Chloride assays against ascorbic acid and R(2) was 0.976 (EC(50)= 77.5 μg/ml). Ferrous reducing power was evaluated by Oyaizu method where R(2) was 0.986. All studies showed that AR possesses antioxidant activity. The results of this study suggest that the antioxidant and free radical scavenging activity of AR may explain its rasayana effect and justify its use as a medicine for age associated diseases.

  10. Estudio fitoquímico y biológico preliminar de la corteza (tallo de vismia cayennensis proveniente del estado Amazonas, Venezuela

    Directory of Open Access Journals (Sweden)

    Marín, Karina

    2017-09-01

    Full Text Available A chemical and biological preliminary study of the species Vismia cayennensis, collected in the Amazonas state, Venezuela. The antibacterial test of plant bark extract showed significant inhibition in Escherichia coli, Shigella sp and Staphylococcus aureus. In addition, three of the five soluble fractions of different polarity solvents, specifically those of chloroform, acetone and water, maintained moderately active against Shigella sp strain. The hydroalcoholic extract of the plant and the fraction soluble in chloroform, exhibited a significant antiinflammatory effect. Cytotoxicity tests performed by the methods of (3-(4,5-dimetiltiazol-2-yl-5-(3-carboximetoxifenil-2-(4-sulfophenyl-2H-tetrazolium/phenazine methosulfate of (MTS/PMS and sulforhodamine B , revealed that only has water soluble cytotoxic effect. Additionally, a study phytochemical obtained information on the presence of some families of secondary metabolites such as flavonoids, saponins, tannins, polyphenols, anthraquinones, triterpenes and sterols. It can be inferred that the stem bark of the plant V. cayennensis, is a promising source of bioactive secondary metabolites

  11. Clove (Syzygium aromaticum ingredients affect lymphocyte subtypes expansion and cytokine profile responses: An in vitro evaluation

    Directory of Open Access Journals (Sweden)

    Shaghayegh Pishkhan Dibazar

    2014-12-01

    Full Text Available Clove (Syzygium aromaticum has been used in folk medicine in many disorders. The present work aimed to investigate effects of clove essential oil as eugenol and water soluble ingredients on mouse splenocytes. Clove extracts were harvested and in different concentrations (0.001–1000 μg/mL were affected to splenocytes and also phytohemagglutinin (PHA = 5 μg/mL and lipopolysaccharide (LPS = 10 μg/mL activated splenocytes; then splenocytes proliferation assayed using the MTT ([3-(4, 5-dimethylthiazole-2-yl -2, 5-diphenyl tetrazolium bromide] method were done. On the culture supernatant interferon (IFN-γ, interleukin (IL-4, IL-10, and transforming growth factor (TGF-β cytokines were measured. Clove ingredients (100 μg/mL and 1000 μg/mL reduced PHA stimulated splenocytes proliferation and enhanced LPS stimulated cells expansion. Treated splenocytes showed suppression of IFN-γ release and induction of IL-4, IL-10, and TGF-β secretion (in the range of 0.1–1000 μg/mL. The results of this study suggest clove extracts could suppress the T cell cellular immunity and enhance humoral immune responses. In clove affection cytokine pattern shifted toward modulatory and Th2 responses and accelerator of humoral immunity cytokines.

  12. High glucose improves healing of periodontal wound by inhibiting proliferation and osteogenetic differentiation of human PDL cells.

    Science.gov (United States)

    Li, Min; Li, Cheng-Zhang

    2016-02-01

    Periodontal ligament (PDL) cells play an important role in wound healing of periodontal tissues. Response of PDL cells' cellular activity to high-glucose concentration levels may be the key in understanding the relationship between periodontal disease and diabetes mellitus. We studied the effect of high-glucose medium on proliferation of PDL cells in vitro. PDL cells were cultured for 1, 4, 7, 10, 14 and 17 days in normal (1100 mg/l) glucose or in high (4500 mg/l) glucose medium. The 3-(4,5-dimethylithiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay for proliferation was performed. In order to evaluate the osteogenetic differentiation of human PDL cells, the cells were induced with normal- or high-glucose medium for 1, 7, 14, 21 and 28 days. The results indicated that high glucose significantly inhibited proliferation of PDL cells. Concerning the mineralised nodule formation, the percentage of calcified area to total culture dish of PDL cells in high glucose level was lower than that in normal glucose medium. The increase in alkaline phosphatase activity and collagen expression could be observed in high-glucose-containing osteogenetic factor. In conclusion, high glucose improves healing of periodontal wound by inhibiting proliferation and differentiation of PDL cells, which could explain for delayed periodontal regeneration and healing in diabetic patients. © 2014 The Authors. International Wound Journal © 2014 Medicalhelplines.com Inc and John Wiley & Sons Ltd.

  13. The electron beam deposition of titanium on polyetheretherketone (PEEK) and the resulting enhanced biological properties.

    Science.gov (United States)

    Han, Cheol-Min; Lee, Eun-Jung; Kim, Hyoun-Ee; Koh, Young-Hag; Kim, Keung N; Ha, Yoon; Kuh, Sung-Uk

    2010-05-01

    The surface of polyetheretherketone (PEEK) was coated with a pure titanium (Ti) layer using an electron beam (e-beam) deposition method in order to enhance its biocompatibility and adhesion to bone tissue. The e-beam deposition method was a low-temperature coating process that formed a dense, uniform and well crystallized Ti layer without deteriorating the characteristics of the PEEK implant. The Ti coating layer strongly adhered to the substrate and remarkably enhanced its wettability. The Ti-coated samples were evaluated in terms of their in vitro cellular behaviors and in vivo osteointegration, and the results were compared to a pure PEEK substrate. The level of proliferation of the cells (MC3T3-E1) was measured using a methoxyphenyl tetrazolium salt (MTS) assay and more than doubled after the Ti coating. The differentiation level of cells was measured using the alkaline phosphatase (ALP) assay and also doubled. Furthermore, the in vivo animal tests showed that the Ti-coated PEEK implants had a much higher bone-in-contact (BIC) ratio than the pure PEEK implants. These in vitro and in vivo results suggested that the e-beam deposited Ti coating significantly improved the potential of PEEK for hard tissue applications. Copyright 2009 Elsevier Ltd. All rights reserved.

