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Sample records for tetrazolium

  1. Microculture tetrazolium assays: a comparison between two new tetrazolium salts, XTT and MTS.

    Science.gov (United States)

    Goodwin, C J; Holt, S J; Downes, S; Marshall, N J

    1995-02-13

    Microculture tetrazolium assays are being widely exploited to investigate the mechanisms of both cell activation and cell damage. They are colorimetric assays which are based upon the bioreduction of a tetrazolium salt to an intensely coloured formazan. We contrast the responses obtainable with two new tetrazolium salts, MTS and XTT, when used on the rat lymphoma cell line (Nb2 cells), which has been activated by human growth hormone. These tetrazolium salts, unlike the more commonly used MTT, form soluble formazans upon bioreduction by the activated cells. This has the advantage that it eliminates the error-prone solubilisation step which is required for the microculture tetrazolium assays which employ MTT. Bioreduction of XTT and MTS usually requires addition of an intermediate electron acceptor, phenazine methosulphate (PMS). We found that the XTT/PMS, but not the MTS/PMS, reagent mixture was unstable. Nucleation and crystal formation in the XTT/PMS reagent mixture, prepared in DPBS, could occur within 1-3 min. This resulted in a decline in XTT-formazan production and manifested itself in the microculture tetrazolium assay as both poor within-assay precision and serious assay drift. Several features of the system suggested that the formation of charge-transfer complexes between XTT and PMS accounted for this instability. No such instability was encountered when MTS and PMS were mixed. We demonstrate that MTS/PMS provides microculture tetrazolium assays for hGH which are free from these serious artefacts and which are uniquely precise. In conclusion we therefore advocate the use of MTS in preference to XTT for the new generation of microculture tetrazolium assays.

  2. Interference of magnesium corrosion with tetrazolium-based cytotoxicity assays.

    Science.gov (United States)

    Fischer, Janine; Prosenc, Marc H; Wolff, Martin; Hort, Norbert; Willumeit, Regine; Feyerabend, Frank

    2010-05-01

    Magnesium (Mg) alloys are promising materials for the development of biodegradable implants. However, the current in vitro test procedures for cytotoxicity, cell viability and proliferation are not always suitable for this class of materials. In this paper we show that tetrazolium-salt-based assays, which are widely used in practice, are influenced by the corrosion products of Mg-based alloys. Corroded Mg converts tetrazolium salts to formazan, leading to a higher background and falsifying the results of cell viability. Tetrazolium-based assays are therefore not a useful tool for testing the cytotoxicity of Mg in static in vitro assays. Copyright (c) 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  3. 1,3- and 1,4-Substituted tetrazolium salts

    International Nuclear Information System (INIS)

    Voitekhovich, Sergei V; Gaponik, Pavel N; Ivashkevich, Oleg A

    2002-01-01

    The published data on the synthesis, physicochemical properties, structures and reactions of 1,3-(1,3,5)- and 1,4-(1,4,5)-substituted tetrazolium salts are systematised and generalised. Their applications as starting compounds in the preparative chemistry of heterocyclic derivatives and some other branches of science and technology are reviewed. The bibliography includes 122 references.

  4. Some Ternary Phenylmethoxybis(tetrazolium) Complexes of Vanadium(IV,V) and Their Constants of Association

    OpenAIRE

    Gavazov, Kiril Blazhev; Racheva, Petya Vasileva; Lekova, Vanya Dimitrova; Dimitrov, Atanas Nikolov; Türkyilmaz, Murat; Genç, Fatma

    2012-01-01

    Several liquid-liquid extraction systems containing vanadium {vanadium(IV) or vanadium(V)}, azoderivative of resorcinol {ADR: 4-(2-pyridylazo)-resorcinol (PAR) or 4-(2-thiazolylazo)-resorcinol (TAR)} and (phenylmethoxibis)tetrazolium salts {MBT: 3,3'-(3,3'-dimetoxy-4,4'-biphenylene)-bis(2,5-diphenyl-2H-tetrazolium) chloride (Blue Tetrazolium, BT) or 3,3'-(3,3'-dimetoxy-4,4'-biphenylene)-bis[2,5-di(4-nitrophenyl)-2H-tetrazolium] chloride (Tetranitroblue Tetrazolium, TNBT)} were studied. The op...

  5. Serial nitroblue tetrazolium tests in the management of infection.

    Science.gov (United States)

    Freeman, R; King, B; Kite, P

    1973-01-01

    Two cases are described in which extensive use was made of the nitroblue tetrazolium (NBT) test. In the first case the advantages of using this technique to diagnose and control infection is shown; in the second the considerable advantage of the speed of the technique. In both of these cases the test made a material contribution to the management of the patient, and it is concluded that the test brings bacteriological control of the patient within the immediate clinical area, thus overcoming one of the principal disadvantages of the classical bacteriological methods.

  6. Nitroblue tetrazolium test in patients with beta-thalassemia major

    International Nuclear Information System (INIS)

    Ghaffari, J.; Vahidshahi, K.; Kosaryan, M.; Karami, H.; Mahdavi, M.; Parvinnejad, N.

    2008-01-01

    Objective to assess the neutrophil function in thalassemia major (TM)patients and compare it with the control group and to recognize its relevantfactors. This was a retrospective cohort study, which was carried out fromOctober 2007 to February 2008 in the Thalassemia research Center in Boo AliSina Hospital in Sari, Mazandaran, North of Iran. The study populationconsisted of TM patients in Boo Ali Sina Teaching Hospital. The method ofsampling in the case group was systematic and it was target based in thecontrol group. The sample size determined was based on previous studies.Thalassemia major was diagnosed based on hemoglobin electrophoresis (casegroup). The control group was their brothers and sisters, who had +-5 yearsof age difference and were of the same gender as the patients. Datacollection was based on interview, investigating demographic characteristicsand also obtaining medical information from medical records of the patients.The neutrophil function was by performing nitroblue tetrazolium (NBT)reduction test. The test was carried out on both groups and the data wereanalyzed by software using SPSS version 13.0. In this study, 39 patients and39 healthy controls were compared. The average age of the patients was21.6+-5.3 years and it was 22.4+-5.1 years in healthy controls (p=0.7). Therewas a significant correlation between the test's results and the patients age(p=0.008). The rate of impaired NBT results in the patients was 36%, while itwas 10% in controls, which were significantly different. The neutrophilactivity based on NBT test was 89.9+-11.6% in the case group and 93.7+-2.51%in the control group (p=0.025). This study indicates that neutrophil activityin thalassemia patients was significantly lower, compared to the normalcontrol group, especially in young patients. Based on the results, evaluationof neutrophil function and pyogenic infections in TM patients seemsnecessary. (author)

  7. Comparison of Tetrazolium Salt Assays for Evaluation of Drug Activity against Leishmania spp.

    Science.gov (United States)

    Ginouves, Marine; Carme, Bernard; Couppie, Pierre

    2014-01-01

    In French Guiana, leishmaniasis is an essentially cutaneous infection. It constitutes a major public health problem, with a real incidence of 0.2 to 0.3%. Leishmania guyanensis is the causal species most frequently encountered in French Guiana. The treatment of leishmaniasis is essentially drug based, but the therapeutic compounds available have major side effects (e.g., liver damage and diabetes) and must be administered parenterally or are costly. The efficacy of some of these agents has declined due to the emergence of resistance in certain strains of Leishmania. There is currently no vaccine against leishmaniasis, and it is therefore both necessary and urgent to identify new compounds effective against Leishmania. The search for new drugs requires effective tests for evaluations of the leishmanicidal activity of a particular molecule or extract. Microculture tetrazolium assays (MTAs) are colorimetric tests based on the use of tetrazolium salts. We compared the efficacies of three tetrazolium salts—3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT), and 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8)—for quantification of the promastigotes of various species of Leishmania. We found that the capacity of Leishmania to metabolize a tetrazolium salt depended on the salt used and the species of Leishmania. WST-8 was the tetrazolium salt best metabolized by L. guyanensis and gave the best sensitivity. PMID:24719447

  8. A critical assessment of the use of microculture tetrazolium assays to measure cell growth and function.

    Science.gov (United States)

    Marshall, N J; Goodwin, C J; Holt, S J

    1995-06-01

    Microculture tetrazolium assays (MTAs) are being widely applied to probe the relationships between cell survival, growth, and differentiation and also to investigate associations between compromised cell metabolism, oxidative stress, and programmed cell death as occurs in apoptosis. MTAs rely upon the cellular reduction of tetrazolium salts to their intensely coloured formazans. The resulting colorimetric assays form the basis of exceptionally precise systems which are technically amenable and capable of a high throughput of samples. As a consequence, MTAs are being used to monitor responses to both extracellular activators and toxic agents in disciplines as diverse as radiobiology and endocrinology. We review the chemistry and histochemical applications of tetrazolium salts and subsequently discuss the criteria for their use in MTAs. These assays are one of the latest examples of the application of the tetrazolium/formazan system to cell biology. We outline current views on the mechanisms of the bioreduction of tetrazolium salts. These probably combine to reflect the integrated pyridine nucleotide dependent redox state of the cell. We try to illustrate how an understanding of these mechanisms helps to avoid some of the pitfalls of the MTA systems. There is now for example, extensive evidence that changes in cell culture environments, such as glucose supply or pH of the medium, influence the reduction of tetrazolium salts and thereby introduce artefacts into MTAs. Finally, we provide examples of situations in which MTAs can be used to complement other more established experimental systems. They then act as unique probes with which to investigate changes in the redox state of the cell. These changes are associated with regulation of cell growth, proliferation and differentiation and conversely, the different pathways leading to cell death.

  9. Tetrazolium Reduction-Malachite Green Method for Assessing the Viability of Filamentous Bacteria in Activated Sludge

    Science.gov (United States)

    Bitton, Gabriel; Koopman, Ben

    1982-01-01

    A method was developed to assess the activity of filamentous bacteria in activated sludge. It involves the incubation of activated sludge with 2(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride followed by staining with malachite green. Both cells and 2(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride-formazan crystals can be observed in prepared specimens by using bright-field microscopy. This procedure allowed us to distinguish between inactive and actively metabolizing filaments after chlorine application to control the bulking of activated sludge. Images PMID:16345999

  10. 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...

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

    African Journals Online (AJOL)

    CD4+ count and Nitro-Blue Tetrazolium reduction rate of neutrophil in newly diagnosed HIV-infected adults in Sokoto Metropolis. U.K. Mustapha, C.C. Onyenekwe, A. Yakubu, B.R. Alkali, M.H. Yeldu, K.M. Hamid, I. Abdullahi, N.M. Bunza, M. Bello, A.B. Ibrahim ...

  12. 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...

  13. Adjustment of tetrazolium methodology for the evaluation of Melanoxylon brauna seeds viability

    Directory of Open Access Journals (Sweden)

    Viviana Borges Corte

    2010-09-01

    Full Text Available The objective of this work was to adjust the tetrazolium test methodology to Melanoxylon brauna seeds. The seeds werepre-conditioned by immersion in distilled water at 25ºC for 24 hours. The tegument of the seeds were removed and the embryos weresubmerged in the tetrazolium solution (pH 6.5 at concentration of 0.05, 0.1 and 0.3%, and were kept in the dark at 25ºC temperaturefor 10 and 24 hours. The embryos were evaluated for the intensity of color, presence of milky areas, aspect of the tissues, and locationof the color. The embryos were individually classified into nine viability classes. They were also submitted to the germination andseedling emergence tests. The best correlations between the standard tests of seed germination of Melanoxylon brauna were those with0.3% concentration for 10 hours. The viability is represented by classes 1, 2 and 3.

  14. Extraction-radiochemical study of the ion-association complex of antimony (V) with tetrazolium violet and its thermal behavior

    International Nuclear Information System (INIS)

    Kostova, S.G.; Boyanov, B.S.

    1995-01-01

    The optimum conditions for extraction of ion-associated complexes (IAS) formed from the tetrazolium salt - tetrazolium violet and Sb(V) in hydrochloric acid medium have been studied. An isotope of antimony ( 125 Sb) was used for determination of the recovery factor (R%) and distribution ratio (D S b). The thermal behavior of the antimony complex with tetrazole violet was studied using differential thermal and thermogravimetric analysis. (author) 12 refs.; 3 figs

  15. Blue tetrazolium as a novel corrosion inhibitor for cold rolled steel in hydrochloric acid solution

    International Nuclear Information System (INIS)

    Li Xianghong; Deng Shuduan; Fu Hui

    2010-01-01

    The inhibition effect of blue tetrazolium (BT) on the corrosion of cold rolled steel (CRS) in 1.0 M HCl solution at 20 o C was investigated by weight loss, potentiodynamic polarization, electrochemical impedance spectroscopy (EIS), and scanning electron microscopy (SEM) methods. The results show that BT is a very good inhibitor, and the adsorption of BT on CRS surface obeys Langmuir adsorption isotherm. Polarization curves reveal that BT acts as a mixed-type inhibitor. EIS spectra exhibit one capacitive loop and confirm the inhibitive ability. The inhibition action of BT is also evidenced by SEM images.

  16. Radiation-induced coloration of nitro blue tetrazolium gel dosimeter for low dose applications

    International Nuclear Information System (INIS)

    Abdel-Fattah, A.A.; Beshir, W.B.; Hassan, H.M.; Soliman, Y.S.

    2017-01-01

    A radiochromic sensor of nitro blue tetrazolium (NBT) in gelatin was evaluated as a new gel dosimeter for radiation applications. The NBT gel has the advantage of visual color change from faint yellow to violet at low absorbed doses (10–1000 Gy). This color change appears as a result of the reduction of NBT to colored formazan then to diformazan species with further increase of absorbed doses. Responses of the gel at different NBT concentrations were analyzed at the absorption maximum centered at 527 nm. An increase of NBT concentrations in the gel enhances the radiation dose sensitivity. Energy dependent study implies the tissue equivalency of the gel in the energy range of 0.15–20 MeV. Dependence of the gel response on irradiation temperature, and color stability before and after irradiation were also studied. The combined uncertainty associated with the dose monitoring (10–1000 Gy) is 6.26% (2σ). Thus, the NBT gel shows its suitability for food irradiation, insect population control, and some food irradiation applications. - Highlights: • Preparation of nitro blue tetrazolium (NBT) gel for the dose range of 10–1000 Gy. • The sensitivity of it increases with increasing NBT concentrations. • The response of irradiated dosimeter is stable after 5 h from irradiation. • The prepared gel dosimeter is a tissue equivalent. • Its combined uncertainty is equal to 6.26% for 10–1000 Gy dose level.

  17. Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay.

    Science.gov (United States)

    Alley, M C; Scudiero, D A; Monks, A; Hursey, M L; Czerwinski, M J; Fine, D L; Abbott, B J; Mayo, J G; Shoemaker, R H; Boyd, M R

    1988-02-01

    For the past 30 years strategies for the preclinical discovery and development of potential anticancer agents have been based largely upon the testing of agents in mice bearing transplantable leukemias and solid tumors derived from a limited number of murine as well as human sources. The feasibility of implementing an alternate approach, namely combined in vitro/in vivo screening for selective cytotoxicity among panels of human tumor cell lines derived from a broad spectrum of human solid tumors is under investigation. A group of 30 cell lines acquired from a variety of sources and representing 8 lung cancer pathologies as well as 76 cell lines representing 10 other categories of human cancer (carcinomas of colon, breast, kidney, prostate, ovary, head and neck; glioma; leukemia; melanoma; and sarcoma) have exhibited acceptable growth characteristics and suitable colorimetric profiles in a single, standard culture medium. Measurements of in vitro growth in microculture wells by cell-mediated reduction of tetrazolium showed excellent correlation (0.89 less than r2 less than 0.98) with measurements of cellular protein in adherent cell line cultures as well as viable cell count in suspension cell line cultures (0.94 less than r2 less than 0.99). Since the microculture tetrazolium assay provides sensitive and reproducible indices of growth as well as drug sensitivity in individual cell lines over the course of multiple passages and several months' cultivation, it appears suitable for initial-stage in vitro drug screening.

  18. Comparison of tetrazolium colorimetric and [3H]-uridine assays for in vitro chemosensitivity testing.

    Science.gov (United States)

    Ford, C H; Richardson, V J; Tsaltas, G

    1989-01-01

    We have routinely used a [3H]-uridine microplate assay for assessing chemosensitivity. A colorimetric assay with the advantages of safety, cost and simplicity has previously been described and relies on the ability of living cells to reduce a soluble tetrazolium salt, 3-4,5-dimethylthiazol-2,5-diphenyl-tetrazolium bromide (MMT), into an insoluble formazan precipitate. We compared the chemosensitivity of 14 human tumour cell lines of colonic, lung and cervical carcinoma origin to doxorubicin, vindesine or vindesine immunoconjugates in both the [3H]-uridine assay and a modified MTT assay to evaluate whether we could change to the non-radiolabelled method. Correlation between the concentration of drug causing 50% inhibition of cell growth (IC50) for these agents between the two assays was very poor. However, taking account of recent reports in the literature, we modified the MTT assay by removing serum-containing medium and using dimethyl sulphoxide to solubilise the formazan precipitate. This considerably improved the correlation between the assays for doxorubicin (r = 0.871; P = 0.001) and vindesine (r = 0.981; P less than 0.001). Our data indicates that the MTT assay can be used to replace the [3H]-uridine assay for chemosensitivity screening, but further modifications are necessary to improve the sensitivity and decrease the problem of cell loss after washing, which was noted with some adherent cell lines.

  19. Aloe vera is non-toxic to cells: A microculture tetrazolium technique colorimetric assay study

    Directory of Open Access Journals (Sweden)

    Devi Gopakumar

    2014-01-01

    Full Text Available Introduction: Aloe vera (Av, a succulent of Liliaceae family is now a widely used medicinal plant. Its′ application covers a wide spectrum of human diseases, including oral mucosa, gastric mucosa and skin. Aloe vera preparations in the form of gel, mouth washes and cream are applied topically for many oral diseases. The applications include oral lichen planus, candidiasis, oral submucous fibrosis, geographic tongue, etc. Aims and Objectives: To evaluate the cytotoxicity of Av on human fibroblasts. Materials and Methods: Aloe vera preparation (70% was applied on the fibroblast cell lineage and the cell viability was evaluated by microculture tetrazolium technique (MTT colorimetric assay. Results: The cell viability at different concentrations was measured. The cells have maintained their viability at different concentrations used in the study. Conclusion: Our study shows the cell viability at different sample concentrations of Av. This could open up wide clinical applications of Av for reactive, inflammatory and potentially malignant oral and other mucocutaneous diseases.

  20. In-vitro antimycobacterial drug susceptibility testing of non-tubercular mycobacteria by tetrazolium microplate assay.

    Science.gov (United States)

    Sankar, Manimuthu Mani; Gopinath, Krishnamoorthy; Singla, Roopak; Singh, Sarman

    2008-07-11

    Non-tubercular mycobacteria (NTM) has not been given due attention till the recent epidemic of HIV. This has led to increasing interest of health care workers in their biology, epidemiology and drug resistance. However, timely detection and drug susceptibility profiling of NTM isolates are always difficult in resource poor settings like India. Furthermore, no standardized methodology or guidelines are available to reproduce the results with clinical concordance. To find an alternative and rapid method for performing the drug susceptibility assay in a resource limited settings like India, we intended to evaluate the utility of Tetrazolium microplate assay (TEMA) in comparison with proportion method for reporting the drug resistance in clinical isolates of NTM. A total of fifty-five NTM isolates were tested for susceptibility against Streptomycin, Rifampicin, Ethambutol, Ciprofloxacin, Ofloxacin, Azithromycin, and Clarithromycin by TEMA and the results were compared with agar proportion method (APM). Of the 55 isolates, 23 (41.8%) were sensitive to all the drugs and the remaining 32 (58.2%) were resistant to at least one drug. TEMA had very good concordance with APM except with minor discrepancies. Susceptibility results were obtained in the median of 5 to 9 days by TEMA. The NTM isolates were highly sensitive against Ofloxacin (98.18% sensitive) and Ciprofloxacin (90.09% sensitive). M. mucogenicum was sensitive only to Clarithromycin and resistant to all the other drugs tested. The concordance between TEMA and APM ranged between 96.4 - 100%. Tetrazolium Microplate Assay is a rapid and highly reproducible method. However, it must be performed only in tertiary level Mycobacteriology laboratories with proper bio-safety conditions.

  1. 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.

  2. Chemosensitivity testing of primary human renal cell carcinoma by a tetrazolium based microculture assay (MTT).

    Science.gov (United States)

    Mickisch, G; Fajta, S; Keilhauer, G; Schlick, E; Tschada, R; Alken, P

    1990-01-01

    MTT staining procedures have been used in chemosensitivity testing of established cell lines of human and other sources as well as of human leukaemias, but only limited information on its application in primary solid human tumors is presently available. We have evaluated MTT staining in primary human Renal Cell Carcinomas (RCCs), studied various factors interfering with the optimal use, and finally applied it in subsequent chemosensitivity testing. The method depends on the conversion of a water-soluble tetrazolium salt (MTT) to a purple colored formazan precipitate, a reaction effected by enzymes active only in living cells. Single cell suspensions of RCCs were obtained either by enzymatic dispersion or by mechanical dissagregation, filtered through gauze, and purified by Ficoll density centrifugation. Tests were carried out in 96-well microculture plates. 10(4) viable tumor cells per well at 4 h incubation time with 20 micrograms MTT/100 microliters total medium volume yielded best results. Formazan crystals were dissolved with DMSO, and the plates were immediately measured on a microculture plate reader at 540 nm. Under these criteria, linearity of the system could be demonstrated. For chemosensitivity testing, cells were continuously exposed to a number of drugs prior to the MTT staining procedure. Reproducibility of results was assessed and confirmed by culturing RCCs in flasks additionally, resubmitting them after 1, 2, and 4 weeks to the MTT assay. We conclude that the semiautomated MTT assay offers a valid, rapid, reliable and simple method to determine the degree of chemoresistance in primary human RCCs.

  3. New visible and selective DNA staining method in gels with tetrazolium salts.

    Science.gov (United States)

    Paredes, Aaron J; Naranjo-Palma, Tatiana; Alfaro-Valdés, Hilda M; Barriga, Andrés; Babul, Jorge; Wilson, Christian A M

    2017-01-15

    DNA staining in gels has historically been carried out using silver staining and fluorescent dyes like ethidium bromide and SYBR Green I (SGI). Using fluorescent dyes allows recovery of the analyte, but requires instruments such as a transilluminator or fluorimeter to visualize the DNA. Here we described a new and simple method that allows DNA visualization to the naked eye by generating a colored precipitate. It works by soaking the acrylamide or agarose DNA gel in SGI and nitro blue tetrazolium (NBT) solution that, when exposed to sunlight, produces a purple insoluble formazan precipitate that remains in the gel after exposure to light. A calibration curve made with a DNA standard established a detection limit of approximately 180 pg/band at 500 bp. Selectivity of this assay was determined using different biomolecules, demonstrating a high selectivity for DNA. Integrity and functionality of the DNA recovered from gels was determined by enzymatic cutting with a restriction enzyme and by transforming competent cells after the different staining methods, respectively. Our method showed the best performance among the dyes employed. Based on its specificity, low cost and its adequacy for field work, this new methodology has enormous potential benefits to research and industry. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. The microculture tetrazolium assay (MTA): another colorimetric method of testing Plasmodium falciparum chemosensitivity.

    Science.gov (United States)

    Delhaes, L; Lazaro, J E; Gay, F; Thellier, M; Danis, M

    1999-01-01

    Malarial lactate dehydrogenase (LDH), which uses 3-acetyl pyridine adenine dinucleotide as coenzyme in a reaction leading to the formation of pyruvate from L-lactate, may be used to study the susceptibility of Plasmodium falciparum to a drug in vitro. Several methods to determine the activity of this enzyme are available. One, the colorimetric method of Makler and colleagues, was modified slightly, by using sodium-2,3-bis-[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5 - carboxanilide (XTT) and following the reaction by measuring the optical density at 450 nm. Using two, culture-adapted strains of P. falciparum, this LDH assay was compared with the unmodified Makler's assay and with the isotopic microtest based on the incorporation of tritium-labelled hypoxanthine. Fresh, clinical P. falciparum isolates were also tested in the presence of several drugs, including chloroquine, mefloquine, quinine, halofantrine, atovaquone and qinghaosu derivatives. The results of the three assays were correlated for all the drugs tested except atovaquone. The two enzymatic assays are non-radioactive, rapid, reliable, inexpensive to perform and semi-automatic. However, they do require an initial parasitaemia of 2% with a haematocrit of 1.8%.

  5. Application of a tetrazolium dye as an indicator of viability in anaerobic bacteria.

    Science.gov (United States)

    Bhupathiraju, V K; Hernandez, M; Landfear, D; Alvarez-Cohen, L

    1999-09-01

    The use of the redox dye 5-cyano-2,3,-ditolyl tetrazolium chloride (CTC) for evaluating the metabolic activity of aerobic bacteria has gained wide application in recent years. In this study, we examined the utility of CTC in capturing the metabolic activity of anaerobic bacteria. In addition, the factors contributing to abiotic reduction of CTC were also examined. CTC was used in conjunction with the fluorochrome 5-(4,6-dichlorotriazinyl) aminofluorescein (DTAF), that targets bacterial cell wall proteins, to quantitate the active fraction of total bacterial numbers. Facultative anaerobic bacteria, including Escherichia coli grown fermentatively, and Pseudomonas chlorophis, P. fluorescens, P. stutzeri, and P. pseudoalcalegenes subsp. pseudoalcalegenes grown under nitrate-reducing conditions, actively reduced CTC during all phases of growth. Greater than 95% of these cells accumulated intracellular CTC-formazan crystals during the exponential phase. Obligate anaerobic bacteria, including Syntrophus aciditrophicus grown fermentatively, Geobacter sulfurreducens grown with fumarate as the electron acceptor, Desulfovibrio desulfuricans subsp. desulfuricans and D. halophilus grown under sulfate-reducing conditions, Methanobacterium formicicum grown on formate, H2 and CO2, and Methanobacterium thermoautotrophicum grown autotrophically on H2 and CO2 all reduced CTC to intracellular CTC-formazan crystals. The optimal CTC concentration for all organisms examined was 5 mM. Anaerobic CTC incubations were not required for quantification of anaerobically grown cells. CTC-formazan production by all cultures examined was proportional to biomass production, and CTC reduction was observed even in the absence of added nutrients. CTC was reduced by culture fluids containing ferric citrate as electron acceptor following growth of either G. metallireducens or G. sulfurreducens. Abiotic reduction of CTC was observed in the presence of ascorbic acid, cysteine hydrochloride, dithiothreitol

  6. The use of intermediate electron acceptors to enhance MTT bioreduction in a microculture tetrazolium assay for human growth hormone.

    Science.gov (United States)

    Goodwin, C J; Holt, S J; Downes, S; Marshall, N J

    1996-01-01

    We contrast the effects of three intermediate electron acceptors (IEAs) on the highly quantitative ESTA bioassay for human growth hormone. This is a microculture tetrazolium assay based upon the in vitro reduction of the tetrazolium salt MTT, by Nb2 cells which have been activated with hGH. Each of the IEAs influenced MTT-formazan production in a distinctive manner. The two quinonoids, namely menadione and co-enzyme Q0 markedly increased the MTT-formazan produced by hormone activated Nb2 cells and thereby amplified the response of our bioassay for human growth hormone (hGH). The exceptionally low bioassay baseline which is characteristic of the unstimulated Nb2 cells when only MTT is added was retained in the presence of CoQ0, but was greatly increased by menadione. Phenazine methosulphate, which is the most widely used redox intermediary in microculture tetrazolium assays, also increased the baseline, but had only a minimal additional effect on MTT reduction by activated Nb2 cells. We conclude that CoQ0 is the preferred IEA for this ESTA bioassay for hGH.

  7. 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.

  8. Investigation of the extraction equilibrium of ternary ionassociation complex of thallium(III) with iodo-nitro-tetrazolium chlorid

    International Nuclear Information System (INIS)

    Alexandrov, A.; Dimitrov, A.

    1976-01-01

    The extraction equilibrium of the ternary ion-association complex of iodo-nitro-tetrazolium [3-(4-iodophenyl)2-(4-nitrophenyl)-5-phenyltetrazolium chloride] with the chlorocomplex of thallium(III) is investigated radiochemically. The molar ratio of the ion-associate is found to be 1:1, the association constant has a value of 3.2x10 3 in aqueous solution and the distribution constant is 8.9. The extraction constant which gives a quantitative characterization of the equilibrium is 2.3x10 4 . From the investigation performed it can be concluded that a quite satisfactory extraction of thallium(III) by means of iodo-nitro-tetrazole in benzene can be carried out. The extraction constant has a relatively high value which allows to use this system conveniently for the extraction-photometric determination of thallium(III). (T.C.)

  9. A novel screening method based on menadione mediated rapid reduction of tetrazolium salt for testing of anti-mycobacterial agents.

    Science.gov (United States)

    Singh, Upasana; Akhtar, Shamim; Mishra, Abhishek; Sarkar, Dhiman

    2011-02-01

    A microplate-based rapid, inexpensive and robust technique is developed by using tetrazolium salt 2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) and menadione to determine the viability of Mycobacterium tuberculosis, Mycobacterium bovis BCG and Mycobacterium smegmatis bacilli in microplate format. In general, XTT reduction is an extremely slow process which takes almost 24 h to produce a detectable signal. Menadione could drastically induce this reduction to an almost equal extent within a few minutes in a dose dependent manner. The reduction of XTT is directly proportional to the cell concentration in the presence of menadione. The standardized protocol used 200 μM of XTT and 60 μM of menadione in 250 μl of cell suspension grown either in aerobic or anaerobic conditions. The cell suspension of M. bovis BCG and M. tuberculosis were incubated for 40 min before reading the optical density at 470 nm whereas M. smegmatis was incubated for 20 min. Calculated Signal/Noise (S/N) ratios obtained by applying this protocol were 5.4, 6.4 and 9.4 using M. bovis BCG, M. tuberculosis and M. smegmatis respectively. The calculated Z' factors were >0.8 for all mycobacterium bacilli indicating the robustness of the XTT Reduction Menadione Assay (XRMA) for rapid screening of inhibitors. The assay protocol was validated by applying 10 standard anti-tubercular agents on M. tuberculosis, M. bovis BCG and M. smegmatis. The Minimum Inhibitory Concentration (MIC) values were found to be similar to reported values from Colony Forming Unit (CFU) and REMA (resazurin microplate assay) assays. Altogether, XRMA is providing a novel anti-tubercular screening protocol which could be useful in high throughput screening programs against different physiological stages of the bacilli. Copyright © 2010 Elsevier B.V. All rights reserved.

  10. 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

  11. Effect of Electron Beam Irradiation on the Structural Properties of Poly (Vinyl Alcohol) Formulations with Triphenyl Tetrazolium Chloride Dye (TTC)

    International Nuclear Information System (INIS)

    Ali, Z.I.; Said, H.M.; Ali, H.E.

    2005-01-01

    Films of poly (vinyl alcohol) (PVA) composites with triphenyl tetrazolium chloride (TTC) dye were prepared and exposed to various radiation doses delivered by accelerated electrons. The results showed that at a low dose of 50 kGy, the color difference (Δ E) of PVA/TTC films was increased by -10 times of the initial value. However, the change in colour differences did not go systematically with increasing the TTC content, in which the composite with 1.5 wt% displayed higher value than that with 3.5 wt%. The differential scanning calorimetry (DSC) showed that the presence of the TTC dye caused a depression in the melting point (Tm) and heat of fusion (Δ Hf) of the PVA bulk polymer. However, the thermogravimetric analysis (TGA) showed that the presence of the TTC dye improved the thermal stability of PVA. Also, the tensile strength at break of PVA/TTC composites was improved after electron beam irradiation. Keywords: PVA, PVA/TTC composites, Electron beam irradiation, color strength, FTIR, thermal and mechanical characterization

  12. 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.

  13. Nitroblue tetrazolium test

    Science.gov (United States)

    ... infections. Normal value ranges may vary slightly from one lab to another. Talk to your doctor about the meaning of your test results. What Abnormal Results Mean If the sample does not change color when NBT is added, ...

  14. Distinction of Gram-positive and -negative bacteria using a colorimetric microbial viability assay based on the reduction of water-soluble tetrazolium salts with a selection medium.

    Science.gov (United States)

    Tsukatani, Tadayuki; Suenaga, Hikaru; Higuchi, Tomoko; Shiga, Masanobu; Noguchi, Katsuya; Matsumoto, Kiyoshi

    2011-01-01

    Bacteria are fundamentally divided into two groups: Gram-positive and Gram-negative. Although the Gram stain and other techniques can be used to differentiate these groups, some issues exist with traditional approaches. In this study, we developed a method for differentiating Gram-positive and -negative bacteria using a colorimetric microbial viability assay based on the reduction of the tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt} (WST-8) via 2-methyl-1,4-napthoquinone with a selection medium. We optimized the composition of the selection medium to allow the growth of Gram-negative bacteria while inhibiting the growth of Gram-positive bacteria. When the colorimetric viability assay was carried out in a selection medium containing 0.5µg/ml crystal violet, 5.0 µg/ml daptomycin, and 5.0µg/ml vancomycin, the reduction in WST-8 by Gram-positive bacteria was inhibited. On the other hand, Gram-negative bacteria produced WST-8-formazan in the selection medium. The proposed method was also applied to determine the Gram staining characteristics of bacteria isolated from various foodstuffs. There was good agreement between the results obtained using the present method and those obtained using a conventional staining method. These results suggest that the WST-8 colorimetric assay with selection medium is a useful technique for accurately differentiating Gram-positive and -negative bacteria.

  15. Determination of water-soluble vitamins using a colorimetric microbial viability assay based on the reduction of water-soluble tetrazolium salts.

    Science.gov (United States)

    Tsukatani, Tadayuki; Suenaga, Hikaru; Ishiyama, Munetaka; Ezoe, Takatoshi; Matsumoto, Kiyoshi

    2011-07-15

    A method for the determination of water-soluble vitamins using a colorimetric microbial viability assay based on the reduction of the tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8)} via 2-methyl-1,4-napthoquinone (NQ) was developed. Measurement conditions were optimized for the microbiological determination of water-soluble vitamins, such as vitamin B(6), biotin, folic acid, niacin, and pantothenic acid, using microorganisms that have a water-soluble vitamin requirement. A linear relationship between absorbance and water-soluble vitamin concentration was obtained. The proposed method was applied to determine the concentration of vitamin B(6) in various foodstuffs. There was good agreement between vitamin B(6) concentrations determined after 24h using the WST-8 colorimetric method and those obtained after 48h using a conventional method. The results suggest that the WST-8 colorimetric assay is a useful method for the rapid determination of water-soluble vitamins in a 96-well microtiter plate. Copyright © 2011 Elsevier Ltd. All rights reserved.

  16. Effect of electron beam irradiation on the structural properties of poly(vinyl alcohol) formulations with triphenyl tetrazolium chloride dye (TTC)

    Science.gov (United States)

    Ali, Z. I.; Said, Hossam M.; Ali, H. E.

    2006-01-01

    Films of poly(vinyl alcohol) (PVA) composites with triphenyl tetrazolium chloride (TTC) dye were prepared and exposed to various radiation doses delivered by accelerated electrons. The results showed that at a low dose of 50 kGy, the colour difference (Δ E*) of PVA/TTC films was increased by ˜10 times of the initial value. However, the change in colour differences did not go systematically with increasing the TTC content, in which the composite with 1.5 wt% displayed higher value than that with 3.5 wt%. The differential scanning calorimetry (DSC) showed that the presence of the TTC dye caused a depression in the melting point ( Tm) and heat of fusion (Δ Hf) of the PVA bulk polymer. However, the thermogravimetric analysis (TGA) showed that the presence of the TTC dye improved the thermal stability of PVA. Also, the tensile strength at break of PVA/TTC composites was improved after electron beam irradiation.

  17. Pré-condicionamento das sementes de mamoneira para o teste de tetrazólio = Preconditioning of castor bean seeds for the tetrazolium test

    Directory of Open Access Journals (Sweden)

    Carolina Maria Gaspar-Oliveira

    2011-04-01

    Full Text Available O objetivo do trabalho foi padronizar a metodologia de pré-condicionamento das sementes de mamoneira (Ricinus communis L. para a avaliação do potencial fisiológico pelo teste de tetrazólio. Testaram-se os seguintes métodos de pré-condicionamento: sementes com tegumento entre papel umedecido a 30, 35 e 40ºC por 6, 8, 10, 12, 14, 16 e 18h; sementes sem tegumento entre papel umedecido e sementes com tegumento imersas em água a 25, 30, 35 e 40º C por 1, 2, 3, 4, 5 e 6h. Após o pré-condicionamento, removeu-se o tegumento das sementes, que foram cortadas no sentido do comprimento e imersas na solução de tetrazólio a 0,2%, por 120 min., a 35ºC. Avaliaram-se a porcentagem de germinação das sementes, o teor de água, antes e após a embebição, e a uniformidade na coloração das sementes após o teste de tetrazólio. O delineamento experimental foi inteiramente casualizado, e a comparação de médias realizada pelo teste de Tukey a 5% de probabilidade. Concluiu-se que o pré-condicionamento para o teste detetrazólio deve ser realizado nas sementes de mamoneira com tegumento, entre papel toalha umedecido, a 35ºC por 12h para que os resultados desse teste assemelhem-se aos obtidos no teste de germinação.This research had the objective of standardizing the methodology for preconditioning of castor bean (Ricinus communis L. seeds for the evaluation of their physiological potential by the tetrazolium test. The evaluated seed preconditioning methods were: seeds with coatbetween moist paper towel at 30, 35 and 40ºC for 6, 8, 10, 12, 14, 16 and 18 hours; and seeds without coat between moist paper towel; and seeds with coat immersed in water, at 25, 30, 35 and 40ºC for 1, 2, 3, 4, 5 and 6 hours. After preconditioning, the seed coat was removed, the seeds were cut lengthwise, and immersed in tetrazolium solution at a concentration of 0.2% for 120 minutes at 35ºC. The seeds’ germination percentage, moisture content before and after

  18. In vitro screening of six anthelmintic plant products against larval Haemonchus contortus with a modified methyl-thiazolyl-tetrazolium reduction assay.

    Science.gov (United States)

    Hördegen, P; Cabaret, J; Hertzberg, H; Langhans, W; Maurer, V

    2006-11-03

    Because of the increasing anthelmintic resistance and the impact of conventional anthelmintics on the environment, it is important to look for alternative strategies against gastrointestinal nematodes. Phytotherapy could be one of the major options to control these pathologies. Extracts or ingredients of six different plant species were tested against exsheathed infective larvae of Haemonchus contortus using a modified methyl-thiazolyl-tetrazolium (MTT) reduction assay. Pyrantel tartrate was used as reference anthelmintic. Bromelain, the enzyme complex of the stem of Ananas comosus (Bromeliaceae), the ethanolic extracts of seeds of Azadirachta indica (Meliaceae), Caesalpinia crista (Caesalpiniaceae) and Vernonia anthelmintica (Asteraceae), and the ethanolic extracts of the whole plant of Fumaria parviflora (Papaveraceae) and of the fruit of Embelia ribes (Myrsinaceae) showed an anthelmintic efficacy of up to 93%, relative to pyrantel tartrate. Based on these results obtained with larval Haemonchus contortus, the modified MTT reduction assay could be a possible method for testing plant products with anthelmintic properties.

  19. Effect of alpha-interferon alone and combined with other antineoplastic agents on renal cell carcinoma determined by the tetrazolium microculture assay.

    Science.gov (United States)

    Homma, Y; Aso, Y

    1994-01-01

    The antiproliferative effect of various alpha-interferons (alpha-IFNs), alone or combined with other agents, on a renal cell carcinoma cell line was evaluated by the tetrazolium microculture assay to examine the rationale for combination therapies. Cells incubated in 96-week microculture plates at 5 x 10(3)/well were exposed to various agents for 3 days. There were no obvious differences in the growth inhibition caused by the 5 kinds of alpha-IFN examined as single agents. The combination of alpha-IFN with the following agents was also assessed: 5-fluorouracil (5FU), methotrexate (MTX), mitomycin C, bleomycin, cis-diaminedichloroplatinum (CDDP), vinblastine, etoposide (ETOP), alpha-IFN, tumor necrosis factor-alpha (TNF), and alpha-difluoromethylornithine. Synergism was observed for the combination of alpha-IFN+TNF, while the other combinations had additive or subadditive effects. No interference or antagonism was found. Trimodal combinations of alpha-IFN+MTX with either 5FU, ETOP, or CDDP all showed subadditive effects. These results indicated that an increased antiproliferative effect, although not necessarily synergistic, was obtained by the combination of alpha-IFN with a variety of antineoplastic agents, providing a rationale to seek for combination therapies including alpha-IFN for treating renal cell carcinoma.

  20. [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.

  1. 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

  2. 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

    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......-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...... on a real-time scale at frequent and continual time intervals for time course experiments, providing more detailed information on the respiratory burst response. The real-time chemiluminescence assay was used to measure respiratory burst activity in macrophage and neutrophilic granulocyte-enriched head...

  3. Critérios para o teste de tetrazólio na estimativa do potencial germinativo em macaúba Criteria for tetrazolium tests in the estimation of the germination potential of macaw palm

    Directory of Open Access Journals (Sweden)

    Leonardo Monteiro Ribeiro

    2010-04-01

    Full Text Available O objetivo deste trabalho foi estabelecer critérios para a aplicação do teste de tetrazólio em embriões de macaúba (Acrocomia aculeata. Para a elaboração do esquema de avaliação, foram realizadas avaliações anatômicas, identificação de padrões de coloração por solução de 2,3,5-trifenil cloreto de tetrazólio a 0,5% por quatro horas e cultivo in vitro de embriões. Em um experimento, foram avaliados três períodos de pré-condicionamento das sementes por imersão em água, por 12 e 24 horas, associados a duas temperaturas de coloração, 35 e 40ºC, em dois lotes de sementes além do controle sem pré-condicionamento. Em outro experimento, foram testadas em três lotes de sementes, três concentrações da solução de tetrazólio (0,5, 0,75 e 1%, associadas a dois tempos de coloração (duas e quatro horas. Utilizou-se o cultivo in vitro de embriões para comparação dos resultados. Um esquema de avaliação com dez padrões de coloração, associados a três classes de vigor, foi definido com base na anatomia dos embriões e no desenvolvimento de plântulas in vitro. O tratamento de pré-condicionamento em água não é necessário, e se deve aplicar o tempo de coloração de quatro horas, em solução de sal de tetrazólio, a 0,5% e 35ºC.The objective of this study was to establish criteria for the tetrazolium test in embryos of macaw palm (Acrocomia aculeata. To develop the evaluation scheme, anatomical analyses were performed, and staining patterns were identified using a 0.5% 2,3,5-triphenyl tetrazolium chloride solution for four hours, and embryo was cultured in vitro. In one experiment, three prior seed-soaking times in water (for 12 and 24 hours, besides a control without seed pre-conditioning, associated with two color temperatures (35 and 40ºC in two seed lots were evaluated. In another experiment, three concentrations of tetrazolium solution (0.5, 0.75 and 1%, associated with two staining times (two and four

  4. Tetrazolium test in the evaluation of watermelon physiological seed quality/ Teste de tetrazólio para avaliação da qualidade fisiológica de sementes de melancia

    Directory of Open Access Journals (Sweden)

    Luciana Magda de Oliveira

    2007-08-01

    Full Text Available The tetrazolium test is one of the most promising methods to estimate in a fast way viability and vigor of seeds. Its usage in watermelon seeds requires some care with the imbibition and handling due to presence of mucilaginous layer adherent to the tegument. The objective of this research work was to define the imbibition time and the method of scarification for removing the mucilage, during pre – conditioning of the tetrazolium test in watermelon. Seeds from different watermellon cultivars were immersed in water at 30°C from 12 to 18 hours, and sloughed in cal, fine and thick sand. After longitudinal cut in the distal portion to the embryonic axis, the tegument was removed and embryos were kept in water for additonal 2 hours at 30°C for hand removal of the remaining membrane. After definition of the ideal pre – conditioning method, the embryos were immersed in tetrazolium solution in concentrations of 0,075%; 0, 5% and 1% for 3 and 4 hours at 30°C. The immersion of watermelon seeds in water at 30°C for 12 hours, mucilage removal with fine sand, longitudinal cut in the distal portion to the embryonic axis, and removal of tegument, followed by the permanence of embryos in water for 2 hours at 30°C for the hand removal of the internal membrane and immersion in the tetrazolium solution at 0,075% for 4 hours at 30°C are adequate procedures for the evaluation of watermelon seed quality.O teste de tetrazólio é um dos métodos mais promissores para estimar, de forma rápida, a viabilidade e o vigor das sementes. Sua utilização em sementes de melancia requer cuidados na embebição e manuseio pela presença de camada mucilaginosa aderente ao tegumento. O objetivo do trabalho foi definir o tempo de embebição e do método de escarificação para retirada da mucilagem, no pré-condicionamento do teste de tetrazólio, sementes de diferentes cultivares de melancia. As sementes foram imersas em água a 30oC por 12 e 18 horas, e escarificadas

  5. 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.