  14. Isolation and characterization of marine Brevibacillus sp. S-1 collected from South China Sea and a novel antitumor peptide produced by the strain.

    Directory of Open Access Journals (Sweden)

    Lanhong Zheng

    Full Text Available A Gram-positive, rod-shaped bacterium, designated as S-1, was isolated from a marine sediment sample collected from South China Sea. Phylogenetic analysis based on 16S rRNA gene sequence showed that S-1 belongs to the genus Brevibacillus. A novel cytotoxic peptide was isolated from the fermentation broth of the marine-derived bacterium Brevibacillus sp. S-1, using ion-exchange chromatography and reverse-phase HPLC chromatography. The molecular weight of this peptide was determined as 1570 Da by MALDI-TOF mass spectrometry, and its structure was proposed as a cyclic peptide elucidated by MALDI-TOF/TOF mass spectrometry and de novo sequencing. 3-(4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2H-tetrazolium bromide (MTT assay showed that this peptide exhibited cytotoxicity against BEL-7402 human hepatocellular carcinoma cells, RKO human colon carcinoma cells, A549 human lung carcinoma cells, U251 human glioma cells and MCF-7 human breast carcinoma cells. Additionally, SBP exhibited low cytotoxicity against HFL1 human normal fibroblast lung cells. The result suggested that the cytotoxic effect of the peptide is specific to tumor cells.

  15. High Efficient Expression, Purification, and Functional Characterization of Native Human Epidermal Growth Factor in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Yi Ma

    2016-01-01

    Full Text Available Human epidermal growth factor (hEGF is a small, mitotic growth polypeptide that promotes the proliferation of various cells and is widely applied in clinical practices. However, high efficient expression of native hEGF in Escherichia coli has not been successful, since three disulfide bonds in monomer hEGF made it unable to fold into correct 3D structure using in vivo system. To tackle this problem, we fused Mxe GyrA intein (Mxe at the C-terminal of hEGF followed by small ubiquitin-related modifier (SUMO and 10x His-tag to construct a chimeric protein hEGF-Mxe-SUMO-H10. The fusion protein was highly expressed at the concentration of 281 mg/L and up to 59.5% of the total cellular soluble proteins. The fusion protein was purified by affinity chromatography and 29.4 mg/L of native hEGF can be released by thiol induced N-terminal cleavage without any proteases. The mitotic activity in Balb/c 3T3 cells is proliferated by commercial and recombinant hEGF measured with methylthiazolyldiphenyl-tetrazolium bromide (MTT assay which indicated that recombinant hEGF protein stimulates the cell proliferation similar to commercial protein. This study significantly improved the yield and reduced the cost of hEGF in the recombinant E. coli system and could be a better strategy to produce native hEGF for pharmaceutical development.

  16. Lab-scale preparations of Candida albicans and dual Candida albicans-Candida glabrata biofilms on the surface of medical-grade polyvinyl chloride (PVC) perfusion tube using a modified gravity-supported free-flow biofilm incubator (GS-FFBI).

    Science.gov (United States)

    Shao, Jing; Lu, KeQiao; Tian, Ge; Cui, YanYan; Yan, YuanYuan; Wang, TianMing; Zhang, XinLong; Wang, ChangZhong

    2015-02-01

    The assembly of a man-made gravity-supported free-flow biofilm incubator (GS-FFBI) was described, which was composed of a gas cushion injector and four incubators. The GS-FFBI had the characteristics of (i) a bottom-up flow direction, and (ii) lab-scale biofilm preparation without the use of a multichannel pump. Two opportunistic fungal strains, namely Candida albicans and Candida glabrata, were employed to incubate C. albicans and dual C. albicans-C. glabrata biofilms on the surface of medical-grade polyvinyl chloride perfusion tube. In terms of the results from {2, 3-bis (2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide} (XTT) assay, dry weight measurement, colony-forming unit counting, susceptibility test, and scanning electron microscopy, it was demonstrated that GS-FFBI could form both stable single and dual Candida biofilms with no significant variations among the four incubators or the three daily incubations within 21h, and could operate for at least 96h smoothly with no contamination of stock medium. The results also indicated, for the first time, that C. albicans and C. glabrata might be co-existent competitively and symbiotically in the dual biofilms with flowing media. GS-FFBI would be a useful device to study in vitro morphological and physiological features of microbial biofilms in the medical settings. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. A novel assay of biofilm antifungal activity reveals that amphotericin B and caspofungin lyse Candida albicans cells in biofilms.

    Science.gov (United States)

    DiDone, Louis; Oga, Duana; Krysan, Damian J

    2011-08-01

    The ability of Candida albicans to form drug-resistant biofilms is an important factor in its contribution to human disease. Assays to identify and characterize molecules with activity against fungal biofilms are crucial for the development of drugs with improved anti-biofilm activity. Here we report the application of an adenylate kinase (AK)-based cytotoxicity assay of fungal cell lysis to the characterization of agents active against C. albicans biofilms. We have developed three protocols for the AK assay. The first measures AK activity in the supernatants of biofilms treated with antifungal drugs and can be performed in parallel with a standard 2,3-bis-(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-caboxanilide-based biofilm susceptibility assay; a second, more sensitive protocol measures the AK activity present within the biofilm matrix; and a third procedure allows the direct visualization of lytic activity toward biofilms formed on catheter material. Amphotericin B and caspofungin, the two most effective anti-biofilm drugs currently used to treat fungal infections, both directly lyse planktonic C. albicans cells in vitro, leading to the release of AK into the culture medium. These studies serve to validate the AK-based lysis assay as a useful addition to the methods for the characterization of antifungal agents active toward biofilms and provide insights into the mode of action of amphotericin B and caspofungin against C. albicans biofilms. Copyright © 2011 John Wiley & Sons, Ltd.