  6. Preparation of C{sup 14}-labelled tetrazolium salts and tracer study of the tetrazene-formazan rearrangement; Preparation de sels de tetrazolium marques au carbone-14 et etude de la transposition tetrazene-formazan, au moyen d'indicateurs radioactifs; Izgotovlenie mechennykh C{sup 14} solej tetrazosoedinenij i issledovanie pri pomoshchi indikatorov peregruppirovok tetrazona-formazana; Preparacion de sales de tetrazolio marcadas con {sup 14}C y estudio de la transposicion tetraceno-formazan con ayuda de trazadores

    Energy Technology Data Exchange (ETDEWEB)

    Marton, Joseph; Meisel, Julia [Central Research Institute for Chemistry of the Hungarian Academy of Sciences, Budapest (Hungary); Gosztonyi, Thomas [Institute of Organic Chemical Technology, Technical University, Budapest (Hungary)

    1962-03-15

    The preparation of [5-C{sup 14}]-TTC, [5, 5'-di-C{sup 14}]NT (neotetrazolium) and [5, 5'-di-C{sup 14}]-BT (tetrazolium blue) starting from benzaldehyde- [1-C{sup 14}] has been accomplished. The yields for both mono- and ditetrazolium salts are high, and the products can be obtained with high sp. activity. The purity of the samples was investigated by paper chromatography. In the case of ditetrazolium salts some impurities could be detected and conclusions drawn as to their structure and quantity. A method has been developed to prepare C{sup 14}- labelled ditetrazolium salts of high purity. The formation of the formazan, the precursor of the tetrazolium salt, goes through an unstable intermediate of tetrazene-type structure which rearranges rapidly in basic medium, to yield the formazan. The tetrazene intermediate can be isolated under suitable conditions. By using C{sup 14}-labelled benzaldehyde phenylhydrazone this rearrangement was investigated and a verification of its intramolecular character given. (author) [French] On a procede a la preparation de [5-C{sup 14}]-TTC, de [5,5'-di-C{sup 14}] NT (neotetrazolium) et de [5,5'-di-C{sup 14}]-VT (bleu de tetrazolium), en partant du benzaldehyde-[1-C{sup 14}]. On a pu obtenir un grand rendement, tant pour les sels de monotetrazolium que pour les sels de ditetrazolium, et des produits d'une activite specifique elevee. La purete des echantillons a ete examinee par chromatographie sur papier. Dans le cas des sels de ditetrazolium, on a pu deceler quelques impuretes et tirer des conclusions quant a leur structure et quantite. On a mis au point une methode permettant de preparer des sels de ditetrazolium marques au carbone-14 et presentant une grande purete. Dans la synthese du formazan, precurseur du sel de tetrazolium, on obtient un produit intermediaire instable d'une structure analogue a celle du tetrazene; ce produit se transforme rapidement en milieu alcalin pour donner du formazan. Le tetrazene intermediaire peut

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

    Science.gov (United States)

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

    2013-05-01

    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 clearance of pathogens and tissue regeneration processes. Therefore, the respiratory burst and associated ROS constitute important indicators of fish health status. This paper compares two methods for quantitation of ROS produced during the respiratory burst in common carp: the widely used, single-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 on a real-time scale at frequent and continual time intervals for time course experiments, providing more detailed information on the respiratory burst response. The real-time chemiluminescence assay was used to measure respiratory burst activity in macrophage and neutrophilic granulocyte-enriched head kidney cell fractions and total head kidney cell suspensions and proved to be a fast, reliable, automated multiwell microplate assay to quantitate fish health status modulated by β-glucans. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. Teste de tetrazólio para avaliação da qualidade fisiológica em sementes de Goiabeira-serrana (Acca sellowiana O. Berg Burret Tetrazolium test to evaluate physiological quality of Brazilian Guava seeds (Acca sellowiana O. Berg Burret

    Directory of Open Access Journals (Sweden)

    Marcelo Benevenga Sarmento

    2013-03-01

    Full Text Available A goiabeira-serrana, espécie frutífera nativa do Sul do Brasil, vem mostrando- se promissora em termos ecológicos e comerciais. O trabalho objetivou avaliar a qualidade fisiológica de dois lotes de sementes de goiabeira-serrana por meio do teste de tetrazólio. O experimento foi dividido em duas etapas. Na primeira, os lotes foram submetidos aos testes de germinação, índice de velocidade de germinação, tempo médio de germinação, emergência em casa de vegetação, índice de velocidade de emergência de plântulas em casa de vegetação, tempo médio de emergência e comprimento de parte aérea. Na segunda, foi realizado o teste de tetrazólio nas concentrações de 0,5 e 1,0, e tempos de embebição (2 h e 4 h. O tratamento 0,5 TZ 4h obteve 73% de sementes viáveis (lote 2007. Para o lote 2008, não houve diferenças entre os tratamentos. O teste de germinação apresentou correlação positiva para os tratamentos 0,5 TZ 2 h e 1 TZ 2 h. Para a emergência de plântulas em casa de vegetação, houve correlação positiva para o tratamento 0,5 TZ 2 h. O teste de tetrazólio permitiu a classificação dos lotes em quatro níveis de viabilidade, confirmando a eficiência do teste na avaliação da viabilidade de sementes de goiabeira-serrana.Brazilian guava, native fruit tree from Southern Brazil, has become relevant due to its potencial use as ecological and comercial. This article aims evaluate the physiological quality of Brazilian guava seeds by tetrazolium test. The work was divided in two parts. In the first, both lots were submitted to the germination test, index of speed germination, mean time of germination, emergency of seedlings in greenhouse, mean time of emergency and shoot length of seedlings. In the second part it was performed the tetrazolium test. Two concentrations of tetrazolium were used (0,5 e 1,0 and two times of imbibition (2 h e 4 h. To the lot 2007, the treatment 0,5 TZ 4 h obtained 73% of viable seeds

  9. 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...

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

    African Journals Online (AJOL)

    A quick analysis of the sludge activity method based on triphenyltetrazolium chloride-dehydrogenase activity (TTC-DHA) was developed to change the rule and status of the biological activity of the activated sludge in tomato paste wastewater treatment. The results indicate that dehydrogenase activity (DHA) can effectively ...

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

    African Journals Online (AJOL)

    1Department of Immunology, Faculty of Medical Laboratory Sciences,. Usmanu ... suppresses both innate and adaptive arms of immune response. .... function test .... manifestations of diseases associated with .... Robbins Basic Pathology (9th.

  12. Experimental and Theoretical Study of 1.5-Diamino-4-H-Tetrazolium Perchlorate

    National Research Council Canada - National Science Library

    Drake, Greg

    2004-01-01

    ... out. Experimental evidence strongly supports the protonation of a nitrogen atom of the tetrazole ring, including the structure observed in a single crystal x-ray diffraction study of the title compound...

  13. 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

    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...... of the method was studied using the newly developed radiochromic dye films as well as already existing ones. (C) 2000 Elsevier Science Ltd. All rights reserved....

  14. Evaluation of tetrazolium-based semiautomatic colorimetric assay for measurement of human antitumor cytotoxicity

    International Nuclear Information System (INIS)

    Heo, D.S.; Park, J.G.; Hata, K.; Day, R.; Herberman, R.B.; Whiteside, T.L.

    1990-01-01

    A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based colorimetric assay was developed and compared with 51Cr release from different adherent tumor cell targets (human squamous cell carcinoma lines of the head and neck established in our laboratory, melanoma, and colorectal carcinoma) using 5-7-day human lymphokine-activated killer cells and monocyte-depleted peripheral blood lymphocytes as effectors. With adherent tumor cell targets, MTT colorimetry was more sensitive than the 51Cr release assay in measuring the antitumor activity of effectors: median, 4385 (range, 988-8144) versus median, 1061 (range, 582-7294) lytic units (the number of effector cells required to lyse 20% of 5 x 10(3) targets)/10(7) effectors (P less than 0.01). Background effects (without effector cells) were comparable in 4-h assays (9% versus 10%) between MTT colorimetry and 51Cr release. In 24-h assays, MTT colorimetry showed higher antitumor activity (70-100% versus 40-60% lysis at 1:1 effector:target cell ratio) but lower background effects (6% versus 38%) than 51Cr release assay. Thus, MTT colorimetry was more sensitive, did not use radiolabeled targets, required fewer effector cells, and was easier, less expensive, and better adaptable to serial monitoring of effector cell function in cancer patients. This colorimetric assay is especially well suited to adherent tumor cell targets. The use of adherent tumor cell monolayers, as opposed to trypsinized single cell suspensions, provides an opportunity to measure interactions of effector cells with enzymatically unaltered solid tumor targets. Because of the greater sensitivity of the colorimetric assay, the transformation of MTT data into lytic units, as commonly used for 51Cr release assays, required an adjustment to avoid the extrapolation based on the exponential fit equation

  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. Reaction rate studies of glucose-6-phosphate dehydrogenase activity in sections of rat liver using four tetrazolium salts

    NARCIS (Netherlands)

    Butcher, R. G.; van Noorden, C. J.

    1985-01-01

    The reaction rate of glucose-6-phosphate dehydrogenase activity in liver sections from fed and starved rats has been monitored by the continuous measurement at 37 degrees C of the reaction product as it is formed using scanning and integrating microdensitometry. Control media lacked either substrate

  17. Pairing Heterocyclic Cations with closo-Icosahedral Borane and Carborane Anions, II: Benchtop Alternative Synthetic Methodologies for Binary Triazolium and Tetrazolium Salts with Significant Water Solubility (POSTPRINT)

    Science.gov (United States)

    2012-01-01

    in the stirbar=recovery flask apparatus, a proton NMR ( dimethylsulfoxide , DMSO -d6, solvent ) performed on the residue indicated essentially complete...of KX by-product that a given dry volume of silica gel might have retained using a reasonable volume of eluting solvent . For KCl, 0.4473 g (6.0mmol...collected after solvent removal. Scheme 2. Formation of the mixed mono-K=triazolium borane salt. WATER-SOLUBLE HETEROCYCLIUM BORANE-BASED SALTS 157 In this

  18. 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.

  19. In vitro screening of six anthelmintic plant products against larval Haemonchus contortus with a modified methyl-thiazolyl-tetrazolium reduction assay

    OpenAIRE

    Hördegen, P.; Cabaret, J.; Hertzberg, H.; Langhans, W.; Maurer, V.

    2006-01-01

    Because of the increasing anthelmintic resistance and the impact of conventional anthelmintics on the environment, it is important to look for alternative strategies against gastrointestinal nematodes. Phytotherapy could be one of the major options to control these pathologies. Extracts or ingredients of six different plant species were tested against exsheathed infective larvae of Haemonchus contortus using a modified methyl-thiazolyltetrazolium (MTT) reduction assay. Pyrantel tartrate was u...

  20. 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

  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. antiplasmodial activity of extracts of tridax procumbens and ...

    African Journals Online (AJOL)

    2011-12-01

    Dec 1, 2011 ... lines using a microculture tetrazolium assay. Cancer Res 1988; 48: 589-601. 17. Scudiero D.A., Shoemaker R.H., Paull K.D.. Monks A., Tierney S., Nofzifer T.H.,et. al. Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumour cell lines.

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

    African Journals Online (AJOL)

    ajl yemi

    2011-11-28

    Nov 28, 2011 ... and the nitroblue tetrazolium (NBT) assay. The cytotoxicity ... The antioxidant activity and cytotoxic effect of the extracts increased with increase ... supplements are concoctions of plants and/or plant .... In vitro antioxidant assay.

  4. Effects of water stress and seed mass on germination and ...

    African Journals Online (AJOL)

    enoh

    2012-03-01

    Mar 1, 2012 ... polyvinylpyrrolidone; TCA, trichloroacetic acid; NBT, nitroblue tetrazolium; TBA ..... seeds had weaker resistances to moderate stress (-0.6. MPa) compared with .... Chen YN,Chen YP,Li WH,Zhang HF (2003). Response of ...

  5. Reconfirmation of antimicrobial activity in the coelomic fluid of the ...

    Indian Academy of Sciences (India)

    Unknown

    723. Keywords. Antimicrobial activity; column chromatography; earthworm; Eisenia fetida andrei; Tetrazolium salt ... fair resistance against E. coli, P. aeruginosa and Arthrobacter sp., respectively. [Pan W, Liu X, Ge F ... 2.2 Test bacteria species.

  6. Attenuation of Neuroinflammatory Responses in Lipopolysaccharide ...

    African Journals Online (AJOL)

    Chenopodiaceae) extract on neuroinflammatory responses induced by lipopolysaccharide (LPS) in BV-2 microglial cells and its antioxidant effects. Methods: Biochemical studies carried out include 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyl-tetrazolium ...

  7. Crop physiological analysis of seed quality variation in common bean (Phaseolus vulgaris L.)

    NARCIS (Netherlands)

    Muasya, R.M.

    2001-01-01

    Keywords : Physiological maturity, harvest maturity, earliness, common bean, Phaseolus vulgaris L., morphological markers, variation, moisture content, dry weight, viability, vigour, electrical conductivity, tetrazolium, seed lot, seed

  8. The effect of Echis coloratus venom on biochemical and molecular ...

    African Journals Online (AJOL)

    Hazem K. Ghneim

    2017-03-28

    induced toxicity. It has been .... yellow tetrazolium salt, by the mitochondrial enzyme succinate ..... progressive loss of viability in a dose-dependent fashion .... and/or mitochondrial receptors, the mechanisms of .... Copper home-.

  9. In vitro germination and growth of babassu (orbygnia phalerata Mart ...

    African Journals Online (AJOL)

    CERRADONLINE

    2012-06-07

    Jun 7, 2012 ... 2Biological Sciences, Instituto Federal Goiano - Campus Rio Verde – GO, Brazil. 3Agricultural .... O. phalerata embryos subjected to the tetrazolium test. Bar = 2 cm. .... temperatures allows for the faster release of water from.

  10. INFLUENCE OF SEASONAL FACTORS ON OYSTER HEMOCYTE KILLING OF VIBRIO PARAHEMOLYTICUS

    Science.gov (United States)

    Seasonal variation of cellular defenses of oyster Crassostrea virginica against Vibrio parahaemolyticus were examined from June 1997 to December 1998 using a recently developed bactericidal assay that utilizes a tetrazolium dye. Mean hemocyte numbers, plasma lysozyme, and P. mari...

  11. Adhesive ability and biofilm metabolic activity of Listeria ...

    African Journals Online (AJOL)

    SWEET

    2012-07-31

    Jul 31, 2012 ... monocytogenes strains were able to adhere to abiotic materials with different degrees. In fact, cold stressed strains ... packaging. Biofilms allow .... reduction of a tetrazolium salt by metabolically active cells to a colored water ...

  12. Microscopic analysis of MTT stained boar sperm cells

    African Journals Online (AJOL)

    tulyasys

    2015-06-08

    2H-tetrazolium bromide is widely used for assessment of cytotoxicity, cell viability, and proliferation studies in cell biology (van Meerloo et al., 2011;. Stockert et al., 2012). The stain is abbreviated as MTT.

  13. Tripolar Mesoionic Compounds

    OpenAIRE

    Shuki, Araki; Jiro, Mizuya; Naomitsu, Aoyama; Yasuo, Butsugan

    1995-01-01

    Tripolar mesoionic compounds have been synthesized by the reaction of (1,3-diphenyltetrazol-5-ylio)cyclopentadienide (or -indenide) with the dicationic ether salts derived from mesoionic olates and trifluoromethanesulfonic anhydride. The structures of the newly prepared mesoionic systems are discussed on the basis of spectroscopic analysis; it is shown that the tripolar [tetrazolium-cyclopentadienide (or indenide)-tetrazolium] canonical structure contributes significantly to the ground state ...

  14. Computational Chemistry Toolkit for Energetic Materials Design

    Science.gov (United States)

    2006-11-01

    industry are aggressively engaged in efforts to develop multiscale modeling and simulation methodologies to model and analyze complex phenomena across...energetic materials design. It is hoped that this toolkit will evolve into a collection of well-integrated multiscale modeling methodologies...Experimenta Theoreticala This Work 1-5-Diamino-4- methyl- tetrazolium nitrate 8.4 41.7 47.5 1-5-Diamino-4- methyl- tetrazolium azide 138.1 161.6

  15. Continuous cytokine exposure of colonic epithelial cells induces DNA damage

    DEFF Research Database (Denmark)

    Seidelin, Jakob B; Nielsen, Ole Haagen

    2005-01-01

    tetrazolium bromide (MTT) test. Production of ROS was determined by the oxidation of 2',7'-dichlorodihydrofluorescein to a fluorescent 2',7'-dichlorofluorescein and measured by fluorescence reading and visualized by fluorescence microscopy. DNA stability was determined by single cell gel electrophoresis...

  16. Enzyme cytochemistry of unfixed leukocytes and bone marrow cells using polyvinyl alcohol for the diagnosis of leukemia

    NARCIS (Netherlands)

    van Noorden, C. J.; Vogels, I. M.; van Wering, E. R.

    1989-01-01

    Cytochemical methods for the demonstration of enzyme activities in blood and bone marrow cells were systematically improved by the addition of an inert polymer, polyvinyl alcohol (PVA), to the incubation medium and by using optimized reaction media. The methods investigated were tetrazolium salt

  17. 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 ...

  18. 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).

  19. Studies on seed germination and in vitro shoot multiplication of ...

    African Journals Online (AJOL)

    Yomi

    2011-12-21

    Dec 21, 2011 ... vitro seed germination and plantlet regeneration of this plant. ... medical books, a few varieties of herbs are described ... Seed viability was determined by tetrazolium (TZ) test. (Perry ..... Dictionary of Drugs and Difficult Terms of 'The Book of ... establishment of stem cuttings of oregano, pepermint and balm.

  20. Carboplatin- and cisplatin-induced potentiation of moderate-dose radiation cytotoxicity in human lung cancer cell lines

    NARCIS (Netherlands)

    Groen, H. J.; Sleijfer, S.; Meijer, C.; Kampinga, H. H.; Konings, A. W. T.; de Vries, E. G. E.; Mulder, N. H.

    1995-01-01

    The interaction between moderate-dose radiation and cisplatin or carboplatin was studied in a cisplatin-sensitive (GLC(4)) and -resistant (GLC(4)-CDDP) human small-cell lung cancer cell line. Cellular toxicity was analysed under oxic conditions with the microculture tetrazolium assay. For the

  1. 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,

  2. 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)

  3. Inhibition of Shiga Toxin 2 (Stx2) in Apple Juices and its Resistance to Pasteurization

    Science.gov (United States)

    In the present study, we evaluated Shiga toxin (Stx2) activity in apple juices by measuring a decrease in dehydrogenase activity of Vero cells with the microculture tetrazolium (MTT) assay. Freshly prepared juice from Red Delicious apples and Golden Delicious apples inhibited the biological activity...

  4. SYNTHESIS AND CYTOTOXICITY OF NOVEL LIGNANS

    NARCIS (Netherlands)

    Middel, O; Woerdenbag, H.J.; van Uden, W.; van Oeveren, A.; Jansen, J.F.G.A.; Feringa, B.L.; Konings, A.WT; Pras, N.; Kellogg, R.M

    1995-01-01

    In this study the syntheses of 11 novel lignans are described. Their cytotoxicities are studied in GLC(4), a human small cell lung carcinoma cell line, using the microculture tetrazolium (MTT) assay. Ten of these compounds were substituted with a menthyloxy group on the 5-position of the lactone.

  5. Cytotoxicity and mode of action of aeroplysinin-1 and a related dienone from the sponge Aplysina aerophoba

    NARCIS (Netherlands)

    Koulman, A; Proksch, P; Ebel, R; Beekman, AC; vanUden, W; Konings, AWT; Pedersen, JA; Pras, N; Woerdenbag, HJ

    Aeroplysinin-1 (1) and the structurally related dienone 2 were cytotoxic to Ehrlich ascites tumor (EAT) cells and HeLa tumor cells in the microculture tetrazolium (MTT) and clonogenic assays. Both compounds are bromotyrosine derivatives, isolated from the marine spong Aplysina aerophoba. As the

  6. Induction of respiratory deficiency in yeast by manganese, copper, cobalt and nickel

    Energy Technology Data Exchange (ETDEWEB)

    Lindegren, C C; Nagai, S; Nagai, H

    1958-08-16

    Among the chemical agents which induce respiratory deficiency in yeasts, acriflavine, triphenyl tetrazolium chloride, p-nitrophenol, and propamidine isethionate are especially effective in producing the deficiency in a large fraction of the surviving population. The present work is a survey of the efficacy of various metallic salts in inducing respiratory deficiency.

  7. In vitro germination and growth of babassu (orbygnia phalerata Mart ...

    African Journals Online (AJOL)

    CERRADONLINE

    2012-06-07

    Jun 7, 2012 ... samples were removed from the dry chambers to assess embryo viability using in vitro germination and the tetrazolium test, which was adapted from the method of Ribeiro et al. (2010). The experimental design was completely randomized, fully factorial (3 drying times × 2 drying temperatures) and used 4 ...

  8. Evaluation of chromosomal aberrations induced by hydralazine in ...

    African Journals Online (AJOL)

    Background and purpose: Hydralazine (HDZ) is a cardiovascular drug that is widely used to treat hypertension. The present study was done to assess the cytogenetic effects of HDZ on Chinese hamster ovary (CHO) cells. Materials and methods: Methylthiazol tetrazolium (MTT) assay was carried out to determine the half ...

  9. Computational Studies of Ionic Liquids

    National Research Council Canada - National Science Library

    Boatz, Jerry

    2004-01-01

    The structures and relative energies of the six possible N-protonated structures of the 1,5-diamino-1,2,3,4-tetrazolium cation have been computed at the B3LYP(3)/6-311G(d,p) and MP2/6-311G(d,p) levels of theory...

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

    African Journals Online (AJOL)

    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 components ...

  12. Effects of Allium hirtifolium (Iranian shallot) and its allicin on ...

    African Journals Online (AJOL)

    effects on nerve cell microtubules. MTs were prepared from sheep brain through two cycles of polymerization and depolymerization. The cell growth inhibition was measured by 3-(4,5-dimethylthiazol 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) after treatment with A. hirtifolium and its allicin on HeLa and MCF-7 and L-929 ...

  13. MODERATE CYTOTOXICITY OF PROANTHOCYANIDINS TO HUMAN TUMOR-CELL LINES

    NARCIS (Netherlands)

    KOLODZIEJ, H; HABERLAND, C; WOERDENBAG, HJ; KONINGS, AWT

    In the present study the cytotoxicity of 16 proanthocyanidins 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 ranging from 18 to >200 mu m following continuous

  14. CYTOTOXICITY OF ARTEMISININ-RELATED ENDOPEROXIDES TO EHRLICH ASCITES TUMOR-CELLS

    NARCIS (Netherlands)

    WOERDENBAG, HJ; MOSKAL, TA; PRAS, N; MALINGRE, TM; ELFERALY, FS; KAMPINGA, HH; KONINGS, AWT

    A series of artemisinin-related endoperoxides was tested for cytotoxicity to Ehrlich ascites tumor (EAT) cells using the microculture tetrazolium (MTT) assay. Artemisinin [1] had an IC50 value of 29.8 muM. Derivatives of dihydroartemisinin [2], being developed as antimalarial drugs (artemether [3],

  15. 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 ...

  16. Een methode ter bepaling van de respiratieaktiviteit in marien plankton

    NARCIS (Netherlands)

    Lambeck, R.H.D.

    1973-01-01

    The usefulness of a method, described by T.T. Packard (1971), for the determination of the potential respiratory rate in marine plankton, based on the use of tetrazolium dye, was tested. Especially the influence of a few aspects of the homogenisation procedure on the final results was investigated.

  17. Quantitative aspects of the cytochemical demonstration of glucose-6-phosphate dehydrogenase with tetranitro BT studied in a model system of polyacrylamide films

    NARCIS (Netherlands)

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

    1980-01-01

    The cytochemical determination of the activity of glucose-6-phosphate dehydrogenase (G6PDH) with tetranitro blue tetrazolium (TNBT) was studied with model films of polyacrylamide gel incorporating purified enzyme. This model system enabled a quantitative study to be made of different parameters

  18. 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 ...

  19. Anti-tumor potential of total alkaloid extract of Prosopis juliflora DC ...

    African Journals Online (AJOL)

    The total alkaloid extract from Prosopis juliflora DC. leaves was obtained using acid/base modified extraction method. The in vitro anti-tumor potential of the extract was evaluated using MTT (3-(4,5- dimethythiazol-2yl)2,5-diphenyl tetrazolium bromide) based cytotoxicity monitoring after 24, 48 and 72 h exposure of the ...