  18. cAMP signalling regulates synchronised growth of symbiotic Epichloë fungi with the host grass Lolium perenne

    Directory of Open Access Journals (Sweden)

    Christine Rosalie Voisey

    2016-10-01

    Full Text Available The seed-transmitted fungal symbiont, Epichloë festucae, colonizes grasses by infecting host tissues as they form on the shoot apical meristem (SAM of the seedling. How this fungus accommodates the complexities of plant development to successfully colonize the leaves and inflorescences is unclear. Since adenosine 3′, 5′-cyclic monophosphate (cAMP-dependent signaling is often essential for host colonization by fungal pathogens, we disrupted the cAMP cascade by insertional mutagenesis of the E. festucae adenylate cyclase gene (acyA. Consistent with deletions of this gene in other fungi, acyA mutants had a slow radial growth rate in culture, and hyphae were convoluted and hyper-branched suggesting that fungal apical dominance had been disrupted. Nitro blue tetrazolium (NBT staining of hyphae showed that cAMP disruption mutants were impaired in their ability to synthesize superoxide, indicating that cAMP signaling regulates accumulation of reactive oxygen species (ROS. Despite significant defects in hyphal growth and ROS production, E. festucae ΔacyA mutants were infectious and capable of forming symbiotic associations with grasses. Plants infected with E. festucae ΔacyA were marginally less robust than the wild-type (WT, however hyphae were hyper-branched, and leaf tissues heavily colonized, indicating that the tight regulation of hyphal growth normally observed in maturing leaves requires functional cAMP signaling.

  19. Optimization of Biodegradability and Toxicity Testing of Degradation Product from Linear Alkyl BenzeneSulfonate (LAS Surfactant as Cleaning Detergent Agent

    Directory of Open Access Journals (Sweden)

    Neera Khairani

    2009-11-01

    Full Text Available Optimization of Biodegradability and Toxicity Testing of Degradation Product from Linear Alkyl BenzeneSulfonate (LAS Surfactant as Cleaning Detergent Agent. Linear Alkyl Benzene Sulfonate (LAS is a surfactantused in laundry detergent as cleaning agent and toxic to aquatic organisms. Results shows, with the LAS concentrationused (20 ppm in medium, adaptation time and Acinetobacter sp. growth has shown better biodegradation ability thanthree other bacteria used Pseudomonas putida, Pseudomonas fluorescence, Bacillaria spp. Thus, Acinetobacter sp isused further for biodegradation process of LAS. Based on its biodegradation half-life using Acinetobacter sp, and withmixed culture (± 52.32% and ± 46.82% respectively could be achieved in 4 (four days, LAS could be categorized as abiodegradable compound. The toxicity assay is based on tetrazolium dye reduction with Rhizobium meliloti as indicatororganism. LAS is more toxic than its intermediate product from biodegradation, with IC50 = 34.35 ppm, and theintermediate product, Ac and Cm, has IC50 = 446.19 ppm and 111.28 ppm respectively. Identification of intermediateproducts using IR and LC-MS analysis shows that the degradation product contains chemicals compounds withfunctional group as follows: benzene, benzoic acid, hydroxyl, and aliphatic carbons with large molecule weight. Untilits half-time degradation time, LAS biodegradation process only occurs at the aliphatic carbon chain, and have not yetreached the stage of aromatic ring opening.

  20. Evaluation of Maltose-Based Cationic Liposomes with Different Hydrophobic Tails for Plasmid DNA Delivery

    Directory of Open Access Journals (Sweden)

    Bo Li

    2017-03-01

    Full Text Available In this paper, three cationic glycolipids with different hydrophobic chains Malt-DiC12MA (IX a, Malt-DiC14MA (IX b and Malt-DiC16MA (IX c were constructed by using maltose as starting material via peracetylation, selective 1-O-deacetylation, trichloroacetimidation, glycosylation, azidation, deacetylation, Staudinger reaction, tertiary amination and quaternization. Target compounds and some intermediates were characterized by 1H-NMR, 13C-NMR, 1H-1H COSY and 1H-13C HSQC. The results of gel electrophoresis assay, atomic force microscopy images (AFM and dynamic light scattering (DLS demonstrate that all the liposomes could efficiently bind and compact DNA (N/P ratio less than 2 into nanoparticles with proper size (88 nm–146 nm, PDI < 0.4 and zeta potential (+15 mV–+26 mV. The transfection efficiency and cellular uptake of glycolipids in HEK293 cell were evaluated through the enhanced green fluorescent protein (EGFP expression and Cy3-labeled pEGFP-C1 (Enhanced Green Fluorescent Protein plasmid images, respectively. Importantly, it indicated that Malt-DiC14MA exhibited high gene transfer efficiency and better uptake capability at N/P ratios of 8:1. Additionally, the result of cell viability showed glycolipids exhibited low biotoxicity and good biocompatibility by thiazolyl blue tetrazolium bromide (MTT assay.

  1. Clinical and molecular findings of chronic granulomatous disease in Oman: family studies.

    Science.gov (United States)

    Al-Zadjali, S; Al-Tamemi, S; Elnour, I; AlKindi, S; Lapoumeroulie, C; Al-Maamari, S; Pathare, A; Dennison, D; Krishnamoorthy, R

    2015-02-01

    Chronic granulomatous disease (CGD), a rare inherited disorder of the innate immune system, results from mutations in any one of the five genes encoding the subunits of the nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) oxidase enzyme, and is characterized by recurrent life-threatening bacterial and fungal infections. Molecular analysis of 14 Omani CGD patients from 10 families, diagnosed to have CGD on clinical (recurrent infections) and biochemical grounds (positive for both the nitroblue tetrazolium (NBT) test and the dihydrorhodamine (DHR-1,2,3 assay), revealed that only one patient had X-linked CGD, with a large deletion involving both the gp91-phox gene (CYBB) and the McLeod gene (XK). The remaining 13 patients were all homozygotes from a previously described c.579G>A (p.Trp193X) mutation in the NCF1 gene on chromosome 7, responsible for autosomal recessive CGD (AR-CGD). Although X-linked CGD is the most common type of CGD disorder in most population groups, AR-CGD is the most prevalent type in Oman. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  2. Bioactivity of the Murex Homeopathic Remedy and of Extracts from an Australian Muricid Mollusc against Human Cancer Cells