  20. Cytophotometry of glucose-6-phosphate dehydrogenase activity in individual cells

    NARCIS (Netherlands)

    van Noorden, C. J.; Tas, J.; Vogels, I. M.

    1983-01-01

    With the aid of thin films of polyacrylamide gel containing purified glucose-6-phosphate dehydrogenase subjected to cytochemical procedures for the enzyme using tetranitro blue tetrazolium, arbitrary units of integrated absorbance obtained with a Barr & Stroud GN5 cytophotometer were converted into

  1. 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 ...

  2. 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...

  3. PHYSIOLOGICAL POTENTIAL AND CONSERVATION OF MURUCI (Byrsonima crassifolia SEEDS

    Directory of Open Access Journals (Sweden)

    CAMILA RIBEIRO DE SOUZA GRZYBOWSKI

    2017-12-01

    Full Text Available ABSTRACT The goal of the present study is to assess the physiological potential of Byrsonima crassifoliaseeds and analyze the procedures of the germination and tetrazolium tests, as well as the conservation of such seeds by testing different combinations of packaging and environment. The experiment used pyrenes of the clone Açu, collected from mother plants in the germplasm collection of muruci plants of Embrapa Amazonia Oriental, in Belém, Brazil. Different temperatures were tested in the germination test (25, 30, 35 and 20 - 30 ºC. The best methodology and assessment dates were determined for the test, and the seedlings of the species were characterized. Viability was assessed with the tetrazolium test, with combinations of hydration methods (by immersion and between paper towels, staining periods (3 and 4 h and concentrations of tetrazolium solution (0.5, 0.7 and 1.0%. Storage of muruci pyrenes was evaluated at three, six and twelve months, in polyethylene and Kraft paper bags, under dry chamber and refrigerator conditions. The study used a randomized block design with four replicates. The data underwent analysis of variance. In conclusion, the physiological potential of seeds of B. crassifoliacan be assessed by the germination test conducted in sand, with alternating temperatures of 20-30 ºC, or with the use of the tetrazolium test (with staining for three hours in the tetrazolium solution at 1.0% as a rapid assessment of viability. Muruci pyrenes can be kept in polythene or paper Kraft bags for up to 12 months, in a dry chamber at 16 ºC and with relative air humidity of 50 to 60%.

  4. Evaluation of 5P12-RANTES analogue expression in Nicotiana benthamiana

    CSIR Research Space (South Africa)

    Mawela, KG

    2012-10-10

    Full Text Available 100 200 300 400 500 600 700 Apoplast Cytosol pICH11599 (-ve control) 5P 12 (? g /k g F W ) 3 dpi 6 dpi 9 dpi (B) Bacterial vector system 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Cytosol ER Chloroplast pICH11599 (-ve... MTS tetrazolium compound, {3-(4,5-dimethyl-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium} Efficacy testing Table 1 Summary of IC50 values of the HIV-1 pseudovirions screened. Sample identification ZM53 (targets CCR5) IC50 (?g...

  5. Leukocytes respiratory burst and lysozyme level in pacu (Piaractus mesopotamicus Holmberg, 1887

    Directory of Open Access Journals (Sweden)

    JS. Abreu

    Full Text Available Innate immune responses are useful to determine the health status of fish and to evaluate the effect of immunomodulatory substances in fish farming. Leukocytes respiratory burst was measured in pacu (Piaractus mesopotamicus using chemiluminescence assay and nitroblue tetrazolium (NBT reduction assay. The nitroblue tetrazolium reduction seemed more adequate than chemiluminescence assay for leukocytes oxidative burst determination, since it was difficult to isolate the blood leucocytes for chemiluminescence assay. Plasma and serum lysozyme were measured using a turbidimetric assay. The heating of serum and plasma samples (56 ºC for 30 minutes for complement system inactivation darkened the plasma samples and interfered in the results. The lysozyme activity in serum was higher than in plasma, suggesting that serum samples are more appropriate for the analysis. This study established protocols that can be useful tools in the study of immune mechanisms of the tropical fish pacu.

  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. Comparison of tests for the analysis of vigor and viability in soybean seeds and their relationship to field emergence - doi: 10.4025/actasciagron.v35i1.14955

    Directory of Open Access Journals (Sweden)

    Márcia Santorum

    2012-08-01

    Full Text Available The objective of this study was to compare tests used to identify vigor in soybean seeds and to establish a model that relates the seeds’ vigor, as determined in the laboratory, with field emergence. Five soybean cultivars were evaluated based on their germination, sand emergence, immersion in sodium hypochlorite, accelerated aging, controlled deterioration, cold test, seedling growth, electrical conductivity, tetrazolium, emergence percentage, seedling length in the field and seedling dry weight. The experimental design was completely randomized and means were compared by the Tukey test at 5% probability. Assays for accelerated aging, controlled deterioration and tetrazolium showed the closest correlation to field emergence. The regression model included the sand emergence speed, germination speed index and index of sand emergence speed as the variables that best correlated with field emergence.

  8. 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.

  9. 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 ...

  10. Pinitol suppresses tumor necrosis factor-α-induced invasion of ...

    African Journals Online (AJOL)

    Methods: Reverse transcription-polymerase chain reaction (RT-PCR) together with Western blot analysis was used to analyze the expression of MMP-9 and nuclear factor-κB (NF-κB) subunits, p65 and p50, in TNF-α- treated LNCaP cells, while 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, ...

  11. Total flavonoid of Litsea coreana leve exerts anti-oxidative effects and alleviates focal cerebral ischemia/reperfusion injury

    OpenAIRE

    Dong, Shuying; Tong, Xuhui; Li, Jun; Huang, Cheng; Hu, Chengmu; Jiao, Hao; Gu, Yuchen

    2013-01-01

    In this study, we hypothesized that total flavonoid of Litsea coreana leve (TFLC) protects against focal cerebral ischemia/reperfusion injury. TFLC (25, 50, 100 mg/kg) was administered orally to a rat model of focal ischemia/reperfusion injury, while the free radical scavenging agent, edaravone, was used as a positive control drug. Results of neurological deficit scoring, 2,3,5-triphenyl tetrazolium chloride staining, hematoxylin-eosin staining and biochemical tests showed that TFLC at differ...

  12. 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 ...

  13. 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...

  14. Activity and Phylogenetic Diversity of Bacterial Cells with High and Low Nucleic Acid Content and Electron Transport System Activity in an Upwelling Ecosystem

    OpenAIRE

    Longnecker, K.; Sherr, B. F.; Sherr, E. B.

    2005-01-01

    We evaluated whether bacteria with higher cell-specific nucleic acid content (HNA) or an active electron transport system, i.e., positive for reduction of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), were responsible for the bulk of bacterioplankton metabolic activity. We also examined whether the phylogenetic diversity of HNA and CTC-positive cells differed from the diversity of Bacteria with low nucleic acid content (LNA). Bacterial assemblages were sampled both in eutrophic shelf waters...

  15. Pre-clinical evaluation of extracts and essential oils from betel-like ...

    African Journals Online (AJOL)

    The extracts and oils were preclinically studied based on cyto - and genotoxicity using microculture tetrazolium (MTT) and comet assays. Results: The crude extracts showed an IC50 in leukocytes and HeLa cells of 58.59 -97.31 mg/ml and 34.91-101.79 mg/ml, the LD50 is higher than 5000 mg/kg. With lower values than the ...

  16. 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 ...

  17. A study on the thermal decomposition behavior of derivatives of 1,5-diamino-1H-tetrazole (DAT): A new family of energetic heterocyclic-based salts

    International Nuclear Information System (INIS)

    Fischer, Gerd; Holl, Gerhard; Klapoetke, Thomas M.; Weigand, Jan J.

    2005-01-01

    The thermal decomposition of the highly energetic 1,5-diamino-4-methyl-1H-tetrazolium nitrate (2b), 1,5-diamino-4-methyl-1H-tetrazolium dinitramide (2c) and 1,5-diamino-4-methyl-1H-tetrazolium azide (2d) were investigated by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). Mass spectrometry and IR spectroscopy were used to identify the gaseous products. Decomposition appears in the cases of 2c and 2d to be initiated by a proton transfer to form the corresponding acid HN 3 and HN 3 O 4 whereas in the case of 2b a methyl group transfer to MeONO 2 is observed as initial process. The gaseous products after the exothermic decomposition are comparable and are in agreement of the possible decomposition pathways discussed for the corresponding compounds. For all processes, possible decomposition schemes are presented. The decomposition temperatures of 2b and 2c are significantly higher than that of 2d and were supported by evaluation the values of the activation energy according the method of Ozawa and Kissinger

  18. 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.

  19. Metabolism in the Uncultivated Giant Sulfide-Oxidizing Bacterium Thiomargarita Namibiensis Assayed Using a Redox-Sensitive Dye

    Science.gov (United States)

    Bailey, J.; Flood, B.; Ricci, E.

    2014-12-01

    The colorless sulfur bacteria are non-photosynthetic chemolithotrophs that live at interfaces between nitrate, or oxygen, and hydrogen sulfide. In sulfidic settings such as cold seeps and oxygen minimum zones, these bacteria are thought to constitute a critical node in the geochemical cycling of carbon, sulfur, nitrogen, and phosphorous. Many of these bacteria remain uncultivated and their metabolisms and physiologies are incompletely understood. Thiomargarita namibiensis is the largest of these sulfur bacteria, with individual cells reaching millimetric diameters. Despite the current inability to maintain a Thiomargarita culture in the lab, their large size allows for individual cells to be followed in time course experiments. Here we report on the novel use of a tetrazolium-based dye that measures the flux of NADH production from catabolic pathways via a colorimetric response. Staining with this dye allows for metabolism to be detected, even in the absence of observable cell division. When coupled to microscopy, this approach also allows for metabolism in Thiomargaritato be differentiated from that of epibionts or contaminants in xenic samples. The results of our tetrazolium dye-based assay suggests that Thiomargarita is the most metabolically versatile under anoxic conditions where it appears capable of using acetate, succinate, formate, thiosulfate, citrate, thiotaurine, hydrogen sulfide, and perhaps hydrogen as electron donors. Under hypoxic conditions, staining results suggest the utilization of acetate, citrate, and hydrogen sulfide. Cells incubated under oxic conditions showed the weakest tetrazolium staining response, and then only to hydrogen sulfide and questionably succinate. These initial results using a redox sensitive dye suggest that Thiomargarita is most metabolically versatile under anaerobic and hypoxic conditions. The results of this assay can be further evaluated using molecular approaches such as transcriptomics, as well as provide cultivation

  20. 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.

  1. Histochemical Localization of Glutathione Dependent NBT-Reductase in Mouse Skin

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective 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. Methods The fresh frozen tissue sections (8m thickness) were prepared and incubated in medium containing NBT, reduced glutathione (GSH) and phosphate buffer. The staining for GSH was performed with mercury orange. Results  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. Conclusion The activity of the NBT-reductase seems to be dependent on the glutathione contents.

  2. Effect of radon inhalations on certain oxyda-reductive enzymes in adrenols of white rats

    International Nuclear Information System (INIS)

    Robaczynski, J.; Kaplonska, J.; Lozinska, E.

    1974-01-01

    Histochemical investigations were carried out on adrenals of white rats after radon inhalations from inhalers in Swieradow-spa. Increased reactions of oxydo-reductive enzymes: NAD tetrazolium reductase, succinic dehydrogenase and glucose-6-phosphate dehydrogenase were observed in the adrenal cortex, particularly in the zona reticularis which was hypertrophied. Raised activity of oxydo-reductive enzymes in the cells of adrenal cortex evidences increased metabolism in these cells which may reflect increased production of hormones. Finding of stimulation of adrenocortical cells after radon inhalations is of essential importance for explanation of the biological mechanism of action of radon used in balneotherapy. (author)

  3. Nickel aggregates produced by radiolysis

    International Nuclear Information System (INIS)

    Marignier, J.L.; Belloni, J.

    1988-01-01

    Nickel aggregates with subcolloidal size and stable in water have been synthesized by inhibiting the corrosion by the medium. The protective effect of the surfactant is discussed in relation with the characteristics of various types of polyvinyl alcohol studied. The reactivity of aggregates towards oxidizing compounds, nitro blue tetrazolium, methylene blue, silver ions, oxygen, methylviologen, enables an estimation of the redox potential of nickel aggregates (E = - 04 ± 0.05 V). It has been applied to quantitative analysis of the particles in presence of nickel ions. 55 refs [fr

  4. Synthesis and In Vitro Antiproliferative Activity of Novel Androst-5-ene Triazolyl and Tetrazolyl Derivatives

    Directory of Open Access Journals (Sweden)

    János Wölfling

    2011-06-01

    Full Text Available A straightforward and reliable method for the regioselective synthesis of steroidal 1,4-disubstituted triazoles and 1,5-disubstituted tetrazoles via copper(I-catalyzed cycloadditions is reported. Heterocycle moieties were efficiently introduced onto the starting azide compound 3β-acetoxy-16β-azidomethylandrost-5-en-17β-ol through use of the “click” chemistry approach. The antiproliferative activities of the newly-synthesized triazoles were determined in vitro on three human gynecological cell lines (HeLa, MCF7 and A2780 using the microculture tetrazolium assay.

  5. Synthesis and Anticancer Activities of Novel 1,4-Disubstituted Phthalazines

    Directory of Open Access Journals (Sweden)

    Ping Gong

    2006-07-01

    Full Text Available A series of novel 1-anilino-4-(arylsulfanylmethylphthalazines were designed and synthesized. The structures of all the compounds were confirmed by IR, 1H-NMR, elemental analysis and MS. The analogues 1-(3-chloro-4-fluoroanilino-4-(3,4- difluorophenylthio-methylphthalazine (12 and 1-(4-fluoro-3-trifluoromethylanilino-4- (3,4-difluorophenyl-thiomethylphthalazine (13 showed higher activity than a cisplatin control when tested in vitro against two different cancer cell lines using the microculture tetrazolium method (MTT method.

  6. Cytotoxic activity of methanol extracts from Basidiomycete mushrooms on murine cancer cell lines.

    Science.gov (United States)

    Tomasi, S; Lohézic-Le Dévéhat, F; Sauleau, P; Bézivin, C; Boustie, J

    2004-04-01

    Crude methanol extracts of 58 mushroom species were screened for their cytotoxic activities against two murine cancer cell lines, L1210 and 3LL, using the tetrazolium assay. A majority of extracts (74%) exhibited IC50 > 100 microg/ml against both cell lines. A most marked activity against one of the cell lines was noted for nine species (14% of the tested species). While Amanitales and Russulales tested were not found active, Polyporales and Boletales gave better results. Four species exhibited a significant cytotoxic activity (IC50 Suillus granulatus, S. luteus). The last one had never been investigated for its cytotoxic compounds before.

  7. Influence of UV-radiation on granulocytic phagocytosis in vitro

    International Nuclear Information System (INIS)

    Walther, T.; Rytter, M.; Gast, W.; Haustein, U.F.

    1987-01-01

    The influence of UV radiation on the vitality, the performance of phagocytosis and the ability to reduce nitro-blue tetrazolium test (NBT) by human granulocytes was investigated in vitro. Already by low doses of UVA (8% UVB) the percentage of phagocytizing granulocytes was decreased more distinctly than their cell vitality. The number of ingested Candida albicans particles was 4.5 particles per granulocyte in the controls. It was reduced to about 1.4 particles per cell by UV radiation independent of the dosis applied. On the other hand the ability of granulocytes to reduce NBT intracellularly remained completely unchanged. (author)

  8. Effect of selected insecticides on SF9 insect cell line

    International Nuclear Information System (INIS)

    Saleh, M.; Rahmo, A.; Hajjar, J.

    2013-01-01

    The toxic effect of three insecticides: dimethoate (organophosphate insecticide), acetamiprid (neonicotinoid insecticide) and deltamethrin (pyrethroid insecticide) were evaluated in vitro on cultured Sf9 cell line. Cell growth inhibition was measured by the 3- (4,5- dimethylthiazol - 2-yl) - 2,5 - diphenyl tetrazolium bromide (MTT) assay. Regression Analysis was used to estimate the 20% inhibition of cells growth (IC 20). The IC 20 values obtained for deltamethrin, acetamipridand dimethoate were: 46.8, 61.6 and 68.9 μM, respectively. The proportion of phagocytic cells was positively correlated with the applied concentrations of the insecticides. (author)

  9. 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.

  10. Effect of coumarins on HL-60 cell differentiation.

    Science.gov (United States)

    Kawaii, S; Tomono, Y; Katase, E; Ogawa, K; Yano, M

    2000-01-01

    Twenty-eight coumarins, including 7 furocoumarins, were examined for their activity of induction of terminal differentiation of human promyelocytic leukemia cells (HL-60) by nitro blue tetrazolium (NBT) reducing, nonspecific esterase, specific esterase and phagocytic activities. Esculetin, nordalbergin, 6,7-dihydroxy-4-methylcoumarin and imperatorin had strong activity among the coumarins examined. HL-60 cells treated with these coumarins differentiated into mature monocyte/macrophage. The structure-activity relationship established from the results revealed that 6,7-dihydroxy moiety had an important role in the induction of differentiation of HL-60.

  11. Isolation of furocoumarins from bergamot fruits as HL-60 differentiation-inducing compounds.

    Science.gov (United States)

    Kawaii, S; Tomono, Y; Katase, E; Ogawa, K; Yano, M

    1999-10-01

    The HL-60 differentiation-inducing compounds in bergamot fruits were isolated with column chromatography and identified as bergamottin, bergapten, and citropten by (1)H and (13)C NMR. Their HL-60 differentiation-inducing activity was measured by examining nitro blue tetrazolium (NBT) reducing, nonspecific acid esterase (NSE), specific esterase (SE), and phagocytic activities, and bergamottin showed the strongest activity among the coumarins isolated from bergamot fruits. The structure-activity relationship obtained from HL-60 differentiation assay suggests that hydrophobicity of furocoumarins is correlated with their activity.

  12. 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...... as the formazan-like dye is released spontaneously into the medium, making it possible to perform several measurements on the same parasite culture without disturbing or killing the parasites. The measurements were shown to reflect the numbers of parasites as confirmed via comparative experiments using...

  13. Inhibition of histone deacetylases prevents cytokine-induced toxicity in beta cells

    DEFF Research Database (Denmark)

    Larsen, L; Tonnesen, M; Ronn, S G

    2007-01-01

    B (NFkappaB) is a critical signalling molecule in inflammation and is required for expression of the gene encoding inducible NO synthase (iNOS) and of pro-apoptotic genes. NFkappaB has recently been shown to associate with chromatin-modifying enzymes histone acetyltransferases and histone...... by immunoblotting and by immunoblotting combined with electrophoretic mobility shift assay, respectively. Viability was analysed by 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl-tetrazolium bromide and apoptosis by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay and histone...

  14. 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.

  15. Biosurfactants prevent in vitro Candida albicans biofilm formation on resins and silicon materials for prosthetic devices.

    Science.gov (United States)

    Cochis, Andrea; Fracchia, Letizia; Martinotti, Maria Giovanna; Rimondini, Lia

    2012-06-01

    The aim of this study was to evaluate in vitro the preventive antiadhesion activity of biosurfactants against Candida albicans biofilm. Disks of silicon and acrylic resin for denture prostheses were precoated with increasing concentrations of biosurfactants obtained from endophyte biofilms selected from Robinia pseudoacacia and from Nerium oleander, and afterward infected with C. albicans cells. The number of biofilm cells were detected by colony-forming unit (CFU) counting, cell viability was established by the 2,3-bis(2-methoxy-4-nitro-5-sulphophenyl)-5-[(phenyl amino)carbonyl]-2H-tetrazolium hydroxide (XTT) assay, and biosurfactant cytotoxicity was evaluated by the [3-(4,5-dimethyliazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium] (MTT) assay. Chlorhexidine was used as control. Precoating with biosurfactants caused a greater reduction (P biosurfactants was observed at low concentrations (78.12 μg/mL and 156.12 μg/mL) which were noncytotoxic. This study demonstrated the preventive antiadhesion activity of biosurfactants against C. albicans biofilm. These agents are amphiphilic, interfere with microbial adhesion, and demonstrate cycompatibility with epithelial cells and fibroblasts. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. A comparative study of three cytotoxicity test methods for nanomaterials using sodium lauryl sulfate.

    Science.gov (United States)

    Kwon, Jae-Sung; Kim, Kwang-Mahn; Kim, Kyoung-Nam

    2014-10-01

    The biocompatibility evaluation of nanomaterials is essential for their medical diagnostic and therapeutic usage, where a cytotoxicity test is the simplest form of biocompatibility evaluation. Three methods have been commonly used in previous studies for the cytotoxicity testing of nanomaterials: trypan blue exclusion, colorimetric assay using water soluble tetrazolium (WST), and imaging under a microscope following calcein AM/ethidium homodimer-1 staining. However, there has yet to be a study to compare each method. Therefore, in this study three methods were compared using the standard reference material of sodium lauryl sulfate (SLS). Each method of the cytotoxicity test was carried out using mouse fibroblasts of L-929 exposed to different concentrations of SLS. Compared to the gold standard trypan blue exclusion test, both colorimetric assay using water soluble tetrazolium (WST) and imaging under microscope with calcein AM/ethidium homodimer-1 staining showed results that were not statistically different. Also, each method exhibited various advantages and disadvantages, which included the need of equipment, time taken for the experiment, and provision of additional information such as cell morphology. Therefore, this study concludes that all three methods of cytotoxicity testing may be valid, though careful consideration will be needed when selecting tests with regard to time, finances, and the amount of information required by the researcher(s).

  17. Menadione-mediated WST1 reduction assay for the determination of metabolic activity of cultured neural cells.

    Science.gov (United States)

    Stapelfeldt, Karsten; Ehrke, Eric; Steinmeier, Johann; Rastedt, Wiebke; Dringen, Ralf

    2017-12-01

    Cellular reduction of tetrazolium salts to their respective formazans is frequently used to determine the metabolic activity of cultured cells as an indicator of cell viability. For membrane-impermeable tetrazolium salts such as WST1 the application of a membrane-permeable electron cycler is usually required to mediate the transfer of intracellular electrons for extracellular WST1 reduction. Here we demonstrate that in addition to the commonly used electron cycler M-PMS, menadione can also serve as an efficient electron cycler for extracellular WST1 reduction in cultured neural cells. The increase in formazan absorbance in glial cell cultures for the WST1 reduction by menadione involves enzymatic menadione reduction and was twice that recorded for the cytosolic enzyme-independent WST1 reduction in the presence of M-PMS. The optimized WST1 reduction assay allowed within 30 min of incubation a highly reliable detection of compromised cell metabolism caused by 3-bromopyruvate and impaired membrane integrity caused by Triton X-100, with a sensitivity as good as that of spectrophotometric assays which determine cellular MTT reduction or lactate dehydrogenase release. The short incubation period of 30 min and the observed good sensitivity make this optimized menadione-mediated WST1 reduction assay a quick and reliable alternative to other viability and toxicity assays. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. 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.

  19. [Contribution of blue-green pigments to hemolytic activity of Pseudomonas aeruginosa cultural fluid].