    Directory of Open Access Journals (Sweden)

    Kirsten Benkendorff

    2011-01-01

    Full Text Available Marine molluscs from the family Muricidae are the source of a homeopathic remedy Murex, which is used to treat a range of conditions, including cancer. The aim of this study was to evaluate the in vitro bioactivity of egg mass extracts of the Australian muricid Dicathais orbita, in comparison to the Murex remedy, against human carcinoma and lymphoma cells. Liquid chromatography coupled with mass spectrometry (LC-MS was used to characterize the chemical composition of the extracts and homeopathic remedy, focusing on biologically active brominated indoles. The MTS (tetrazolium salt colorimetric assay was used to determine effects on cell viability, while necrosis and apoptosis induction were investigated using flow cytometry (propidium iodide and Annexin-V staining, resp.. Cells were treated with varying concentrations (1–0.01 mg/mL of crude and semi-purified extracts or preparations (dilute 1 M and concentrated 4 mg/mL from the Murex remedy (4 h. The Murex remedy showed little biological activity against the majority of cell lines tested. In contrast, the D. orbita egg extracts significantly decreased cell viability in the majority of carcinoma cell lines. Flow cytometry revealed these extracts induce necrosis in HT29 colorectal cancer cells, whereas apoptosis was induced in Jurkat cells. These findings highlight the biomedical potential of Muricidae extracts in the development of a natural therapy for the treatment of neoplastic tumors and lymphomas.

  3. Cytotoxic and genotoxic effects of abamectin, chlorfenapyr, and imidacloprid on CHOK1 cells.

    Science.gov (United States)

    Al-Sarar, Ali S; Abobakr, Yasser; Bayoumi, Alaa E; Hussein, Hamdy I

    2015-11-01

    The cytotoxicity and genotoxicity of abamectin, chlorfenapyr, and imidacloprid have been evaluated on the Chinese hamster ovary (CHOK1) cells. Neutral red incorporation (NRI), total cellular protein content (TCP), and methyl tetrazolium (MTT) assays were followed to estimate the mid-point cytotoxicity values, NRI50, TCP50, and MTT50, respectively. The effects of the sublethal concentration (NRI25) on glutathione S-transferase (GST), glutathione reductase (GRD), glutathione peroxidase (GPX), and total glutathione content have been evaluated in the presence and absence of reduced glutathione (GSH), vitamin C, and vitamin E. The genotoxicity was evaluated using chromosomal aberrations (CA), micronucleus (MN) formation, and DNA fragmentation techniques in the presence and absence of the metabolic activation system, S9 mix. Abamectin was the most cytotoxic pesticide followed by chlorfenapyr, while imidacloprid was the least cytotoxic one. The glutathione redox cycle components were altered by the tested pesticides in the absence and presence of the tested antioxidants. The results of genotoxicity indicate that abamectin, chlorfenapyr, and imidacloprid have potential genotoxic effects on CHOK1 cells under the experimental conditions.

  4. The endoplasmic reticulum stress inducer thapsigargin enhances the toxicity of ZnO nanoparticles to macrophages and macrophage-endothelial co-culture.

    Science.gov (United States)

    Chen, Gui; Shen, Yuexin; Li, Xiyue; Jiang, Qin; Cheng, Shanshan; Gu, Yuxiu; Liu, Liangliang; Cao, Yi

    2017-03-01

    It was recently shown that exposure to ZnO nanoparticles (NPs) could induce endoplasmic reticulum (ER) stress both in vivo and in vitro, but the role of ER stress in ZnO NP induced toxicity remains unclear. Because macrophages are sensitive to ER stress, we hypothesized that stressing macrophages with ER stress inducer could enhance the toxicity of ZnO NPs. In this study, the effects of ER stress inducer thapsigargin (TG) on the toxicity of ZnO NPs to THP-1 macrophages were investigated. The results showed that TG enhanced ZnO NP induced cytotoxicity as revealed by water soluble tetrazolium-1 (WST-1) and neutral red uptake assays, but not lactate dehydrogenase (LDH) assay. ZnO NPs dose-dependently enhanced the accumulation of intracellular Zn ions without the induction of reactive oxygen species (ROS), and the presence of TG did not significantly affect these effects. In the co-culture, exposure of THP-1 macrophages in the upper chamber to ZnO NPs and TG significantly reduced the viability of human umbilical vein endothelial cells (HUVECs) in the lower chamber, but the release of tumor necrosis factor α (TNFα) was not induced. In summary, our data showed that stressing THP-1 macrophages with TG enhanced the cytotoxicity of ZnO NPs to macrophages and macrophage-endothelial co-cultures. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Aquatic toxicity assessment of the additive 6-methylcoumarine using four experimental systems.

    Science.gov (United States)

    Jos, A; Repetto, G; Ríos, J C; Del Peso, A; Salguero, M; Cameán, A M

    2009-01-01

    The toxicity assessment of chemicals is one of the main issues in the current policies in order to protect the health of the environment and human beings. Food and cosmetic additives have been extensively studied in relation to their toxicity to humans, but data about their ecotoxicological effects are scarce. The aim of this study was to evaluate the toxic effects of the additive 6-methylcoumarine in the aquatic milieu using a test battery comprising experimental model systems from different trophic levels. The inhibition of bioluminiscence was studied in the bacteria Vibrio fischeri (decomposer), the inhibition of growth was evaluated in the alga Chlorella vulgaris (producer) and immobilization was studied in the cladoceran Daphnia magna (first consumer). Finally, several end points were evaluated in the RTG-2 salmonid fish cell line, including neutral red uptake, protein content, methylthiazol tetrazolium salt metabolization, glucose-6-phosphate dehydrogenase activity, lactate dehydrogenase activity and leakage, and morphology. The sensitivity of the test systems employed was as follows: V. fischeri > D. magna > C. vulgaris > RTG-2 cell line. The results show that 6-methylcoumarine is not expected to produce acute toxic effects on the aquatic biota. However, chronic and synergistic effects with other chemicals cannot be excluded and should be further investigated.