    Science.gov (United States)

    Pyzh, A É; Nikandrov, V N

    2011-01-01

    To assess the contribution of blue-green pigments of Pseudomonas aeruginosa to hemolytic activity of its cultural fluid. MATERIALS AND METHODS. Eight hospital strains and reference strain ATCC 15442 were used. Growth dynamics of strains as well as features of accumulation of hemolytic and phospholipase activity were studied. Purified samples of pyoverdin and pyocyanin were extracted by gel-chromatography and chloroform extraction methods. Hemolytic and lecitinase activities of the samples as well as effect of active oxygen scavengers and chelating agents on these activities were studied. Dynamics of accumulation of hemolytic activity significantly differed from that of phospholipase activity when strains were grown in liquid medium. Chromatographic separation of the pigments from cultural fluid supernatants sharply reduced its hemolytic activity. Purified samples of pyoverdin and pyocyanin were capable to lyse erythrocytes and chicken egg lecitin. These characteristics of the pigments were inhibited by nitroblue tetrazolium and sensitive to chelating agents. Conclusion. Pyoverdin and pyocyanin of pathogenic strains of P. aeruginosa are capable to lyse erythrocytes and suspension of purified chicken egg lecitin, they contribute to total hemolytic activity of pathogenic strains of Pseudomonas, which is not determined only by phospholipase C produced by microorganism. Lytic activity of the pigments is blocked by nitroblue tetrazolium and susceptible to some chelating agents. Apparently, this activity is mediated by superoxide radical and determined by presence of metals with transient valence in pigments' molecules.

  20. A new microtitre plate screening method for evaluating the viability of aerobic respiring bacteria in high surface biofilms.

    Science.gov (United States)

    Pérez, L M; Alvarez, B L; Codony, F; Fittipaldi, M; Adrados, B; Peñuela, G; Morató, J

    2010-09-01

    It is difficult to determine the effects of bactericidal compounds against bacteria in a biofilm because classical procedures for determining cell viability require several working days, multiple complicated steps and are frequently only applicable to cells in suspension. We attempt to develop a compact, inexpensive and versatile system to measure directly the extent of biofilm formation from water systems and to determine the viability of respiring bacteria in high surface biofilms. It has been reported that the reduction of tetrazolium sodium salts, such as XTT (sodium 3,3'-[1-[(phenylamino)carbonyl]-3,4-tetrazolium]Bis(4-methoxy)-6-nitro)benzene sulfonic acid hydrate), during active bacterial metabolism can be incorporated into a colorimetric method for quantifying cell viability. XTT is reduced to a soluble formazan compound during bacterial aerobic metabolism such that the amount of formazan generated is proportional to the bacterial biomass. We show here, for the first time, that this colorimetric approach can be used to determine the metabolic activity of adherent aerobic bacteria in a biofilm as a measure of cell viability. This technique has been used to estimate viability and proliferation of bacteria in suspension, but this is the first application to microbial communities in a real undisturbed biofilm. This simple new system can be used to evaluate the complex biofilm community without separating the bacteria from their support. Thus, the results obtained by this practice may be more representative of the circumstances in a natural system, opening the possibility to multiple potential applications.

  1. A novel, colorimetric neutralization assay for measuring antibodies to influenza viruses.

    Science.gov (United States)

    Lehtoranta, Liisa; Villberg, Anja; Santanen, Riitta; Ziegler, Thedi

    2009-08-01

    A colorimetric cell proliferation assay for measuring neutralizing antibodies to influenza viruses in human sera is described. Following a 90-min incubation, the serum-virus mixture was transferred to Madin-Darby canine kidney cells cultured in 96-well plates. After further incubation for three days, a tetrazolium salt was added to the wells. Cellular mitochondrial dehydrogenases cleave the tetrazolium salt to formazan, and the resulting color change is read by a spectrophotometer. The absorbance values correlate directly to the number of viable cells in the assay well and thus also to the neutralizing activity of influenza-specific antibodies present in the serum. With the few hands-on manipulations required, this assay allows simultaneous testing of a considerable number of sera, offers opportunities for automation, and is suitable for use under biosafety level-3 conditions. The test was used to study the antibody response after the administration of seasonal, inactivated, trivalent influenza vaccine. Antibody titers determined by the neutralization test in pre- and post-vaccination serum pairs were compared with those obtained by the hemagglutination inhibition assay. The neutralization test yielded higher pre- and post-vaccination titers and a larger number of significant increases in post-vaccination antibody titer than the hemagglutination inhibition test. This new test format could serve as a valuable laboratory tool for influenza vaccine studies.

  2. The MTT assays of bovine retinal pericytes and human microvascular endothelial cells on DLC and Si-DLC-coated TCPS.

    Science.gov (United States)

    Okpalugo, T I T; McKenna, E; Magee, A C; McLaughlin, J; Brown, N M D

    2004-11-01

    MTT (Tetrazolium)-assay suggests that diamond-like carbon (DLC) and silicon-doped DLC (Si-DLC) films obtained under appropriate deposition parameters are not toxic to bovine retinal pericytes, and human microvascular endothelial cells (HMEC). The observed frequency distributions of the optical density (OD) values indicative of cell viability are near Gaussian-normal distribution. One-way ANOVA indicates that at 0.05 levels the population means are not significantly different for the coated and control samples. The observed OD values depend on the cell line (cell growth/metabolic rate), possibly cell cycle stage, the deposition parameters-bias voltage, ion energy, pressure, argon precleaning, and the dopant. For colored thin films like DLC with room temperature photoconductivity and photoelectric effects, it is important to account for the OD contribution from the coating itself. MTT assay, not surprisingly, seems not to be highly sensitive to interfacial cellular interaction resulting from the change in the film's nanostructure, because the tetrazolium metabolism is mainly intracellular and not interfacial. The thin films were synthesized by 13.56 MHz RF-PECVD using argon and acetylene as source gases, with tetramethylsilane (TMS) vapor introduced for silicon doping. This study could be relevant to biomedical application of the films in the eye, peri-vascular, vascular compartments, and for cell-tissue engineering. (c) 2004 Wiley Periodicals, Inc.

  3. Oxygen-Poor Microzones as Potential Sites of Microbial N2 Fixation in Nitrogen-Depleted Aerobic Marine Waters

    Science.gov (United States)

    Paerl, Hans W.; Prufert, Leslie E.

    1987-01-01

    The nitrogen-deficient coastal waters of North Carolina contain suspended bacteria potentially able to fix N2. Bioassays aimed at identifying environmental factors controlling the development and proliferation of N2 fixation showed that dissolved organic carbon (as simple sugars and sugar alcohols) and particulate organic carbon (derived from Spartina alterniflora) additions elicited and enhanced N2 fixation (nitrogenase activity) in these waters. Nitrogenase activity occurred in samples containing flocculent, mucilage-covered bacterial aggregates. Cyanobacterium-bacterium aggregates also revealed N2 fixation. In all cases bacterial N2 fixation occurred in association with surficial microenvironments or microzones. Since nitrogenase is oxygen labile, we hypothesized that the aggregates themselves protected their constituent microbes from O2. Microelectrode O2 profiles revealed that aggregates had lower internal O2 tensions than surrounding waters. Tetrazolium salt (2,3,5-triphenyl-3-tetrazolium chloride) reduction revealed that patchy zones existed both within microbes and extracellularly in the mucilage surrounding microbes where free O2 was excluded. Triphenyltetrazolium chloride reduction also strongly inhibited nitrogenase activity. These findings suggest that N2 fixation is mediated by the availability of the appropriate types of reduced microzones. Organic carbon enrichment appears to serve as an energy and structural source for aggregate formation, both of which were required for eliciting N2 fixation responses of these waters. Images PMID:16347337

  4. 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.

  5. 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

  6. 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.

  7. Antibacterial Activity of Azadirachta indica, Pongamia pinnata, Psidium guajava, and Mangifera indica and their mechanism of action against Streptococcus mutans.

    Science.gov (United States)

    Bodiba, Dikonketso Cathrine; Prasad, Preety; Srivastava, Ajay; Crampton, Brigdet; Lall, Namrita Sharan

    2018-01-01

    Curative plants have reportedly been used to make chewing sticks/toothbrushes intended for the treatment of oral diseases. The in vitro antibacterial activities of Azadirachta indica , Pongamia pinnata , Psidium guajava , and Mangifera indica were evaluated against Streptococcus mutans , along with the cytotoxicity and antioxidant and synergistic potentials. The effect of M. indica on the expression of crucial virulence genes spaP and gtfB of S. mutans was determined. The antibacterial activity was determined using a modified microdilution method. The antioxidant potential was evaluated using diphenyl picrylhydrazyl (DPPH), Griess reagent, and nitroblue tetrazolium calorimetric assays. The synergistic activity was investigated using a modified checkerboard method, while the cytotoxicity was determined according to a cell proliferation 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt assay. Reverse transcription was the chosen method for determining the difference in expression of the spaP and gtfB genes after treatment with the plant sample. M. indica and A. indica had the highest antibacterial activity at concentrations of 0.3 mg/ml and 6.25 mg/ml, respectively. A. indica had the best free radical scavenging of DPPH, exhibiting 50% inhibition at 28.72 μg/ml; while M. indica showed better superoxide scavenging potential than the positive control quercetin. Both M. indica and A. indica had adequate activity against the nitric oxide-free radical (12.87 and 18.89 μg/ml, respectively). M. indica selectively reduced the expression of the gtfB gene, indicating a mechanism involving Glucotranferases, specifically targeting bacterial attachment. Mangifera indica and Azadirachta indica had very good antibacterial activity against Streptococcus mutans and moderate toxicity against Vero cells M. indica had the best antioxidant capacity overall M. indica reduced the expression of gtfB gene at 0.5 mg/ml. Abbreviations used : AA: Ascorbic acid; BHI

  8. 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.

  9. 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.

  10. Microstructure and osteoblast response of gradient bioceramic coating on titanium alloy fabricated by laser cladding

    International Nuclear Information System (INIS)

    Zheng Min; Fan Ding; Li Xiukun; Li Wenfei; Liu Qibin; Zhang Jianbin

    2008-01-01

    To construct a bioactive interface between metal implant and the surrounding bone tissue, the gradient calcium phosphate bioceramic coating on titanium alloy (Ti-6Al-4V) was designed and fabricated by laser cladding. The results demonstrated that the gradient bioceramic coating was metallurgically bonded to the titanium alloy substrate. The appearance of hydroxyapatite and β-tricalcium phosphate indicated that the bioactive phases were synthesized on the surface of coating. The microhardness gradually decreased from the coating to substrate, which could help stress relaxation between coating and bone tissue. Furthermore, the methyl thiazolyl tetrazolium (MTT) assay of cell proliferation revealed that the laser-cladded bioceramic coating had more favorable osteoblast response compared with the surface of untreated titanium alloy substrate

  11. Bioactivity of calcium phosphate bioceramic coating fabricated by laser cladding

    Science.gov (United States)

    Zhu, Yizhi; Liu, Qibin; Xu, Peng; Li, Long; Jiang, Haibing; Bai, Yang

    2016-05-01

    There were always strong expectations for suitable biomaterials used for bone regeneration. In this study, to improve the biocompatiblity of titanium alloy, calcium phosphate bioceramic coating was obtained by laser cladding technology. The microstructure, phases, bioactivity, cell differentiation, morphology and resorption lacunae were investigated by optical microscope (OM), x-ray diffraction (XRD), methyl thiazolyl tetrazolium (MTT) assay, tartrate-resistant acid phosphatase (TRAP) staining and scanning electronic microscope (SEM), respectively. The results show that bioceramic coating consists of three layers, which are a substrate, an alloyed layer and a ceramic layer. Bioactive phases of β-tricalcium phosphate (β-TCP) and hydroxyapatite (HA) were found in ceramic coating. Osteoclast precursors have excellent proliferation on the bioceramic surface. The bioceramics coating could be digested by osteoclasts, which led to the resorption lacunae formed on its surface. It revealed that the gradient bioceramic coating has an excellent bioactivity.

  12. Bioactivity of calcium phosphate bioceramic coating fabricated by laser cladding

    International Nuclear Information System (INIS)

    Zhu, Yizhi; Liu, Qibin; Xu, Peng; Li, Long; Jiang, Haibing; Bai, Yang

    2016-01-01

    There were always strong expectations for suitable biomaterials used for bone regeneration. In this study, to improve the biocompatiblity of titanium alloy, calcium phosphate bioceramic coating was obtained by laser cladding technology. The microstructure, phases, bioactivity, cell differentiation, morphology and resorption lacunae were investigated by optical microscope (OM), x-ray diffraction (XRD), methyl thiazolyl tetrazolium (MTT) assay, tartrate-resistant acid phosphatase (TRAP) staining and scanning electronic microscope (SEM), respectively. The results show that bioceramic coating consists of three layers, which are a substrate, an alloyed layer and a ceramic layer. Bioactive phases of β-tricalcium phosphate (β-TCP) and hydroxyapatite (HA) were found in ceramic coating. Osteoclast precursors have excellent proliferation on the bioceramic surface. The bioceramics coating could be digested by osteoclasts, which led to the resorption lacunae formed on its surface. It revealed that the gradient bioceramic coating has an excellent bioactivity. (letter)

  13. 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.

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

    International Nuclear Information System (INIS)

    Vieira, J.M.B.D.; Seabra, S.H.; Vallim, D.C.; Americo, M.A.; Fracallanza, S.E.L.; Vommaro, R.C.; Domingues, R.M.C.P.

    2009-01-01

    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.

  15. Viability test of fish scale collagen (Oshpronemus gouramy on baby hamster kidney fibroblasts-21 fibroblast cell culture

    Directory of Open Access Journals (Sweden)

    Chiquita Prahasanti

    2018-04-01

    Full Text Available Aim: This study aims to examine the toxicity of collagen extracted from gouramy fish scales (Oshpronemus gouramy by evaluating its viability against baby hamster kidney fibroblasts-21. Materials and Methods: Collagen was extracted from gouramy fish scales (O. gouramy with 6% acetic acid. Its results were analyzed using Fourier-transform infrared spectroscopy and freeze-dried technique. Its morphology then was analyzed with scanning electron microscope. Afterward, 3-(4.5-dimethylthiazole-2-yl2.5-diphenyl tetrazolium bromide assay was conducted to compare cells with and without fish scale collagen treatment. Results: Collagen extracted from gouramy fish scales had no influence statistically on cultured fibroblast cells with a statistical significance (2-tailed value of 0.754 (p>00025. Conclusion: Collagen extracted from gouramy fish scales has high viability against BHK21 fibroblast cells.

  16. Cytotoxic activity of ethanolic extract of the marine sponge Aaptos suberitoides against T47D cell

    Science.gov (United States)

    Nurhayati, Awik Puji Dyah; Prastiwi, Rarastoeti; Sukardiman, Wahyuningsih, Tri

    2018-04-01

    Aaptos suberitoides marine sponge produce many kinds of secondary metabolites. The purpose of this study were to examine the cytotoxic, proliferation inhibition and apoptosis induction of marine sponge A.suberitoides. The sponge was extracted with 96 % ethanol. Ethanol extract cytotoxicity assay were performed with MTT method (Microculture Tetrazolium) against to cell line of T47D. The proliferation inhibition were done by doubling time. The apoptosis induction by observing the treated cell morphology after staining with acrydine orange. The results show that cytotoxic activity of the ethanol extract was 153.109 µg/mL, inhibits cell proliferation cell lines of T47D at 24 hours of incubation and apoptosis induction.

  17. Preliminary in vitro evaluation of the anti-proliferative activity of guanylhydrazone derivatives.

    Science.gov (United States)

    França, Paulo H B; Da Silva-Júnior, Edeildo F; Aquino, Pedro G V; Santana, Antônio E G; Ferro, Jamylle N S; De Oliveira Barreto, Emiliano; Do Ó Pessoa, Cláudia; Meira, Assuero Silva; De Aquino, Thiago M; Alexandre-Moreira, Magna S; Schmitt, Martine; De Araújo-Júnior, João X

    2016-03-01

    Guanylhydrazones have shown promising antitumor activity in preclinical tumor models in several studies. In this study, we aimed at evaluating the cytotoxic effect of a series of synthetic guanylhydrazones. Different human tumor cell lines, by including HCT-8 (colon carcinoma), MDA-MB-435 (melanoma) and SF-295 (glioblastoma) were continuous exposed to guanylhydrazone derivatives for 72 hours and growth inhibition of tumor cell lines and macrophages J774 was measured using tetrazolium salt (MTT) assay. Compounds 7, 11, 16 and 17 showed strong cytotoxic activity with IC50 values lower than 10 μmol L(-1) against four tumor cell lines. Among them, 7 was less toxic to non-tumor cells. Finally, obtained data suggest that guanylhydrazones may be regarded as potential lead compounds for the design of novel anticancer agents.

  18. Preliminary in vitro evaluation of the anti-proliferative activity of guanylhydrazone derivatives

    Directory of Open Access Journals (Sweden)

    França Paulo H. B.

    2016-03-01

    Full Text Available Guanylhydrazones have shown promising antitumor activity in preclinical tumor models in several studies. In this study, we aimed at evaluating the cytotoxic effect of a series of synthetic guanylhydrazones. Different human tumor cell lines, by including HCT-8 (colon carcinoma, MDA-MB-435 (melanoma and SF-295 (glioblastoma were continuous exposed to guanylhydrazone derivatives for 72 hours and growth inhibition of tumor cell lines and macrophages J774 was measured using tetrazolium salt (MTT assay. Compounds 7, 11, 16 and 17 showed strong cytotoxic activity with IC50 values lower than 10 μmol L−1 against four tumor cell lines. Among them, 7 was less toxic to non-tumor cells. Finally, obtained data suggest that guanylhydrazones may be regarded as potential lead compounds for the design of novel anticancer agents.

  19. Corrosion assessment and enhanced biocompatibility analysis of biodegradable magnesium-based alloys

    Science.gov (United States)

    Pompa, Luis Enrique

    Magnesium alloys have raised immense interest to many researchers because of its evolution as a new third generation material. Due to their biocompatibility, density, and mechanical properties, magnesium alloys are frequently reported as prospective biodegradable implant materials. Moreover, magnesium based alloys experience a natural phenomena to biodegrade in aqueous solutions due to its corrosive activity, which is excellent for orthopedic and cardiovascular applications. However, major concerns with such alloys are fast and non-uniform corrosion degradation. Controlling the degradation rate in the physiological environment determines the success of an implant. In this investigation, three grades of magnesium alloys: AZ31B, AZ91E and ZK60A were studied for their corrosion resistance and biocompatibility. Scanning electron microscopy, energy dispersive spectroscopy, atomic force microscopy and contact angle meter are used to study surface morphology, chemistry, roughness and wettability, respectively. Additionally, the cytotoxicity of the leached metal ions was evaluated by a tetrazolium based bio-assay, MTS.

  20. Chemical composition of the essential oil from basil (Ocimum basilicum Linn.) and its in vitro cytotoxicity against HeLa and HEp-2 human cancer cell lines and NIH 3T3 mouse embryonic fibroblasts.

    Science.gov (United States)

    Kathirvel, Poonkodi; Ravi, Subban

    2012-01-01

    This study examines the chemical composition and in vitro anticancer activity of the essential oil from Ocimum basilicum Linn. (Lamiaceae), cultivated in the Western Ghats of South India. The chemical compositions of basil fresh leaves were identified by GC-MS: 11 components were identified. The major constituents were found to be methyl cinnamate (70.1%), linalool (17.5%), β-elemene (2.6%) and camphor (1.52%). The results revealed that this plant may belong to the methyl cinnamate and linalool chemotype. A methyl thiazol tetrazolium assay was used for in vitro cytotoxicity screening against the human cervical cancer cell line (HeLa), human laryngeal epithelial carcinoma cell line (HEp-2) and NIH 3T3 mouse embryonic fibroblasts. The IC(50) values obtained were 90.5 and 96.3 µg mL(-1), respectively, and the results revealed that basil oil has potent cytotoxicity.

  1. [Comparative studies on toxicity of various dielectrics, petroleum derivatives, used in electroerosion technology. IV. Morphological and cytoenzymatic changes in the lungs and acid-base imbalance in rats chronically exposed to petroleum hydrocarbons].

    Science.gov (United States)

    Starek, A; Kamiński, M

    1981-01-01

    In rats exposed to odourless kerosene of 75 and 300 mg/m3 concentration, for 14 weeks, morphologic and cytoenzymatic examinations of lungs have been carried out and acid-base equilibrium indices in blood have been determined. Passive congestion of lung parenchyma, subpleural blood extravasation, atelectasis foci, thickened interalveolar septa with infiltrates from neutrophils, lymphocytes, eosinophils and macrophages have been found. In addition a decrease in succinic dehydrogenase activity, NADPH -- tetrazolium reductase, and Mg++-ATP-ase and increase in acid phosphatase activity have been revealed. Those have been focal changes, involving, apart from bronchial tree (low exposure -- 75 mg/m3), the remaining lung parenchyma segments (high exposure -- 300 mg/m3). In addition, disturbances in acid-base equilibrium in form of compensated metabolic alkalosis (75 mg/m3) and compensated metabolic acidosis (300 mg/m3) have occurred. The obtained results demonstrate toxic effects of kerosene hydrocarbons on the function and structure of lungs.

  2. Shedding Light on the Oxygen Reduction Reaction Mechanism in Ether-Based Electrolyte Solutions: A Study Using Operando UV-Vis Spectroscopy.

    Science.gov (United States)

    Hirshberg, Daniel; Sharon, Daniel; Afri, Michal; Lavi, Ronit; Frimer, Aryeh A; Metoki, Noa; Eliaz, Noam; Kwak, Won-Jin; Sun, Yang-Kook; Aurbach, Doron

    2018-04-04

    Using UV-vis spectroscopy in conjunction with various electrochemical techniques, we have developed a new effective operando methodology for investigating the oxygen reduction reactions (ORRs) and their mechanisms in nonaqueous solutions. We can follow the in situ formation and presence of superoxide moieties during ORR as a function of solvent, cations, anions, and additives in the solution. Thus, using operando UV-vis spectroscopy, we found evidence for the formation of superoxide radical anions during oxygen reduction in LiTFSI/diglyme electrolyte solutions. Nitro blue tetrazolium (NBT) was used to indicate the presence of superoxide moieties based on its unique spectral response. Indeed, the spectral response of NBT containing solutions undergoing ORR could provide a direct indication for the level of association of the Li cations with the electrolyte anions.

  3. Investigation of the proinflammatory activity of bronchoalveolar lavage fluid in lung cancer and inflammatory diseases of respiratory organs

    Directory of Open Access Journals (Sweden)

    O. P. Makarova

    2014-01-01

    Full Text Available The proinflammatory activity of bronchoalveolar lavage fluid (BALF was investigated using the biological test system (donor blood in 71 patients with lung cancer and inflammatory disease of respiratory organs (lung abscess, infiltrative tuberculosis, tuberculoma, fibrocavernous tuberculosis. The supernatant of BALF was purified by filtration through millipores and added to blood, and a nitroblue tetrazolium recovery test (HBT was carried out. Hank’s balanced salt solution was used in the control. The proinflammatory effect of BALF was defined as a diformazan-positive neutrophil count ratio in the experimental and control samples (stimulation index. The inflammatory process developing in lung cancer was active, as suggested by the accumulation of neutrophils in the lung and by the high proinflammatory activity of BALF, which were comparable with the similar indicators in lung abscess and fibrocavernous tuberculosis.

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

    International Nuclear Information System (INIS)

    Huang, Heng-Li; Chang, Yin-Yu; Chen, Hung-Jui; Chou, Yu-Kai; Lai, Chih-Ho; Chen, Michael Y. C.