  6. The New Semisynthetic Cardenolide Analog 3β-[2-(1-Amantadine)-1-on-ethylamine]-digitoxigenin (AMANTADIG) Efficiently Suppresses Cell Growth in Human Leukemia and Urological Tumor Cell Lines.

    Science.gov (United States)

    Nolte, Elke; Sobel, Anna; Wach, Sven; Hertlein, Heidi; Ebert, Nadja; Müller-Uri, Frieder; Slany, Robert; Taubert, Helge; Wullich, Bernd; Kreis, Wolfgang

    2015-10-01

    The use of cardenolides in the treatment of cardiac insufficiency is well-established. However, the potential of cardenolides in tumor therapy has not been comprehensively studied. The aim of the present study was to characterize the cytotoxic effects of the new semisynthetic cardenolide analog AMANTADIG (3β-[2-(1-amantadine)-1-on-ethylamine]-digitoxigenin), and the cardenolide digitoxin on leukemia and urological tumor cell lines. The anti-proliferative effects of AMANTADIG and digitoxin on leukemia and urological cancer cell lines were analyzed using (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction viability assay. AMANTADIG and digitoxin exhibited anti-proliferative activities against the leukemia cell lines in the low nanomolar range. The prostate cancer and renal cell carcinoma cell lines were equally sensitive to AMANTADIG and digitoxin, however, the leukemia cell lines were more sensitive to both cardenolides. The new cardenolide analog AMANTADIG appears effective in cell growth inhibition of leukemia and urological tumor cell lines. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  7. Dithizone and its oxidation products: a DFT, spectroscopic, and X-ray structural study.

    Science.gov (United States)

    von Eschwege, Karel G; Conradie, Jeanet; Kuhn, Annemarie

    2011-12-29

    Air oxidation of ortho-fluorodithizone resulted in the first X-ray resolved structure of a disulfide of dithizone, validating the last outstanding X-ray structure in the oxidation of dithizone, H(2)Dz, which proceeds via the disulfide, (HDz)(2), to the deprotonated dehydrodithizone tetrazolium salt, Dz. Density functional theory calculations established the energetically favored tautomers along the entire pathway; in gas phase and in polar as well as nonpolar solvent environments. DFT calculations using the classic pure OLYP and PW91, or the newer B3LYP hybrid functional, as well as MP2 calculations, yielded the lowest energy structures in agreement with corresponding experimental X-ray crystallographic results. Atomic charge distribution patterns confirmed the cyclization reaction pathway and crystal packing of Dz. Time dependent DFT for the first time gave satisfactory explanation for the solvatochromic properties of dithizone, pointing to different tautomers that give rise to the observed orange color in methanol and green in dichloromethane. Concentratochromism of H(2)Dz was observed in methanol. Computed orbitals and oscillators are in close agreement with UV-visible spectroscopic experimental results. © 2011 American Chemical Society

  8. Induction of differentiation and apoptosis by dithizone in human myeloid leukemia cell lines.

    Science.gov (United States)

    Kohroki, J; Muto, N; Tanaka, T; Itoh, N; Inada, A; Tanaka, K

    1998-05-01

    We investigated the effect of diphenylthiocarbazone (dithizone) and its structurally related compounds on the differentiation and apoptosis of two human myeloid leukemia cell lines. Dithizone caused a time- and concentration-dependent induction of differentiation in both the promyelocytic leukemia cell line HL-60 cells and the myeloblastic leukemia cell line ML-1 cells, as measured by nitroblue tetrazolium (NBT) reducing activity. Morphological changes and esterase activities confirmed that this differentiation took place. The induction of differentiation required the addition of dithizone to the culture medium for at least 12 h. The differentiation inducing activity was inhibited by the preincubation of dithizone with various metal ions such as Pb2+, Zn2+, Cu2+ and Mn2+ ions, but not with Fe3+ and Mg2+ ions. In addition, the DNA extracted from dithizone-treated HL-60 cells showed a typical ladder pattern characteristic of apoptosis in agarose gel electrophoresis. A quantitative analysis of DNA fragmentation revealed that this apoptosis was concentration- and time-dependent in both the HL-60 and ML-1 cells. Dithizone-induced apoptosis was also inhibited by preincubation with Mn2+ ions, but not with Mg2+ ions. These results indicate that dithizone induces both differentiation and apoptosis in HL-60 and ML-1 cells through a unique mechanism including metal chelation.

  9. The antitumor immune response in HER-2 positive, metastatic breast cancer patients

    Directory of Open Access Journals (Sweden)

    Stanojevic-Bakic Nevenka

    2005-03-01

    Full Text Available Abstract The aim of this study was to determine the basis for anti-tumor immune reactivity observed in patients with human epidermal growth factor receptor-2 (HER-2 (3+ breast carcinoma using an in vitro model in which the role of the HER-2-specific monoclonal antibody Herceptin was also investigated. Patients with metastatic breast cancer who had their primary tumor resected were included in this study. Peripheral blood mononuclear cell (PBMC-dependent cytotoxicity in the presence or absence of Herceptin were assessed using the survival of target breast adenocarcinoma MDA-MB-361 cells as a parameter in a (3-(4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2H-tetrazolium bromide (MTT test. We observed a significant increase in PBMC-dependent cytotoxicity when autologous serum was introduced in the assay. Furthermore, the addition of Herceptin significantly increases their cytotoxicity. These data suggest that autologous serum constitutively contains factors that might affect PBMC-dependent cytotoxic activity against HER-2 positive cancer cells.

  10. BH3 mimetics inhibit growth of chondrosarcoma--a novel targeted-therapy for candidate models.

    Science.gov (United States)

    Morii, Takeshi; Ohtsuka, Kouki; Ohnishi, Hiroaki; Mochizuki, Kazuo; Yoshiyama, Akira; Aoyagi, Takayuki; Hornicek, Francis J; Ichimura, Shoichi

    2014-11-01

    Chondrosarcoma is refractory to conventional chemotherapy. BH-3 mimetics ABT-737 and ABT-263 are synthetic small-molecule inhibitors of anti-apoptotic proteins B-cell lymphoma-2 (Bcl2) and Bcl-xL, which play a critical role in survival of chondrosarcoma cells. Chondrosarcoma cell lines SW-1353 and CS-1 were used as the disease model. We used immunoblotting to assess the expression of target molecules Bcl2 and Bcl-xL, and the apoptotic inducers Bcl2-associated X (Bax) and Bcl2-antagonist/killer (Bak). In vitro growth inhibition by BH-3 mimetics was confirmed by photomicroscopic cell counting and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. Apoptotic induction was confirmed by Enzyme-Linked ImmunoSorbent Assay (ELISA). In vivo growth inhibition was assessed in a non-obese diabetic/severe combined immunodeficient (NOD/SCID) mouse model. Expression of the target and effector molecules was confirmed in chondrosarcoma cell lines. BH3 mimetics significantly inhibited cell growth and induced apoptosis in vitro. Administration of ABT-263 inhibited chondrosarcoma growth and improved survival in a mouse model. BH3 mimetics represent a novel treatment modality for chondrosarcoma. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  11. Biocompatibility and Corrosion Protection Behaviour of Hydroxyapatite Sol-Gel-Derived Coatings on Ti6Al4V Alloy.

    Science.gov (United States)

    El Hadad, Amir A; Peón, Eduardo; García-Galván, Federico R; Barranco, Violeta; Parra, Juan; Jiménez-Morales, Antonia; Galván, Juan Carlos

    2017-01-24

    The aim of this work was to prepare hydroxyapatite coatings (HAp) by a sol-gel method on Ti6Al4V alloy and to study the bioactivity, biocompatibility and corrosion protection behaviour of these coatings in presence of simulated body fluids (SBFs). Thermogravimetric/Differential Thermal Analyses (TG/DTA) and X-ray Diffraction (XRD) have been applied to obtain information about the phase transformations, mass loss, identification of the phases developed, crystallite size and degree of crystallinity of the obtained HAp powders. Fourier Transformer Infrared Spectroscopy (FTIR) has been utilized for studying the functional groups of the prepared structures. The surface morphology of the resulting HAp coatings was studied by Scanning Electron Microscopy (SEM). The bioactivity was evaluated by soaking the HAp-coatings/Ti6Al4V system in Kokubo's Simulated Body Fluid (SBF) applying Inductively Coupled Plasma (ICP) spectrometry. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and Alamar blue cell viability assays were used to study the biocompatibility. Finally, the corrosion behaviour of HAp-coatings/Ti6Al4V system was researched by means of Electrochemical Impedance Spectroscopy (EIS). The obtained results showed that the prepared powders were nanocrystalline HAp with little deviations from that present in the human bone. All the prepared HAp coatings deposited on Ti6Al4V showed well-behaved biocompatibility, good bioactivity and corrosion protection properties.

  12. Use of the caco-2 model in the screening of polluting substance toxicity.

    Science.gov (United States)

    Velarde, G; Ait-Aissa, S; Gillet, C; Rogerieux, F; Lambre, C; Vindimian, E; Porcher, J M

    1999-01-01

    The aim of this work was to investigate the oral toxicity of representative chemicals chosen from each class of the list of 132 substances present in industrial effluents after the EEC Directive 76-464. Owing to its characterization as a model of the intestinal epithelium, the CaCo-2 cell line model was chosen. Cytotoxicity was assayed using the tetrazolium blue (MTT) test. For most of the substances, a linear correlation was observed between the octanol/water partition coefficient (log Kw) and the median inhibition concentration (IC(50)). This relationship between lipophilicity and toxicity is the hallmark of a narcotic mechanism of action. However, diethylamine appeared more toxic than the correlation would predict. Other amines were then tested (tert-butylamine, n-butylamine and benzylamine). All of these did not fit into the baseline correlation. The IC(50) were corrected by taking into account only the non-ionized, lipid insoluble, concentration at pH7.3. The amines still did not fit into the correlation, reinforcing the idea of a non-narcotic mechanism. The toxicity of a large number of substances can thus be predicted from their physico-chemical properties only when the substances exert a direct and non-specific effect. The amines appeared more toxic than substances with the same partition coefficient, showing that knowledge of the only lipophilicity is too restrictive to predict toxicity.

  13. Protective effect of the ultra-filtration extract from Xin Mai Jia on human aortic smooth muscle cell injury induced by hydrogen peroxide

    Science.gov (United States)

    WAN, JIA; YIN, YALING; SUN, RUILI; PAN, GUOPIN; LI, PENG; JIA, YANLONG; WAN, GUANGRUI; LIU, ZHANG-SUO

    2014-01-01

    The aim of the present study was to explore whether an ultra-filtration extract from Xin Mai Jia (XMJ), a Chinese medicinal formulation, has a protective effect on human aortic smooth muscle cell (HASMC) injury models induced by hydrogen peroxide (H2O2), and to consider the mechanism and efficacy of the therapeutic action of XMJ on atherosclerosis. HASMCs were injured by H2O2 and then exposed to various concentrations of XMJ. The morphological changes, growth, proliferation, migration and cytokine release of HASMCs were detected using 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT), an enzyme-linked immunosorbent assay and a scratch adhesion test. H2O2 significantly promoted the proliferation of HASMCs. The ultra-filtration extract from XMJ was observed to significantly attenuate the morphological changes of injured HASMCs, reduce the expression levels of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), interleukin (IL)-1, IL-6 and nuclear factor (NF)-κB, and increase the expression levels of matrix metalloproteinase (MMP)-2 and tissue inhibitor of metalloproteinase (TIMP). XMJ has clear anti-inflammatory and antioxidant effects, and significantly inhibits the proliferation and migration of HASMCs. PMID:24348756