    2014-01-01

    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 2 O 5 and Ta 2 O 5 -Ag coatings with 12.5 at. % of Ag exhibit improved antibacterial effects against S. aureus and have good skin fibroblast cell cellular biocompatibility

  5. [Ursodeoxycholic acid induced apoptosis of human hepatoma cells HepG2 and SMMC-7721 bymitochondrial-mediated pathway].

    Science.gov (United States)

    Wu, Duan; Zhou, Jianyin; Yin, Zhenyu; Liu, Pingguo; Zhao, Yilin; Liu, Jianming; Wang, Xiaomin

    2014-12-02

    To explore the effects and underlying mechanisms of ursodeoxycholic acid on human hepatoma cells. HepG2 and SMMC-7721 HCC cell lines were respectively treated with ursodeoxycholic acid. And cell proliferation, apoptosis and the expression of Bax/Bcl-2 gene were detected by methyl thiazolyl tetrazolium (MTT), inverted microscopy, fluorescent microscopy, flow cytometry and Western blot. Ursodeoxycholic acid significantly inhibited the proliferation of human hepatoma cells in a concentration- and time-dependent manner. The half maximal inhibitory concentrations (IC50) of HepG2 and SMMC-7721 were 397.3 and 387.7 µg/ml respectively after a 48-hour treatment of 400 µg /ml ursodeoxycholic acid. And it also induced the apoptosis of HepG2 and SMMC-7721 cells, up-regulated Bax gene and down-regulated Bcl-2 gene. Ursodeoxycholic acid inhibits the proliferation of hepatoma cells and induce apoptosis by mitochondrial-mediated pathway.

  6. 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)Cycadaceae) showed low activity against amastigote stage (IC(50) around 50 microg/ml). Of those only Tabernaemontana sananho displayed also good activity on promastigotes (IC(50)<10 microg/ml). Results are discussed herein, in relation with the traditional use of the plants and compared with other data from the relevant literature.

  7. 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.

  8. 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.

  9. Non-thermal effects of 94 GHz radiation on bacterial metabolism

    Science.gov (United States)

    Raitt, Brittany J.

    Bacillus subtilis, Escherichia coli, Staphylococcus aureus, and Klebsiella pneumoniae were used to investigate the non-thermal effects of terahertz (THz) radiation exposure on bacterial cells. The THz source used was a 94 GHz (0.94 THz) Millitech Gunn Diode Oscillator with a power density of 1.3 mW/cm2. The cultures were placed in the middle sixty wells of two 96-well microplates, one serving as the experimental plate and one serving as a control. The experimental plate was placed on the radiation source for either two, eighteen, or twenty-four hours and the metabolism of the cells was measured in a spectrophotometer using the tetrazolium dye XTT. The results showed no consistent significant differences in either the growth rates or the metabolism of any of the bacterial species at this frequency and power density.

  10. UTP reduces infarct size and improves mice heart function after myocardial infarct via P2Y2 receptor

    DEFF Research Database (Denmark)

    Cohen, A; Shainberg, Asher; Hochhauser, E

    2011-01-01

    Pyrimidine nucleotides are signaling molecules, which activate G protein-coupled membrane receptors of the P2Y family. P2Y(2) and P2Y(4) receptors are part of the P2Y family, which is composed of 8 subtypes that have been cloned and functionally defined. We have previously found that uridine-5......'-triphosphate (UTP) reduces infarct size and improves cardiac function following myocardial infarct (MI). The aim of the present study was to determine the role of P2Y(2) receptor in cardiac protection following MI using knockout (KO) mice, in vivo and wild type (WT) for controls. In both experimental groups...... used (WT and P2Y(2)(-/-) receptor KO mice) there were 3 subgroups: sham, MI, and MI+UTP. 24h post MI we performed echocardiography and measured infarct size using triphenyl tetrazolium chloride (TTC) staining on all mice. Fractional shortening (FS) was higher in WT UTP-treated mice than the MI group...

  11. 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.

  12. Microstructure and osteoblast response of gradient bioceramic coating on titanium alloy fabricated by laser cladding

    Energy Technology Data Exchange (ETDEWEB)

    Zheng Min [State Key Laboratory of Gansu Advanced Non-ferrous Metal Materials, Lanzhou University of Technology, Lanzhou 730050 (China)], E-mail: zhminmin@sina.com; Fan Ding; Li Xiukun [State Key Laboratory of Gansu Advanced Non-ferrous Metal Materials, Lanzhou University of Technology, Lanzhou 730050 (China); Li Wenfei; Liu Qibin [College of Materials Science and Engineering, Guizhou University, Guiyang 550003 (China); Zhang Jianbin [State Key Laboratory of Gansu Advanced Non-ferrous Metal Materials, Lanzhou University of Technology, Lanzhou 730050 (China)

    2008-11-15

    To construct a bioactive interface between metal implant and the surrounding bone tissue, the gradient calcium phosphate bioceramic coating on titanium alloy (Ti-6Al-4V) was designed and fabricated by laser cladding. The results demonstrated that the gradient bioceramic coating was metallurgically bonded to the titanium alloy substrate. The appearance of hydroxyapatite and {beta}-tricalcium phosphate indicated that the bioactive phases were synthesized on the surface of coating. The microhardness gradually decreased from the coating to substrate, which could help stress relaxation between coating and bone tissue. Furthermore, the methyl thiazolyl tetrazolium (MTT) assay of cell proliferation revealed that the laser-cladded bioceramic coating had more favorable osteoblast response compared with the surface of untreated titanium alloy substrate.

  13. Metalloantibiotic Mn(II)-bacitracin complex mimicking manganese superoxide dismutase

    International Nuclear Information System (INIS)

    Piacham, Theeraphon; Isarankura-Na-Ayudhya, Chartchalerm; Nantasenamat, Chanin; Yainoy, Sakda; Ye Lei; Buelow, Leif; Prachayasittikul, Virapong

    2006-01-01

    Superoxide dismutase (SOD) activities of various metallobacitracin complexes were evaluated using the riboflavin-methionine-nitro blue tetrazolium assay. The radical scavenging activity of various metallobacitracin complexes was shown to be higher than those of the negative controls, e.g., free transition metal ions and metal-free bacitracin. The SOD activity of the complex was found to be in the order of Mn(II) > Cu(II) > Co(II) > Ni(II). Furthermore, the effect of bacitracin and their complexation to metals on various microorganisms was assessed by antibiotic susceptibility testing. Moreover, molecular modeling and quantum chemical calculation of the metallobacitracin complex was performed to evaluate the correlation of electrostatic charge of transition metal ions on the SOD activity

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

    Science.gov (United States)

    Zhou, Yuanyuan; Yang, Bingyou; Jiang, Yanqiu; Liu, Zhaoxi; Liu, Yuxin; Wang, Xiaoli; Kuang, Haixue

    2015-08-26

    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.

  15. Effects of Plasma Rich in Growth Factors and Platelet-Rich Fibrin on Proliferation and Viability of Human Gingival Fibroblasts

    Science.gov (United States)

    Vahabi, Surena; Vaziri, Shahram; Torshabi, Maryam

    2015-01-01

    Objectives: Platelet preparations are commonly used to enhance bone and soft tissue regeneration. Considering the existing controversies on the efficacy of platelet products for tissue regeneration, more in vitro studies are required. The aim of the present study was to compare the in vitro effects of plasma rich in growth factors (PRGF) and platelet-rich fibrin (PRF) on proliferation and viability of human gingival fibroblasts (HGFs). Materials and Methods: Anitua’s PRGF and Choukran’s PRF were prepared according to the standard protocols. After culture periods of 24, 48 and 72 hours, proliferation of HGFs was evaluated by the methyl thiazol tetrazolium assay. Statistical analysis was performed using one-way ANOVA followed by Tukey-Kramer’s multiple comparisons and P-valuesPRGF treatment induced statistically significant (PPRGF than in PRF group (PPRGF had a strong stimulatory effect on HGF cell viability and proliferation compared to PRF. PMID:26877740

  16. Superoxide radical formation, superoxide dismutase and glutathione reductase activity in the brain of irradiated rats

    International Nuclear Information System (INIS)

    Stanimirovic, D.; Ivanovic, L.; Simovic, M.; Cernak, I.; Savic, J.

    1989-01-01

    In the forebrain cortex, basal ganglia and hippocampus of irradiated rats (whole body, X-ray, 9 Gy), nitroblue-tetrazolium (NBT) reduction was measured as a probe of superoxide radical formation 1 hr, 6 hrs, 24 hrs and 72 hrs after irradiation. Increased superoxide radical formation was found in parallel with increase of superoxide dismutase (SOD) activity and marked decrease of glutathione reductase (GR) activity which is the most pronounced in basal ganglia. The results indicate that in the postradiation period disproportion among free radical production and capacity of brain antioxidative system occurs. This disbalance is more expressed in the brain regions known as selective vulnerable (basal ganglia, hippocampus). (author). 10 refs.; 2 tabs

  17. Determination of pollen quality and quantity in mulberry (morus alba l.)

    International Nuclear Information System (INIS)

    Erdogan, U.

    2015-01-01

    In this study, the pollen grains of eight different mulberry types in I.spir and Pazaryolu districts of Erzurum were tested for the determination of viability, germination rates, pollen production levels and morphologically homogeneity. Viability of the pollens was determined by TTC (2, 3, 5-triphenly tetrazolium chlorid) and IKI (iodine + potassium iodide) tests. Pollen germination experiments were performed with Agar-plate (1%) method in sucrose solutions of 0, 5, 10, 15, 20 and 25% concentrations. In addition, pollen production and morphologically homogeneity were determined by the Hemacytometric method. The pollen viability of all types used in this study was obtained in high ratios. Pollen germination rates were the highest for 15% and 20% sucrose solutions. The highest pollen production level was obtained from the genotype 6. The morphologically homogeneity levels of pollens changed from 97.36 to 98.86% in types. (author)

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

    International Nuclear Information System (INIS)

    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

    2012-01-01

    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.

  19. Partial transformation from fast to slow muscle fibers induced by deafferentation of capsaicin-sensitive muscle afferents.

    Science.gov (United States)

    Brunetti, O; Barazzoni, A M; Della Torre, G; Clavenzani, P; Pettorossi, V E; Bortolami, R

    1997-11-01

    Mechanical and histochemical characteristics of the lateral gastrocnemius (LG) muscle of the rat were examined 21 days after capsaicin injection into the LG muscle. The capsaicin caused a decrease in generation rate of twitch and tetanic tension and an increase in fatigue resistance of LG muscle. The histochemical muscle fiber profile evaluated by myosin adenosine triphosphatase and reduced nicotinamide adenine dinucleotide tetrazolium reductase methods showed an increase of type I and IIC fibers and a decrease of the type IIB in whole muscle, and a decrease of the IIA, IIX fibers in the red part accompanied by their increase in the white part. Therefore the capsaicin treatment, which selectively eliminated fibers belonging to the III and IV groups of muscle afferents, induced muscle fiber transformation from fast contracting fatiguing fibers to slowly contracting nonfatiguing ones.

  20. 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)Urea 2 , Cu(II)Ser 2 and CuCl 2 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.

  1. 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

  2. In vitro preliminary cytotoxicity testing of vegetal extracts, using colorimetric methods

    Directory of Open Access Journals (Sweden)

    Claudia Patricia Cordero Camacho

    2002-01-01

    Full Text Available To advance in the study of the Colombian vegetal biodiversity, considered as a potential source of pharmacologically active products, the establishment of biological activity evaluation systems is necessary, which allow the detection of active products against pathologies with high social and economical impact, such as cancer. This work describes the implementation of a preliminary in vitro methodology for the determination of potential anticancer activity in vegetal extracts, by cytotoxicity testing upon human tumor cell lines, measuring the cellular mass indirectly with the colorimetric assays of MTT (methyl tetrazolium tiazole reduction and SRB (sulforhodamine Bstaining. HT-29, MCF-7, SiHa and HEp-2 cell lines cultures were adapted, MTT concentration, cellular density and treatment period parameters for the cytotoxicity assay were selected. Cell lines sensitivity to the chemotherapeutic agent Doxorubicin HCl was determined. Colombian vegetal species extracts cytotoxicity was tested and usefulness of the assay as a tool to bioguide the search of active products was evidenced.

  3. 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.

  4. Rapid Bioassay-Guided Isolation of Antibacterial Clerodane Type Diterpenoid from Dodonaea viscosa (L. Jaeq.

    Directory of Open Access Journals (Sweden)

    Muhammad Khurram

    2015-08-01

    Full Text Available Plant extracts are complex matrices and, although crude extracts are widely in use, purified compounds are pivotal in drug discovery. This study describes the application of automated preparative-HPLC combined with a rapid off-line bacterial bioassay, using reduction of a tetrazolium salt as an indicator of bacterial metabolism. This approach enabled the identification of fractions from Dodonaea viscosa that were active against Staphylococcus aureus and Escherichia coli, which, ultimately, resulted in the identification of a clerodane type diterpenoid, 6β-hydroxy-15,16-epoxy-5β, 8β, 9β, 10α-cleroda-3, 13(16, 14-trien-18-oic acid, showing bacteriostatic activity (minimum inhibitory concentration (MIC = 64–128 µg/mL against test bacteria. To the best of our knowledge, this is the first report on antibacterial activity of this metabolite from D. viscosa.

  5. Inhibition of Shiga toxin 2 (Stx2) in apple juices and its resistance to pasteurization.

    Science.gov (United States)

    Rasooly, Reuven; Do, Paula M; Levin, Carol E; Friedman, Mendel

    2010-06-01

    In the present study, we evaluated Shiga toxin (Stx2) activity in apple juices by measuring a decrease in dehydrogenase activity of Vero cells with the microculture tetrazolium (MTT) assay. Freshly prepared juice from Red Delicious apples and Golden Delicious apples inhibited the biological activity of the bacterial toxin Stx2 produced by E. coli O157:H7 strains. Studies with immunomagnetic beads bearing specific antibodies against the toxin revealed that Stx2 activity was restored when removed from the apple juice. SDS gel electrophoresis revealed no difference (P pasteurize apple juice, but lost all activity when exposed to 100 degrees C for 5 min. The results suggest that pasteurization of apple juice used to inactivate E. coli O157:H7 has no effect on Stx2, and that food-compatible and safe antitoxin compounds can be used to inhibit the biological activity of the Shiga toxin.

  6. 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.

  7. The toxicity study of functionalized CNT from fermented tapioca on neuroblastoma cell

    Science.gov (United States)

    Nurulhuda, I.; Mazatulikhma, M. Z.; Alrokayan, S.; Khan, H.; Rusop, M.

    2018-05-01

    Carbon nanotubes known as one of the most interesting types of nanomaterials, especially use in application directly to cells. Somehow the use should take into consideration regarding the potential adverse impact on human health. Current study, the carbon nanotube was synthesized from fermented tapioca and functionalized with polyethylene glycol and directly test on the neuroblastoma cells in vitro. The toxicity effect on cells was assessed by 3(4, 5-dimethylthiazol-2-yl)-2, 5-tetrazolium bromide assays. It showed a dose-and time-dependent less toxic effect on functionalized carbon nanotube compared to non-functionalized. This leads us to the conclusion that functionalized carbon nanotube can be use for drug delivery in future.

  8. Attachment, proliferation and differentiation of BMSCs on gas-jet/electrospun nHAP/PHB fibrous scaffolds

    International Nuclear Information System (INIS)

    Guan Donghua; Chen Zhiqing; Huang Chunpeng; Lin Yinghe

    2008-01-01

    In this study, poly(3-hydroxybutyrate) (PHB)-based scaffolds containing nanosized hydroxyapatite (nHAP) were manufactured by gas-jet/electrospinning. The morphologies of the scaffolds were characterized. The effect of the scaffolds on attachment, proliferation and differentiation of the bone marrow stroma cells (BMSCs) were accessed by using scanning electron microscopy (SEM), methylthiazol tetrazolium (MTT) assay and alkaline phosphatase (ALP) activity. The results show that the gas-jet/electrospun scaffolds possess an extracellular matrix-like topography. In vitro studies describe that the scaffolds have positive effects on attachment, proliferation and differentiation of BMSCs in vitro. It can be concluded that the scaffolds combing the unique structural features generated by gas-jet/electrospinning with functional factors, have the potential to be used in bone tissue engineering

  9. 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.

  10. [The effect of magnetotherapy on the immunobiochemical indices of subjects with diseases of the periodontal tissues and joints].

    Science.gov (United States)

    Samoĭlovich, V A

    1999-01-01

    Kept under medical surveillance in a health resort setting were 52 patients with disorders of the parodontium and large joints. All patients were given a complex therapy involving dietotherapy, therapeutic exercise, hydrotherapy, mud-treatment. Those patients having parodontium diseases were also prescribed topical treatment (chloride-sodium mouth baths and mud applications to the gingiva area). The main group subjects were also exposed to VMF using the unit for low-frequency therapy "Gradient-1". Laboratory means were also made use of, as a complex of biochemical tests characterizing changes in lipid metabolism. The level of the natural bodily resistance was determined by nitroblue tetrazolium test (NBT-test). The condition of the parodontium was evaluated by the Loë-Silness index. Adaptive reactions were studied by the lymphocytes-to-segmented neutrophils ratio. Adoption of therapy involving physiobalneofactors in patients with afflictions of the parodontium tissues and large joints makes for development of favourable in prognostic respect adaptive reactions.

  11. Rapid differentiation between gamma-irradiated and non irradiated potato tubers

    International Nuclear Information System (INIS)

    Jona, R.; Fronda, A.

    1990-01-01

    The use of gamma irradiation as commercial method for the preservation of fruits and vegetables calls for methods of differentiation between irradiated and non-irradiated foodstuffs. In a previous research, the polysaccharidic content of cell walls of irradiated tissue has been investigated, but it required rather long time to reach the result. A method devised to ascertain the vitality of cells has been applied to distinguish irradiated from non-irradiated potato tubers. 500 mg of tissue excised from tubers have been infiltrated with tetrazolium chloride 0.6% in phosphate buffer, pH 7.4. After 15 hrs of incubation at 30 0 C the treated tissues have been extracted with 95% ethanol whose O.D. has been measured at 530 mμ wavelength. The colour intensity of the alcohol allowed a very clearcut recognition of the irradiated tubers. (author)

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

    International Nuclear Information System (INIS)

    Yan Penghua; Wang Jinqing; Wang Lin; Liu Bin; Lei Ziqiang; Yang Shengrong

    2011-01-01

    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.

  13. 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.

  14. A study on superoxide dismutase activity of some model compounds.

    Science.gov (United States)

    Liao, Z; Liu, W; Liu, J; Jiang, Y; Shi, J; Liu, C

    1994-08-15

    The synthesis and characteristics of a binuclear ligand N,N,N',N'-tetrakis (2'-benzimidazolyl methyl)-1,4-diethylene amino glycol ether (EGTB) and its series of coordination compounds containing copper(II), iron(III), and manganese(II) with and without exogenous bridging ligand which was imidazolate ion (Im-), bipyridine (bpy), or 1,10-phenanthroline (phen) are reported. Depending on the redox potentials by cyclic voltammetry, the coordination compounds can act as catalysts for the dismutation of superoxide radicals (O2-). The detection of the rate constant of the reaction of superoxide ion with nitroblue tetrazolium (NBT) which is inhibited by superoxide dismutase (SOD) and its model compounds of the EGTB system has been performed by a modified illumination method. The rate constants kQ of the catalytic dismutation have been obtained.

  15. Test of Antifibrotic Drugs in a Cellular Model of Fibrosis Based on Muscle-Derived Fibroblasts from Duchenne Muscular Dystrophy Patients.

    Science.gov (United States)

    Zanotti, Simona; Mora, Marina

    2018-01-01

    An in vitro model of muscle fibrosis, based on the use of primary human fibroblasts isolated from muscle biopsies of patients affected by Duchenne muscular dystrophies (DMD) and cultivated in monolayer and 3D conditions, is used to test the potential antifibrotic activity of pirfenidone (PFD). This in vitro model may be usefully also to evaluate the toxicity and efficacy of other candidate molecules for the treatment of fibrosis. The drug toxicity is evaluated using a colorimetric assay based on the conversion of tetrazolium salt (MTT) to insoluble formazan, while the effect of the drug on cell proliferation is measured with the bromodeoxyuridine incorporation assay. The efficacy of the drug is evaluated in fibroblast monolayers by quantitating synthesis and deposition of intracellular collagen with a spectrophotometric picrosirius red-based assay, and by quantitating cell migration using a "scratch" assay. The efficacy of PFD as antifibrotic drug is also evaluated in a 3D fibroblast model by measuring diameters and number of nodules.

  16. PEG-phospholipid-encapsulated bismuth sulfide and CdSe/ZnS quantum dot core–shell nanoparticle and its computed tomography/fluorescence performance

    International Nuclear Information System (INIS)

    Chen, Jun; Yang, Xiao-Quan; Qin, Meng-Yao; Zhang, Xiao-Shuai; Xuan, Yang; Zhao, Yuan-Di

    2015-01-01

    In this paper, polyethylene glycol-phospholipid structure is used to synthesize hybrid cluster of 40–50 nm diameter that contains hydrophobic bismuth sulfide nanoparticles and CdSe/ZnS quantum dots. The composite probe’s toxicity, CT imaging, and fluorescence imaging performance are also studied. Experimental results show that the nanocomposite hybrid cluster has obvious CT contrast enhancement and fluorescence imaging capability in vitro even after cellular uptake. It gives a CT number of 700 (Hounsfield units) at 15 mg/mL, higher than that of the current iobitridol CT contrast agent. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide experiment reveals that it has low cytotoxicity at concentration up to of 3.14 mg/mL of Bi, indicating the composite probe has potential ability for CT and fluorescence bimodal imaging

  17. In vitro preliminary cytotoxicity testing of vegetal extracts, using colorimetric methods

    Directory of Open Access Journals (Sweden)

    Claudia Patricia Cordero Camacho

    2011-12-01

    Full Text Available To advance in the study of the Colombian vegetal biodiversity, considered as a potential source of pharmacologically active products, the establishment of biological activity evaluation systems is necessary, which allow the detection of active products against pathologies with high social and economical impact, such as cancer. This work describes the implementation of a preliminary in vitro methodology for the determination of potential anticancer activity in vegetal extracts, by cytotoxicity testing upon human tumor cell lines, measuring the cellular mass indirectly with the colorimetric assays of MTT (methyl tetrazolium tiazole reduction and SRB (sulforhodamine Bstaining. HT-29, MCF-7, SiHa and HEp-2 cell lines cultures were adapted, MTT concentration, cellular density and treatment period parameters for the cytotoxicity assay were selected. Cell lines sensitivity to the chemotherapeutic agent Doxorubicin HCl was determined. Colombian vegetal species extracts cytotoxicity was tested and usefulness of the assay as a tool to bioguide the search of active products was evidenced.