  14. A porphyrin-based metal–organic framework as a pH-responsive drug carrier

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Wenxin [State Key Laboratory of Silicon Materials, Cyrus Tang Center for Sensor Materials and Applications, School of Materials Science & Engineering, Zhejiang University, Hangzhou 310027 (China); Hu, Quan [Department of Pharmacy, School of Medicine, Hangzhou Normal University, Hangzhou 310036 (China); Jiang, Ke; Yang, Yanyu [State Key Laboratory of Silicon Materials, Cyrus Tang Center for Sensor Materials and Applications, School of Materials Science & Engineering, Zhejiang University, Hangzhou 310027 (China); Yang, Yu, E-mail: yuyang@zju.edu.cn [State Key Laboratory of Silicon Materials, Cyrus Tang Center for Sensor Materials and Applications, School of Materials Science & Engineering, Zhejiang University, Hangzhou 310027 (China); Cui, Yuanjing [State Key Laboratory of Silicon Materials, Cyrus Tang Center for Sensor Materials and Applications, School of Materials Science & Engineering, Zhejiang University, Hangzhou 310027 (China); Qian, Guodong, E-mail: gdqian@zju.edu.cn [State Key Laboratory of Silicon Materials, Cyrus Tang Center for Sensor Materials and Applications, School of Materials Science & Engineering, Zhejiang University, Hangzhou 310027 (China)

    2016-05-15

    A low cytotoxic porphyrin-based metal–organic framework (MOF) PCN-221, which exhibited high PC12 cell viability via 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyl tetrazolium (MTT) assay, was selected as an oral drug carrier. Methotrexate (MTX) was chosen as the model drug molecule which was absorbed into inner pores and channels of MOFs by diffusion. PCN-221 showed high drug loading and sustained release behavior under physiological environment without “burst effect”. The controlled pH-responsive release of drugs by PCN-221 revealed its promising application in oral drug delivery. - Graphical abstract: The porous crystals PCN-221 with pore openings (MOF) PCN-221 was prepared exhibiting low cytotoxicity. PCN-221 showed high drug Methotrexate loading and controlled pH-responsive release of Methotrexate. - Highlights: • A porphyrin-based metal–organic framework (MOF) PCN-221 was prepared showing low cytotoxicity. • PCN-221 showed high drug Methotrexate loading. • PCN-221 showed controlled pH-responsive release of Methotrexate.

  15. Synthesis, characterization and biological evaluation of ruthenium flavanol complexes against breast cancer

    Science.gov (United States)

    Singh, Ashok Kumar; Saxena, Gunjan; Sahabjada; Arshad, M.

    2017-06-01

    Four Ru(II) DMSO complexes (M1R-M4R) having substituted flavones viz. 3-Hydroxy-2-(4-methoxyphenyl)-4H-chromen-4-one (HL1), 3-Hydroxy-2-(4-nitrophenyl)-4H-chromen-4-one (HL2), 3-Hydroxy-2-(4-dimethylaminophenyl)-4H-chromen-4-one (HL3) and 3-Hydroxy-2-(4-chlorophenyl)-4H-chromen-4-one (HL4) were synthesized and characterized by elemental analysis, IR, UV-Vis, 1H NMR spectroscopies and ESI-MS. The molecular structures of the complexes were investigated by integrated spectroscopic and computational techniques (DFT). Both ligands as well as their complexes were screened for anticancer activities against breast cancer cell lines MCF-7. Cytotoxicity was assayed by MTT [3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] assay. All ligands and their complexes exhibited significant cytotoxic potential of 5-40 μM concentration at incubation period of 24 h. The cell cytotoxicity increased significantly in a concentration-dependent manner. In this series of compounds, HL2 (IC50 17.2 μM) and its complex M2R (IC50 16 μM) induced the highest cytotoxicity.

  16. Candida albicans Biofilms Do Not Trigger Reactive Oxygen Species and Evade Neutrophil Killing

    Science.gov (United States)

    Xie, Zhihong; Thompson, Angela; Sobue, Takanori; Kashleva, Helena; Xu, Hongbin; Vasilakos, John; Dongari-Bagtzoglou, Anna

    2012-01-01

    Neutrophils are found within Candida albicans biofilms in vivo and could play a crucial role in clearing the pathogen from biofilms forming on catheters and mucosal surfaces. Our goal was to compare the antimicrobial activity of neutrophils against developing and mature C. albicans biofilms and identify biofilm-specific properties mediating resistance to immune cells. Antibiofilm activity was measured with the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)2H-tetrazolium-5-carboxanilide assay and a molecular Candida viability assay. Reactive oxygen species generation was assessed by measuring fluorescence of 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester in preloaded neutrophils. We found that mature biofilms were resistant to leukocytic killing and did not trigger reactive oxygen species, even though neutrophils retained their viability and functional activation potential. Beta-glucans found in the extracellular matrix negatively affected antibiofilm activities. We conclude that these polymers act as a decoy mechanism to prevent neutrophil activation and that this represents an important innate immune evasion mechanism of C. albicans biofilms. PMID:23033146

  17. Ultrastructural and metabolic changes in osteoblasts exposed to uranyl nitrate

    Energy Technology Data Exchange (ETDEWEB)

    Tasat, D.R. [Universidad Nacional de San Martin, Escuela de Ciencia y Tecnologia, Pcia de Bs.As. (Argentina); Universidad de Buenos Aires, Catedra de Histologia y Embriologia, Facultad de Odontologia, Buenos Aires (Argentina); Orona, N.S. [Universidad Nacional de San Martin, Escuela de Ciencia y Tecnologia, Pcia de Bs.As. (Argentina); Mandalunis, P.M. [Universidad de Buenos Aires, Catedra de Histologia y Embriologia, Facultad de Odontologia, Buenos Aires (Argentina); Cabrini, R.L. [Comision Nacional de Energia Atomica, Departamento de Radiobiologia, Buenos Aires (Argentina); Ubios, A.M. [Comision Nacional de Energia Atomica, Departamento de Radiobiologia, Buenos Aires (Argentina); Universidad de Buenos Aires, Catedra de Histologia y Embriologia, Facultad de Odontologia, Buenos Aires (Argentina)

    2007-05-15

    Exposure to uranium is an occupational hazard to workers who continually handle uranium and an environmental risk to the population at large. Since the cellular and molecular pathways of uranium toxicity in osteoblast cells are still unknown, the aim of the present work was to evaluate the adverse effects of uranyl nitrate (UN) on osteoblasts both in vivo and in vitro. Herein we studied the osteoblastic ultrastructural changes induced by UN in vivo and analyzed cell proliferation, generation of reactive oxygen species (ROS), apoptosis, and alkaline phosphatase (APh) activity in osteoblasts exposed to various UN concentrations (0.1, 1, 10, and 100 {mu}M) in vitro. Cell proliferation was quantified by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, ROS was determined using the nitro blue tetrazolium test, apoptosis was morphologically determined using Hoechst 3332 and APh activity was assayed spectrophotometrically. Electron microscopy revealed that the ultrastructure of active and inactive osteoblasts exposed to uranium presented cytoplasmic and nuclear alterations. In vitro, 1-100 {mu}M UN failed to modify cell proliferation ratio and to induce apoptosis. ROS generation increased in a dose-dependent manner in all tested doses. APh activity was found to decrease in 1-100 {mu}M UN-treated cells vs. controls. Our results show that UN modifies osteoblast cell metabolism by increasing ROS generation and reducing APh activity, suggesting that ROS may play a more complex role in cell physiology than simply causing oxidative damage. (orig.)

  18. ACTIVITY OF SUPEROXIDE DISMUTASE MIMIC OF [Mn(SALENOAc] COMPLEX COMPOUND NON-ENZYMATICALLY IN VITRO THROUGH RIBOFLAVIN PHOTOREDUCTION

    Directory of Open Access Journals (Sweden)

    Yusi Deawati

    2017-05-01

    Full Text Available The complex compound of [Mn(salenOAc] can serve as mSOD and its activity has been determined non-enzymatically in vitro through riboflavin photoreduction. The complex was synthesized from Mn(OAc2.4H2O and H2salen. Based on the elemental analysis, the C=56.69%; H=4.21%; and N=7.52% content are corresponding to the chemical formula of MnC18H17N2O4. The functional groups and ionic species in the complex have been analyzed by infrared spectroscopy and ESI-MS. SOD activity was determined by mixing complex at various concentrations with riboflavin and nitroblue tetrazolium (NBT, then the mixture was lighted with 20 Watt tungsten lamp for 15 minutes in a closed box. Then the reduced NBT absorptions were measured at λ 560 nm. The difference of absorbance between standard and sample solutions (without and with riboflavin, respectively was multiplied by 100% to obtain %inhibition of each various sample concentration against NBT. SOD activity was obtained from IC50 data defined as a 50% inhibition of the plot curve of % inhibition to the concentration of the complex. The result obtained for this compound is IC50 = 2.7 ± 0.05 µM as well as enzymatic method. Therefore, this method can be used to determine the SOD activity by giving more stability and accuracy of IC50 value.

  19. Clove (Syzygium aromaticum) ingredients affect lymphocyte subtypes expansion and cytokine profile responses: An in vitro evaluation.

    Science.gov (United States)

    Dibazar, Shaghayegh Pishkhan; Fateh, Shirin; Daneshmandi, Saeed

    2014-12-01

    Clove (Syzygium aromaticum) has been used in folk medicine in many disorders. The present work aimed to investigate effects of clove essential oil as eugenol and water soluble ingredients on mouse splenocytes. Clove extracts were harvested and in different concentrations (0.001-1000 μg/mL) were affected to splenocytes and also phytohemagglutinin (PHA = 5 μg/mL) and lipopolysaccharide (LPS = 10 μg/mL) activated splenocytes; then splenocytes proliferation assayed using the MTT ([3-(4, 5-dimethylthiazole-2-yl) -2, 5-diphenyl tetrazolium bromide]) method were done. On the culture supernatant interferon (IFN)-γ, interleukin (IL)-4, IL-10, and transforming growth factor (TGF)-β cytokines were measured. Clove ingredients (100 μg/mL and 1000 μg/mL) reduced PHA stimulated splenocytes proliferation and enhanced LPS stimulated cells expansion. Treated splenocytes showed suppression of IFN-γ release and induction of IL-4, IL-10, and TGF-β secretion (in the range of 0.1-1000 μg/mL). The results of this study suggest clove extracts could suppress the T cell cellular immunity and enhance humoral immune responses. In clove affection cytokine pattern shifted toward modulatory and Th2 responses and accelerator of humoral immunity cytokines. Copyright © 2014. Published by Elsevier B.V.

  20. Metabolic activity of tree saps of different origin towards cultured human cells in the light of grade correspondence analysis and multiple regression modeling

    Directory of Open Access Journals (Sweden)

    Artur Wnorowski

    2017-06-01

    Full Text Available Tree saps are nourishing biological media commonly used for beverage and syrup production. Although the nutritional aspect of tree saps is widely acknowledged, the exact relationship between the sap composition, origin, and effect on the metabolic rate of human cells is still elusive. Thus, we collected saps from seven different tree species and conducted composition-activity analysis. Saps from trees of Betulaceae, but not from Salicaceae, Sapindaceae, nor Juglandaceae families, were increasing the metabolic rate of HepG2 cells, as measured using tetrazolium-based assay. Content of glucose, fructose, sucrose, chlorides, nitrates, sulphates, fumarates, malates, and succinates in sap samples varied across different tree species. Grade correspondence analysis clustered trees based on the saps’ chemical footprint indicating its usability in chemotaxonomy. Multiple regression modeling showed that glucose and fumarate present in saps from silver birch (Betula pendula Roth., black alder (Alnus glutinosa Gaertn., and European hornbeam (Carpinus betulus L. are positively affecting the metabolic activity of HepG2 cells.