  18. Benzylidene derivatives of andrographolide inhibit growth of breast and colon cancer cells in vitro by inducing G1 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

    2008-01-01

    Background and purpose: 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. Experimental approach: 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. Key results: 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 G1 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. Conclusion and implications: 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 G1 phase cell cycle arrest, coupled with induction of apoptosis. PMID:18806812

  19. The prosurvival role of autophagy in Resveratrol-induced cytotoxicity in human U251 glioma cells

    International Nuclear Information System (INIS)

    Li, Jun; Qin, Zhenghong; Liang, Zhongqin

    2009-01-01

    Previous study reported that resveratrol has anti-tumor activity. In this study, we investigated the involvement of autophagy in the resveratrol-induced apoptotic death of human U251 glioma cells. The growth inhibition of U251 cells induced by resveratrol was assessed with methyl thiazolyl tetrazolium (MTT). The activation of autophagy and proapoptotic effect were characterized by monodansylcadaverine labeling and Hoechst stain, respectively. Mitochondrialtransmembrane potential (ΔΨm) was measured as a function of drug treatment using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). The role of autophagy and apoptosis in the resveratrol-induced death of U251 cells was assessed using autophagic and caspase inhibitors. Immunofluorescence, flow cytometry, and Western blot analysis were used to study the apoptotic and autophagic mechanisms. Methyl thiazolyl tetrazolium (MTT) assays indicated that resveratrol decreased the viability of U251 cells in a dose- and time-dependent manner. Flow cytometry analysis indicated that resveratrol increased cell population at sub-G1 phase, an index of apoptosis. Furthermore, resveratrol-induced cell death was associated with a collapse of the mitochondrial membrane potential. The pan-caspase inhibitor Z-VAD-fmk suppressed resveratrol-induced U251 cell death. Resveratrol stimulated autophagy was evidenced by punctuate monodansylcadaverine(MDC) staining and microtubule-associated protein light chain 3 (LC3) immunoreactivty. Resveratrol also increased protein levels of beclin 1 and membrane form LC3 (LC3-II). Autophagy inhibitors 3-methylademine (3-MA) and bafilomycin A1 sensitized the cytotoxicity of resveratrol. Together, these findings indicate that resveratrol induces autophagy in human U251 glioma cells and autophagy suppressed resveratrol-induced apoptosis. This study thus suggests that autophagy inhibitors can increase the cytotoxicity of resveratrol to glioma cells

  20. 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.

  1. 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

  2. Influence of constant, alternating and cyclotron low-intensity electromagnetic fields on fibroblast proliferative activity in vitro.

    Science.gov (United States)

    Afinogenov, Gennadi; Afinogenova, Anna; Kalinin, Andrey

    2009-12-16

    Available data allow assuming the presence of stimulation of reparative processes under influence of low-intensity electromagnetic field, commensurable with a magnetic field of the Earth. Research of effects of low-intensity electromagnetic fields on fibroblast proliferative activity in human lungs in cell culture was performed.The influence of a constant electromagnetic field, an alternating electromagnetic field by frequency of 50 Hz and cyclotron electromagnetic field with identical intensity for all kinds of fields - 80 mcTl - on value of cellular mass and a correlation of live and dead cells in culture is investigated in three series of experiments. We used the universal electromagnetic radiator generating all three kinds of fields and supplied by a magnetometer which allows measuring the intensity of accurate within 0.1 mcTl including taking into account the Earth's magnetic field intensity.The peak value for stimulation cellular proliferation in the present experiences was two-hour influence by any of the specified kinds of electromagnetic fields. The irradiation by cyclotron electromagnetic field conducts positive dynamics in growth of live cells (up to 206+/-22%) and decreases the number of dead cells (down to 31+/-6%). Application of cyclotron magnetic fields promoted creation of optimum conditions for proliferation. As a result of researches we observed the reliable 30% increase of nitro-tetrazolium index (in nitro-tetrazolium blue test) after irradiation by cyclotron electromagnetic field in experience that testifies to strengthening of the cell breathing of living cells.In our opinion, it is necessary to pay attention not only to a pure gain of cells, but also to reduction of number dead cells that can be criterion of creation of optimum conditions for their specific development and valuable functioning.

  3. Synergistic growth inhibition by sorafenib and vitamin K2 in human hepatocellular carcinoma cells.

    Science.gov (United States)

    Zhang, Yafei; Zhang, Bicheng; Zhang, Anran; Zhao, Yong; Zhao, Jie; Liu, Jian; Gao, Jianfei; Fang, Dianchun; Rao, Zhiguo

    2012-09-01

    Sorafenib is an oral multikinase inhibitor that has been proven effective as a single-agent therapy in hepatocellular carcinoma, and there is a strong rationale for investigating its use in combination with other agents. Vitamin K2 is nearly non-toxic to humans and has been shown to inhibit the growth of hepatocellular carcinoma. In this study, we evaluated the effects of a combination of sorafenib and vitamin K2 on the growth of hepatocellular carcinoma cells. Flow cytometry, 3-(4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2H-tetrazolium bromide) and nude mouse xenograft assays were used to examine the effects of sorafenib and vitamin K2 on the growth of hepatocellular carcinoma cells. Western blotting was used to elucidate the possible mechanisms underlying these effects. Assays for 3-(4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2H-tetrazolium bromide) revealed a strong synergistic growth-inhibitory effect between sorafenib and vitamin K2. Flow cytometry showed an increase in cell cycle arrest and apoptosis after treatment with a combination of these two drugs at low concentrations. Sorafenib-mediated inhibition of extracellular signal-regulated kinase phosphorylation was promoted by vitamin K2, and downregulation of Mcl-1, which is required for sorafenib-induced apoptosis, was observed after combined treatment. Vitamin K2 also attenuated the downregulation of p21 expression induced by sorafenib, which may represent the mechanism by which vitamin K2 promotes the inhibitory effects of sorafenib on cell proliferation. Moreover, the combination of sorafenib and vitamin K2 significantly inhibited the growth of hepatocellular carcinoma xenografts in nude mice. Our results determined that combined treatment with sorafenib and vitamin K2 can work synergistically to inhibit the growth of hepatocellular carcinoma cells. This finding raises the possibility that this combined treatment strategy might be promising as a new therapy against hepatocellular carcinoma, especially for patients

  4. Arachidonic acid triggers an oxidative burst in leukocytes

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    Pompeia C.

    2003-01-01

    Full Text Available The change in cellular reducing potential, most likely reflecting an oxidative burst, was investigated in arachidonic acid- (AA stimulated leukocytes. The cells studied included the human leukemia cell lines HL-60 (undifferentiated and differentiated into macrophage-like and polymorphonuclear-like cells, Jurkat and Raji, and thymocytes and macrophages from rat primary cultures. The oxidative burst was assessed by nitroblue tetrazolium reduction. AA increased the oxidative burst until an optimum AA concentration was reached and the burst decreased thereafter. In the leukemia cell lines, optimum concentration ranged from 200 to 400 µM (up to 16-fold, whereas in rat cells it varied from 10 to 20 µM. Initial rates of superoxide generation were high, decreasing steadily and ceasing about 2 h post-treatment. The continuous presence of AA was not needed to stimulate superoxide generation. It seems that the NADPH oxidase system participates in AA-stimulated superoxide production in these cells since the oxidative burst was stimulated by NADPH and inhibited by N-ethylmaleimide, diphenyleneiodonium and superoxide dismutase. Some of the effects of AA on the oxidative burst may be due to its detergent action. There apparently was no contribution of other superoxide-generating systems such as xanthine-xanthine oxidase, cytochromes P-450 and mitochondrial electron transport chain, as assessed by the use of inhibitors. Eicosanoids and nitric oxide also do not seem to interfere with the AA-stimulated oxidative burst since there was no systematic effect of cyclooxygenase, lipoxygenase or nitric oxide synthase inhibitors, but lipid peroxides may play a role, as indicated by the inhibition of nitroblue tetrazolium reduction promoted by tocopherol.

  5. Potencial fisiológico de sementes de soja e sua relação com a emergência das plântulas em campo = Soybean physiological seed potential and the relationship with seedlings emergence in field

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    Sandra Regina Pelegrinello Schuab

    2006-10-01

    Full Text Available Objetivou-se, nesse estudo, comparar os diferentes testes empregados na avaliação do vigor das sementes de soja e verificar a sua relação com a emergência das plântulas em campo. Assim, o vigor das sementes de soja de dez cultivares foi avaliado por meio dos seguintes testes: emergência das plântulas em campo; germinação; emergência dasplântulas em leito de areia; velocidade de emergência em areia; envelhecimento acelerado; frio modificado; tetrazólio (1-3 e 1-5 e condutividade elétrica. Foi possível observar que os testes de envelhecimento acelerado, germinação, tetrazólio e condutividade elétrica foram os que melhor se correlacionaram com a emergência em campo. Além disso, a interpretação dos resultados obtidos entre os testes de laboratório e de emergência em campo não deve considerar apenas a análise de correlação.The objective of this work was to compare different tests used to evaluate soybean seed vigour and to verify their relationship with seedlings emergence in field. Thus, the soybean seed vigour of ten cultivars was evaluated by the following tests: seedling emergence in field, standard germination, seedlings emergence in sand seedbed, speed of emergence in sand seedbed, accelerated aging, modified cold test, tetrazolium (1-3 and 1-5 and electrical conductivity. The accelerating aging, standard germination, tetrazolium and electrical conductivity tests showed higher correlations with field emergence. The results obtained from laboratory tests and seedlings emergence in field should not consider only the correlation analysis.

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

    Science.gov (United States)

    Bonetti, Keila A P; Quoirin, Marguerite; Quisen, Regina C; Lima, Suelen C S

    2016-01-01

    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.

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

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    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.

  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. 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

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

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    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.

  11. Biocompatibility Assessment of Novel Bioresorbable Alloys Mg-Zn-Se and Mg-Zn-Cu for Endovascular Applications: In- Vitro Studies.

    Science.gov (United States)

    Persaud-Sharma, Dharam; Budiansky, Noah; McGoron, Anthony J

    2013-01-01

    Previous studies have shown that using biodegradable magnesium alloys such as Mg-Zn and Mg-Zn-Al possess the appropriate mechanical properties and biocompatibility to serve in a multitude of biological applications ranging from endovascular to orthopedic and fixation devices. The objective of this study was to evaluate the biocompatibility of novel as-cast magnesium alloys Mg-1Zn-1Cu wt.% and Mg-1Zn-1Se wt.% as potential implantable biomedical materials, and compare their biologically effective properties to a binary Mg-Zn alloy. The cytotoxicity of these experimental alloys was evaluated using a tetrazolium based- MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay and a lactate dehydrogenase membrane integrity assay (LDH). The MTS assay was performed on extract solutions obtained from a 30-day period of alloy immersion and agitation in simulated body fluid to evaluate the major degradation products eluted from the alloy materials. Human foreskin fibroblast cell growth on the experimental magnesium alloys was evaluated for a 72 hour period, and cell death was quantified by measuring lactate dehydrogenase concentrations. Both Mg-Zn-Se and Mg-Zn-Cu alloys exhibit low cytotoxicity levels which are suitable for biomaterial applications. The Mg-Zn-Cu alloy was found to completely degrade within 72 hours, resulting in lower human foreskin fibroblast cell viability. The Mg-Zn-Se alloy was shown to be less cytotoxic than both the Mg-Zn-Cu and Mg-Zn alloys.

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

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    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

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

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    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.

  14. The optimal condition of performing MTT assay for the determination of radiation sensitivity

    International Nuclear Information System (INIS)

    Hong, Semie; Kim, Il Han

    2001-01-01

    The measurement of radiation survival using a clonogenic assay, the established standard, can be difficult and time consuming. In this study, We have used the MTT assay, based on the reduction of a tetrazolium salt to a purple formazan precipitate by living cells, as a substitution for clonogenic assay and have examined the optimal condition for performing this assay in determination of radiation sensitivity. Four human cancer cell lines - PCI-1, SNU-1066, NCI-H63O and RKO cells have been used. For each cell line, a clonogenic assay and a MTT assay using Premix WST-1 solution, which is one of the tetrazolium salts and does not require washing or solubilization of the precipitate were carried out after irradiation of 0, 2, 4, 6, 8, 10 Gy, For clonogenic assay, cells in 25 cm 2 flasks were irradiated after overnight incubation and the resultant colonies containing more than 50 cells were scored after culturing the cells for 10-14 days, For MTT assay, the relationship between absorbance and cell number, optimal seeding cell number, and optimal timing of assay was determined. Then, MTT assay was performed when the irradiated cells had regained exponential growth or when the non-irradiated cells had undergone four or more doubling times. There was minimal variation in the values gained from these two methods with the standard deviation generally less than 5%, and there were no statistically significant differences between two methods according to t-test in low radiation dose (below 6 Gy). The regression analyses showed high linear correlation with the R 2 value of 0.975-0.992 between data from the two different methods. The optimal cell numbers for MTT assay were found to be dependent on plating efficiency of used cell line. Less than 300 cells/well were appropriate for cells with high plating efficiency (more than 30%). For cells with low plating efficiency (less than 30%), 500 cells/well or more were appropriate for assay. The optimal time for MTT assay was alter 6

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

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    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

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

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    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.

  18. Effect of leaching residual methyl methacrylate concentrations on in vitro cytotoxicity of heat polymerized denture base acrylic resin processed with different polymerization cycles

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    Canan Bural

    2011-08-01

    Full Text Available OBJECTIVES: Residual methyl methacrylate (MMA may leach from the acrylic resin denture bases and have adverse effects on the oral mucosa. This in vitro study evaluated and correlated the effect of the leaching residual MMA concentrations ([MMA]r on in vitro cytotoxicity of L-929 fibroblasts. MATERIAL AND METHODS: A total of 144 heat-polymerized acrylic resin specimens were fabricated using 4 different polymerization cycles: (1 at 74ºC for 9 h, (2 at 74ºC for 9 h and terminal boiling (at 100ºC for 30 min, (3 at 74ºC for 9 h and terminal boiling for 3 h, (4 at 74ºC for 30 min and terminal boiling for 30 min. Specimens were eluted in a complete cell culture medium at 37ºC for 1, 2, 5 and 7 days. [MMA]r in eluates was measured using high-performance liquid chromatography. In vitro cytotoxicity of eluates on L-929 fibroblasts was evaluated by means of cell proliferation using a tetrazolium salt XTT (sodium 3´-[1-phenyl-aminocarbonyl-3,4-tetrazolium]bis(4-methoxy-6-nitrobenzenesulphonic acid assay. Differences in [MMA]r of eluates and cell proliferation values between polymerization cycles were statistically analyzed by Kruskal-Wallis, Friedman and Dunn's multiple comparison tests. The correlation between [MMA]r of eluates and cell proliferation was analyzed by Pearson's correlation test (p<0.05. RESULTS: [MMA]r was significantly (p<0.001 higher in eluates of specimens polymerized with cycle without terminal boiling after elution of 1 and 2 days. Cell proliferation values for all cycles were significantly (p<0.01 lower in eluates of 1 day than those of 2 days. The correlation between [MMA]r and cell proliferation values was negative after all elution periods, showing significance (p<0.05 for elution of 1 and 2 days. MMA continued to leach from acrylic resin throughout 7 days and leaching concentrations markedly reduced after elution of 1 and 2 days. CONCLUSION: Due to reduction of leaching residual MMA concentrations, use of terminal boiling in

  19. Inhibition of Pro-inflammatory Mediators and Cytokines by Chlorella Vulgaris Extracts.

    Science.gov (United States)

    Sibi, G; Rabina, Santa

    2016-01-01

    The aim of this study was to determine the in vitro anti-inflammatory activities of solvent fractions from Chlorella vulgaris by inhibiting the production of pro-inflammatory mediators and cytokines. Methanolic extracts (80%) of C. vulgaris were prepared and partitioned with solvents of increasing polarity viz., n-hexane, chloroform, ethanol, and water. Various concentrations of the fractions were tested for cytotoxicity in RAW 264.7 cells using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and the concentrations inducing cell growth inhibition by about 50% (IC50) were chosen for further studies. Lipopolysaccharide (LPS) stimulated RAW 264.7 cells were treated with varying concentrations of C. vulgaris fractions and examined for its effects on nitric oxide (NO) production by Griess assay. The release of prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), and interleukin 6 (IL-6) were quantified using enzyme-linked immunosorbent assay using Celecoxib and polymyxin B as positive controls. MTT assay revealed all the solvent fractions that inhibited cell growth in a dose-dependent manner. Of all the extracts, 80% methanolic extract exhibited the strongest anti-inflammatory activity by inhibiting NO production (P < 0.01), PGE2 (P < 0.05), TNF-α, and IL-6 (P < 0.001) release in LPS induced RAW 264.7 cells. Both hexane and chloroform fractions recorded a significant (P < 0.05) and dose-dependent inhibition of LPS induced inflammatory mediators and cytokines in vitro. The anti-inflammatory effect of ethanol and aqueous extracts was not significant in the study. The significant inhibition of inflammatory mediators and cytokines by fractions from C. vulgaris suggests that this microalga would be a potential source of developing anti-inflammatory agents and a good alternate for conventional steroidal and nonsteroidal anti-inflammatory drugs. C. vulgaris extracts have potential anti-inflammatory activitySolvent extraction using methanol

  20. 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

  1. The effects of cynodon dactylon on the immune response of NMRI-MICE after challenge with REV1

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    Behrooz Ilkhanizadeh

    2016-10-01

    Full Text Available Cynodon dactylon is used in Iranian traditional medicine as a healing agent for reducing the complications of diabetes mellitus. We proposed that Cynodon dactylon may perform its effects through moderating humoral and cellular immune responses. We aimed to determine the possible effects of hydroalcholic extract of Cynodon dactylonon humoral and cellular immune responses following the Rev1 challenge in the mouse model. 20 NMRI male mice were randomly grouped in two equal groups and immunized with Rev1[0.1 ml Rev1+0.9 PBS[. Mice in the treatment group orally received 400 mg/kg hydroalcoholic extract of Cynodon dactylon every day from the beginning of the study for 2 weeks. Blood samples were obtained from the animals 5 days after the last injection. Moreover, 48 hr before bleeding time, Rev1[0.1 ml Rev1+0.9 PBS[was injected into the left hind foot pad of mice. The levels of anti-Rev1 antibody and the specific cellular immune responses were measured by microhemagglutination test and footpad thickness, respectively. Moreover, susceptibility of macrophages respiratory burst and proliferation of immune cells were measured in order withNitroblue tetrazolium[NBT] and Microculture Tetrazolium Assay [MTT]. The concentrations of IL-1, TNFα, Il-6, and IL-10 in the serum were determined using commercially available ELISA kits. We found a significant increase in anti-Rev1 antibody levels and simultaneously a significant decrease in the level of cellular immunity[DTH] in the treatment group compared to the control group. Lymphocyte proliferation index in splenocytes was significantly increased in the treatment group. However, the level of respiratory burst in phagocytic population of splenocytes dramatically decreased in the treatment group compared to the control. A significant decrease in IL-6, TNF-α , IL-1 and increaseIl-10 serum levels were also seen in the treatment group. Cynodon dactylon extract could have an anti-inflammatory effect through

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

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    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

  3. Effect of hepatic blood flow alteration on the therapeutic effect of cryoablation in VX2 hepatic tumor rabbit: an experimental study

    International Nuclear Information System (INIS)

    Guo Zhi; Ni Hong; Li Baoguo; Hu Yonghua; Xing Wenge; Liu Fang

    2008-01-01

    Objective: To investigate the effect of alteration of blood flow in the hepatic artery on the therapeutic effect of cryoablation in VX2 hepatic tumor rabbit model. Methods: Thirty rabbits with VX2 hepatic tumor were divided into three groups according to hepatic artery blood flow: complete occlusion of the hepatic artery(group A), partial occlusion of the hepatic artery (group B), and no occlusion of the hepatic artery (group C). With conventional CT scan and perfusion scan, the values of blood flow (BF) and blood volume(BV) of VX 2 tumor were computed and the differences among the three groups were analyzed. After cryoablation, the animals were euthanized and the livers were removed. The hepatic tissue from the cryoablation area and surrounding area underwent both methyl thiazolyl tetrazolium (MTY) diaphorase staining and triphenyl tetrazolium chloride (TTC) staining. The gross pathology and histopathological changes were observed. Results: (1)The BF and BV in the three groups were: (7.23 + 2. 15 ) ml·100 g -1 ·min -1 and (1.63±0.52) ml/100 g in group A; (32.65±6.12) ml·100 g -1 ·min -1 and (9.32±2.63) ml/100 g in group B; (61.34±12.15) ml·100 g -1 ·min -1 and (17.51± 3.14) ml/100 g in group C, respectively. There were significant differences among the three groups in the BF and BV (F value was 452.16 and 421.33 in the BF and BV, respectively, P <0.01); (2) The maximum diameter of cryoablation-induced necrosis was (2.3±0.3)cm in group A, (1.5±0.2) cm in group B, and (0.8±0.1) cm in group C, respectively. The difference was significant among the groups (F value was 315.32,P <0.01). (3) There were well-defined frozen areas, bordering areas and normal surrounding areas in MTT staining. In group C, positive staining around some blood vessels could be seen. Conclusion: Alteration of the blood flow in the hepatic artery can affect the cryoablation efficacy. With the decrease of hepatic artery blood flow, the efficacy of cryoablation on liver tumor

  4. Concentration- and time-dependent response of human gingival fibroblasts to fibroblast growth factor 2 immobilized on titanium dental implants

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    Ma Q

    2012-04-01

    Full Text Available Qianli Ma1*, Wei Wang1*, Paul K Chu2, Shenglin Mei1,2, Kun Ji3, Lei Jin4, Yumei Zhang11Department of Prosthetic Dentistry, School of Stomatology, Fourth Military Medical University, Xi'an, People's Republic of China; 2Department of Physics and Materials Science, City University of Hong Kong, Kowloon, Hong Kong, People's Republic of China; 3Department of Pediatric Dentistry, School of Stomatology, Fourth Military Medical University, Xi'an, People's Republic of China; 4Stomatology Department, Jinling Hospital, School of Medicine, Southern Medical University, Nanjing, People's Republic of China*These authors contributed equally to this workBackground: Titanium (Ti implants are widely used clinically, but peri-implantitis remains one of the most common and serious complications. Healthy integration between gingival tissue and the implant surface is critical to long-term success in dental implant therapy. The objective of this study was to investigate how different concentrations of immobilized fibroblast growth factor 2 (FGF2 on the titania nanotubular surface influence the response of human gingival fibroblasts (HGFs.Methods: Pure Ti metal was anodized at 20 V to form a vertically organized titanium dioxide nanotube array on which three concentrations of FGF2 (250 ng/mL, 500 ng/mL, or 1000 ng/mL were immobilized by repeated lyophilization. Surface topography was observed and FGF2 elution was detected using enzyme-linked immunosorbent assay. The bioactivity changes of dissolvable immobilized FGF2 were measured by methyl-thiazolyl-tetrazolium assay. Behavior of HGFs was evaluated using adhesion and methyl-thiazolyl-tetrazolium bromide assays.Results: The FGF2 remained for several days on the modified surface on which HGFs were cultured. Over 90% of the dissolvable immobilized FGF2 had been eluted by Day 9, whereas the FGF2 activity was found to diminish gradually from Day 1 to Day 9. The titania nanotubular surface with an optimal preparing

  5. Measurement of infarct size and percentage myocardium infarcted in a dog preparation with single photon-emission computed tomography, thallium-201, and indium 111-monoclonal antimyosin Fab

    International Nuclear Information System (INIS)

    Johnson, L.L.; Lerrick, K.S.; Coromilas, J.

    1987-01-01

    Single photon-emission tomography (SPECT) and indium 111-labeled monoclonal antimyosin Fab fragments were used to measure myocardial infarct size in 12 dogs, six subjected to balloon catheter-induced coronary artery occlusion for 6 hr (late reperfusion) and six subjected to occlusion with reperfusion at 2 hr (early reperfusion). Tomographic imaging was performed 24 hr after the intravenous injection of labeled Fab fragments with the use of a dual-head SPECT camera with medium-energy collimators. Immediately after the first tomographic scan, thallium-201 was injected into nine of 12 dogs and imaging was repeated. Estimated infarct size in grams was calculated from transaxially reconstructed, normalized, and background-corrected indium SPECT images with the use of a threshold technique for edge detection. Estimated noninfarcted myocardium in grams was calculated from obliquely reconstructed thallium SPECT images by a similar method. The animals were killed and infarct size in grams and true infarct size as a percentage of total left ventricular myocardial volume were measured by triphenyl tetrazolium chloride staining. Estimated infarct size from indium SPECT images showed an excellent correlation with true infarct size (r = .95, SEE = 4.1 g). Estimated percentage myocardium infarcted was calculated by dividing estimated infarct size from indium images by the sum of estimated infarct size plus estimated noninfarcted myocardium obtained from thallium images. Correlation between the estimated percentage of myocardium infarcted and true percentage of myocardium infarcted was excellent

  6. TTC- Based Test as an Efficient Method to Determine Antibiofilm Activity of Silver Nanoparticles

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    Chojniak Joanna

    2017-01-01

    Full Text Available Among metal nanoparticles, silver nanoparticles are a widely used in various life sectors such as in biomedical applications, air and water purification, food production, cosmetics, garments and in various household products. There are several methods for production of silver nanoparticles. Generally, silver nanoparticles can be prepared by chemical methods such as chemical reduction and electrochemical techniques, physical methods, and biological methods such as the use of microorganisms. The biological route of synthesis provides a great diversity in choice for its raw materials such as bacteria, algae, fungi and plants. The aim of the study was to evaluate the tetrazolium/formazan test as a method to determine antibiofilm activity of biological synthetized silver nanoparticles. In this study Bacillus subtilis grown on brewery effluent and produced biosurfactant was used for silver nanoparticles (Ag-NPs synthesis. The culture supernatants were used in synthesis of Ag-NPs. The formation of nanoparticles accompanied by colour changes of the used reaction system was confirmed by UV-vis spectroscopy. The bacteria isolated from the biofilm of water supply system were used in the evaluation of the antibiofilm activity of biologically synthetized Ag-NPs. To compare the results the commonly used crystal violet assay (CV for biofilm analysis was applied.

  7. LncRNA Taurine-Upregulated Gene 1 Promotes Cell Proliferation by Inhibiting MicroRNA-9 in MCF-7 Cells.

    Science.gov (United States)

    Zhao, Xiao-Bo; Ren, Guo-Sheng

    2016-12-01

    This study was designed to investigate the role of taurine-upregulated gene 1 ( TUG1 ) in MCF-7 breast cancer cells and the molecular mechanism involved in the regulation of microRNA-9 (miR-9). The expression of TUG1 in breast cancer tissues and cells was evaluated using quantitative reverse transcription polymerase chain reaction. Cell viability was examined using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay; cell cycle progression and apoptosis were analyzed using flow cytometry. A dual luciferase reporter assay was used to detect the relationship between TUG1 and miR-9. The expression of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) was measured by western blot. Higher expression of TUG1 was observed in breast cancer tissues and cell lines than in the corresponding controls. TUG1 knockdown reduced proliferation, suppressed cell cycle progression, and promoted apoptosis of MCF-7 cells. The dual luciferase reporter assay showed that TUG1 could negatively regulate the expression of miR-9. MiR-9 inhibition abrogated the effect of TUG1 knockdown on the proliferation, cell cycle progression, and apoptosis of MCF-7 cells. TUG1 positively regulated the expression of MTHFD2 in breast cancer cells. TUG1 knockdown was significantly associated with decreased cell proliferation and it promoted apoptosis of breast cancer cells through the regulation of miR-9.

  8. An in vitro study of the long non-coding RNA TUG1 in tongue squamous cell carcinoma.

    Science.gov (United States)

    Li, Zhi-Qiang; Zou, Rui; Ouyang, Ke-Xiong; Ai, Wei-Jian

    2017-11-01

    This study sought to study the expression of the long non-coding RNA (lncRNA) taurine-upregulated gene 1 (TUG1) in tongue squamous cell carcinoma (TSCC) and reveal its possible function. qRT-PCR was used to evaluate 27 samples of fresh TSCC tissues and adjacent normal tongue tissues. siRNA technology was employed to downregulate TUG1 expression in CAL-27 and SCC-9 cell lines. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay was utilized to assess cell proliferation ability; apoptosis and cell-cycle phases were analysed via flow cytometry. qRT-PCR findings indicated that the lncRNA TUG1 was upregulated in TSCC tissues compared with adjacent normal tongue tissues (PTUG1 expression was downregulated using siRNA technology, cell proliferation was significantly inhibited (PTUG1 may represent a potential oncogene in TSCC. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  9. A Novel Photosensitizer 31,131-phenylhydrazine -Mppa (BPHM and Its in Vitro Photodynamic Therapy against HeLa Cells

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    Wenting Li

    2016-04-01

    Full Text Available Photodynamic therapy (PDT has attracted widespread attention due to its potential in the treatment of various cancers. Porphyrinic pyropheophorbide-a (PPa has been shown to be a potent photosensitizer in PDT experiments. In this paper, a C-31,131 bisphenylhydrazone modified methyl pyropheophorbide-a (BPHM was designed and synthesized with the consideration that phenylhydrazone structure may extend absorption wavelength of methyl pyro-pheophorbide-a (Mppa, and make the photosensitizer potential in deep tumor treatment. The synthesis, spectral properties and in vitro photodynamic therapy (PDT against human HeLa cervical cancer cell line was studied. Methyl thiazolyl tetrazolium (MTT assay showed the title compound could achieve strong inhibition of cervical cancer cell viability under visible light (675 nm, 25 J/cm2. Cell uptake experiments were performed on HeLa cells. Morphological changes were examined and analyzed by fluorescent inverted microscope. In addition, the mechanism of the photochemical processes of PDT was investigated, which showed that the formation of singlet oxygen after treatment with PDT played a moderate important role.

  10. A Novel Photosensitizer 3¹,13¹-phenylhydrazine -Mppa (BPHM) and Its in Vitro Photodynamic Therapy against HeLa Cells.

    Science.gov (United States)

    Li, Wenting; Tan, Guanghui; Cheng, Jianjun; Zhao, Lishuang; Wang, Zhiqiang; Jin, Yingxue

    2016-04-29

    Photodynamic therapy (PDT) has attracted widespread attention due to its potential in the treatment of various cancers. Porphyrinic pyropheophorbide-a (PPa) has been shown to be a potent photosensitizer in PDT experiments. In this paper, a C-3¹,13¹ bisphenylhydrazone modified methyl pyropheophorbide-a (BPHM) was designed and synthesized with the consideration that phenylhydrazone structure may extend absorption wavelength of methyl pyro-pheophorbide-a (Mppa), and make the photosensitizer potential in deep tumor treatment. The synthesis, spectral properties and in vitro photodynamic therapy (PDT) against human HeLa cervical cancer cell line was studied. Methyl thiazolyl tetrazolium (MTT) assay showed the title compound could achieve strong inhibition of cervical cancer cell viability under visible light (675 nm, 25 J/cm²). Cell uptake experiments were performed on HeLa cells. Morphological changes were examined and analyzed by fluorescent inverted microscope. In addition, the mechanism of the photochemical processes of PDT was investigated, which showed that the formation of singlet oxygen after treatment with PDT played a moderate important role.

  11. Andrographolide radiosensitizes human esophageal cancer cell line ECA109 to radiation in vitro.

    Science.gov (United States)

    Wang, Z-M; Kang, Y-H; Yang, X; Wang, J-F; Zhang, Q; Yang, B-X; Zhao, K-L; Xu, L-P; Yang, L-P; Ma, J-X; Huang, G-H; Cai, J; Sun, X-C

    2016-01-01

    To explore the radiosensitivity of andrographolide on esophageal cancer cell line ECA109. The inhibition effects of andrographolide were measured using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) assay. Clonogenic survival assay was used to evaluate the effects of andrographolide on the radiosensitivity of esophageal cancer cells. Immunofluorescence was employed to examine Bax expression. The changes in cell cycle distribution and apoptosis were assayed using flow cytometry. The expression of NF-κb/Cleaved-Caspase3/Bax/Bcl-2 was measured using Western blot analysis. DNA damage was detected via γ-H2AX foci counting. With a clear dose and time effects, andrographolide was found to inhibit the proliferation of esophageal cell line ECA109. The results of the clonogenic survival assay show that andrographolide could markedly enhance radiosensitivity (P Andrographolide caused a dose-dependent increase in Cleaved-Caspase3/Bax protein expression and a decrease in Bcl-2/NF-κb expression. Apoptosis in andrographolide-treated ECA-109 increased significantly compared with the apoptosis in the simple drug and radiation combined with drug groups (P andrographolide combined with radiation group increased the number of DNA double chain breaks. Andrographolide can increase the radiosensitivity of esophageal cell line ECA109. This result may be associated with the decrease in the NF-κb level and the induced apoptosis of esophageal cancer cells. © 2014 International Society for Diseases of the Esophagus.

  12. CONSERVATION OF THE VIABILITY AND VIGOR OF Araucaria angustifolia (Bert. O. Kuntze SEEDS DURING THE STORAGE

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    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.

  13. 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.

  14. Effects of Krill Oil on serum lipids of hyperlipidemic rats and human SW480 cells

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    Qian Wen-Bin

    2008-08-01

    Full Text Available Abstract Background Cardiovascular disease (CVD and colon cancer incidence are known to be closely related to dietary factors. This article evaluated effects of krill oil (KO on serum lipids of hyperlipidemia rats and human colon cancer cells (SW480. Serum lipids of rats fed with high fat diet (HFD and different doses of KO were measured by automatic analyzer. Effect of KO on viability of cells was determined by methyl thiazolyl tetrazolium (MTT assay. Results Except for higher dose group, body weights decreased significantly. Total cholesterol (TC, LDL-cholesterol (LDL-C of all dose groups, Triglycerides (TG of low and mid dose groups descended significantly, while there were no significant differences of HDL-cholesterol (HDL-C, compared with control group. Treatment of colon cancer cells with KO also resulted in time-dependent inhibition of cell growth. Conclusion Our findings indicated that the consumption of KO may provide benefits to control serum lipid levels in certain diseases and inhibit growth of colon cancer cells. Therefore, KO may be a good candidate for development as a functional food and nutraceutical.

  15. Mannotriose regulates learning and memory signal transduction in the hippocampus

    Institute of Scientific and Technical Information of China (English)

    Lina Zhang; Weiwei Dai; Xueli Zhang; Zhangbin Gong; Guoqin Jin

    2013-01-01

    Rehmannia is a commonly used Chinese herb, which improves learning and memory. However, the crucial components of the signal transduction pathway associated with this effect remain elusive. Pri-mary hippocampal neurons were cultured in vitro, insulted with high-concentration (1 × 10-4 mol/L) cor-ticosterone, and treated with 1 × 10-4 mol/L mannotriose. Thiazolyl blue tetrazolium bromide assay and western blot analysis showed that hippocampal neuron survival rates and protein levels of glucocorti-coid receptor, serum and glucocorticoid-regulated protein kinase, and brain-derived neurotrophic factor were al dramatical y decreased after high-concentration corticosterone-induced injury. This effect was reversed by mannotriose, to a similar level as RU38486 and donepezil. Our findings indicate that mannotriose could protect hippocampal neurons from high-concentration corticosterone-induced injury. The mechanism by which this occurred was associated with levels of glucocorticoid receptor protein, serum and glucocorticoid-regulated protein kinase, and brain-derived neurotrophic factor.

  16. Synthesis and Cell Seeding Assessment of Novel Biphasic Nano Powder in the CaO–MgO–SiO2 System for Bone Implant Application

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    Kazem Marzban

    2017-02-01

    Full Text Available Objective(s: CaO–MgO–SiO2 system bioceramics possess good characteristics for hard tissue engineering applications. The aim of the study was to synthesize the nano powder by using a sol-gel method and evaluate of bioactivity in the cells culture. Methods: To characterize of powder X-ray diffraction (XRD, transmission electron microscopy (TEM and to evaluate the bioactivity sample cell seeding and methylthiazol tetrazolium (MTT assay were performed. Results: X-ray diffraction (XRD analysis showed that the biphasic powder was obtained at 1300°C for 2 h by using a sol-gel method. Transmission electron microscopy (TEM image showed that powder particle size was about 45 nm. Besides, cell culture results indicated that the percentage of viability values was increased by the extension of period. Conclusions: found that the sample is cytocompatible and has cell proliferation potential in culture medium. The present study demonstrates that, the biphasic CaO–MgO–SiO2 system can be used to achieve novel bioactive materials for bone implant application.

  17. 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.

    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. PMID:28042599

  18. Bacterial Endophytes Isolated from Plants in Natural Oil Seep Soils with Chronic Hydrocarbon Contamination.

    Science.gov (United States)

    Lumactud, Rhea; Shen, Shu Yi; Lau, Mimas; Fulthorpe, Roberta

    2016-01-01

    The bacterial endophytic communities of four plants growing abundantly in soils highly contaminated by hydrocarbons were analyzed through culturable and culture-independent means. Given their tolerance to the high levels of petroleum contamination at our study site, we sought evidence that Achillea millefolium, Solidago canadensis, Trifolium aureum, and Dactylis glomerata support high levels of hydrocarbon degrading endophytes. A total of 190 isolates were isolated from four plant species. The isolates were identified by partial 16S rDNA sequence analysis, with class Actinobacteria as the dominant group in all species except S. canadensis, which was dominated by Gammaproteobacteria. Microbacterium foliorum and Plantibacter flavus were present in all the plants, with M. foliorum showing predominance in D. glomerata and both endophytic bacterial species dominated T. aureum. More than 50% of the isolates demonstrated degradative capabilities for octanol, toluene, naphthalene, kerosene, or motor oil based on sole carbon source growth screens involving the reduction of tetrazolium dye. P. flavus isolates from all the sampled plants showed growth on all the petroleum hydrocarbons (PHCs) substrates tested. Mineralization of toluene and naphthalene was confirmed using gas-chromatography. 16S based terminal restriction fragment length polymorphism analysis revealed significant differences between the endophytic bacterial communities showing them to be plant host specific at this site. To our knowledge, this is the first account of the degradation potential of bacterial endophytes in these commonly occurring pioneer plants that were not previously known as phytoremediating plants.

  19. Noscapine protects OLN-93 oligodendrocytes from ischemia-reperfusion damage: Calcium and nitric oxide involvement.

    Science.gov (United States)

    Nadjafi, S; Ebrahimi, S-A; Rahbar-Roshandel, N

    2015-12-01

    This study was carried out to evaluate the effects of noscapine, a benzylisoquinoline alkaloid from opium poppy, on oligodendrocyte during ischemia/reperfusion-induced excitotoxic injury. Changes in intracellular calcium levels due to chemical ischemia and nitric oxide (NO) production during ischemia/reperfusion were evaluated as the hallmarks of ischemia-derived excitotoxic event. OLN-93 cell line (a permanent immature rat oligodendrocyte) was used as a model of oligodendrocyte. 30- or 60-minute-oxygen-glucose deprivation/24 hours reperfusion were used to induce excitotoxicity. MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay was used to evaluate cell viability. Ratiometric fluorescence microscopy using Ca(2+)-sensitive indicator Fura-2/AM was utilized to assess intracellular calcium levels. NO production was evaluated by Griess method. Noscapine (4 μM) significantly attenuated intracellular Ca(2+) elevation (P < 0.001). Also, noscapine significantly decreased NO production during a 30-minute oxygen-glucose deprivation/reperfusion (P < 0.01). The inhibitory effect of noscapine (4 μM) on intracellular Ca(2+) was greater than ionotropic glutamate receptors antagonists. Noscapine is protective against ischemia/reperfusion-induced excitotoxic injury in OLN-93 oligodendrocyte. This protective effect seems to be related to attenuation of intracellular Ca(2+) overload and NO production.

  20. 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.

  1. Variation of weigh and viability of seeds of Pinus tropicalis from different populations

    Directory of Open Access Journals (Sweden)

    Marta Bonilla Vichot

    2014-06-01

    Full Text Available Pinus tropicalis Morelet is endemic in the province of Pinar del Rio and Isla de la Juventud. In this paper the variation is evaluated in weight and viability of seeds from 4 seed areas of the province of Pinar del Rio, Cuba, which were harvested in July and stored for five months at room temperature until They were made to the corresponding analysis. Samples of each of the sources weretaken to determine the weight of 1000 seeds, as established methodology ISTA (1993. Viability was also determined from the tetrazolium Test. It was observed that the individual weight of the seeds of Pinus tropicalis has 0,0051g values to 0,050g, while the average weight of 1000 seeds shows variation by origin, showing the greatest weight to the seeds from the seed mass Ceja del Negro (orchard seedling genetically enhanced trees, which directly influences the quality of seeds. The viability was also superior in the origin of Ceja del Negro.

  2. 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.

  3. Detection of irradiated mushrooms by GC/MS analysis of lipid-derived hydrocarbons

    International Nuclear Information System (INIS)

    Delincee, H.; Koller, W.D.

    1993-01-01

    A number of methods has been developed for the detection of irradiated foods in recent years, and in the case of mushrooms several methods have been proposed, of which the thermoluminescence (TL) measurements seem to be the most valuable. However, in several cases mineral contamination of fresh mushrooms is so extremely low that not enough minerals can be isolated for TL analysis. In that case an alternative method is needed to detect the radiation treatment of mushrooms. Several methods including TTC (2,3,5-triphenyl-tetrazolium-chloride) staining, kinetin treatment, dropping out of spores and mirco-gel electrophoresis of spores, were tested, but the most promising method was the GC/MS analysis of radiation-induced lipid-derived hydrocarbons in spite of the low fat content - around 0.2-0.3% - of mushrooms. Successful results were achieved by GC/MS analysis of the radiolytic hydrocarbons. Although mushrooms have a low fat content, by extracting a large quantity, in this case 500 g of mushrooms, about 1.2-1.5 g of fat could be obtained. The main fatty acids of mushroom fat and some of their expected cleavage products on irradiation - the c n-1 hydrocarbon which has one C atom less than the parent fatty acid and the C n-2:1 hydrocarbon, which has two C atoms less and an additional double bond in position 1 - are given. (orig./Vhe)

  4. Characterization of chemical compounds for dosimetry of the radiation in industrial processes; Caracterizacao de compostos quimicos para dosimetria das radiacaoes em processos industriais

    Energy Technology Data Exchange (ETDEWEB)

    Galante, Ana Maria Sisti

    1999-07-01

    Different chemical compounds have been studied to optimize dosimetric systems in irradiation processes. In this study 2,3,5 Triphenyl -2H- Tetrazolium Chloride, Brilliant Cresyl Blue, Bromocresol Green and Potassium Nitrate were investigated for their merits or faults, for {sup 60} Co gamma field, in order to verify if can be considered as dosimeters. Fricke solution was used as reference dosimeter to determine absorption dose rates at the gamma facilities.Only Bromocresol Green and Potassium Nitrate are recommended for dosimetry purposes since the main characteristics were achieved. The other two compounds could be used in dosimetry with changes in their formulation. Bromocresol Green and potassium Nitrate are reproducible and radiation sensitive for absorbed doses from 300 Gy to 150 kGy Bromocresol Green was used in liquid form and Potassium Nitrate was prepared in solid pellets form. Spectrophotometry in the visible region was used as the main detection technique, which allows relating optical absorption, before and after irradiation, with the absorbed dose. The maximum absorption wavelength for each compound was observed at 450-460nm for bromocresol Green and 546nm for Potassium Nitrate. Dose calibration curves are linear for both compounds in all dose intervals. When irradiated with accelerated electrons, with energies between o,9 MeV and 1,5MeV, optical absorption intensification, of about 2,6 times, was observed when comparing results for Potassium Nitrate, with those for gamma rays. All the evaluations are presented in this work. (author)

  5. 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.

  6. 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.

  7. 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.

  8. A three-stage-integrative approach for the identification of potential hepatotoxic compounds from botanical products.

    Science.gov (United States)

    Jin, Yecheng; Zhao, Xiaoping; Zhang, Yufeng; Li, Xiang; Nie, Xiaojing; Wang, Yi; Fan, Xiaohui

    2011-05-01

    With the increasing use of herbal medicines and dietary supplements, intensive concerns about their potential toxicities have been raised. Screening and identifying the toxic compounds from these botanical products composed by hundreds of components have become a critical but challenging problem. In this study, 3 methods, including fraction separation, an in-house-developed fluorescein diacetate-based automatic microscopy screening (FAMS) platform, and liquid chromatography-mass spectrometry-based compounds identification were integrated within the Three-Stage-Integrative (TSI) approach for the identification of potential hepatotoxicants from botanical products. The sensitivity and linear range of FAMS assay was validated and compared with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay by previously reported hepatotoxic compounds. The success of TSI approach was further demonstrated by its application to Fructus aristolochiae. Aristolochic acid IVa and aristolodione were tentatively identified to be potential hepatotoxicants in this plant. These applications suggested that our TSI approach provides an effective tool for identifying potential toxic compounds from botanical products.

  9. Assessment of cellular viability on calcium sulphate/hydroxyapatite injectable scaffolds

    Directory of Open Access Journals (Sweden)

    Randa Alfotawi

    2013-10-01

    Full Text Available Cements for maxillofacial reconstruction of jaw defects through calcification of rotated muscle have been tested. The objective of this study was to investigate the visibility of loading of two types of commercially available cements, Cerament™ Spine Support and Cerament Bone Void Filler with mesenchymal cells and cytokines (bone morphogenetic protein to act as a biomimetic scaffolding for future clinical application. Determination of basic biocompatibility (cell viability using methyl thiazolyl tetrazolium and live/dead assay was carried out using MG-63 cells at various time points. Next, in order to inform potential subsequent in vivo experiments, a collagen tissue mimic was used for characterization of rabbit mesenchymal stromal cells using immunofluorescent cytoskeleton staining, and simultaneous and then sequential injection of Cerament Spine Support cement and cells into collagen gels. Results indicated that Cerament Spine Support was more biocompatible and that sequential injection of cement and then rabbit mesenchymal stromal cells into the tissue mimics is an optimal approach for clinical applications.

  10. Effect of essential oils prepared from Thai culinary herbs on sessile Candida albicans cultures.

    Science.gov (United States)

    Hovijitra, Ray S; Choonharuangdej, Suwan; Srithavaj, Theerathavaj

    2016-01-01

    Although medicinal herbs with fungicidal effects have been ubiquitously employed in traditional medicine, such effects of culinary herbs and spices still have to be elucidated. Therefore, it is noteworthy to determine the antifungal efficacy of some edible herbs used in Thai cuisine against sessile Candida albicans cultures, and to inquire if they can be further utilized as naturally-derived antifungals. Fourteen essential oils extracted from Thai culinary herbs and spices were tested for their antifungal activity against C. albicans using the agar disk diffusion method followed by broth micro-dilution method for the determination of minimum inhibitory concentration (MIC) and minimum fungicidal concentration. The oils with potent antifungal effects against planktonic fungi were then assessed for their effect against sessile fungus (adherent organisms and established biofilm culture). MIC of the oils against sessile C. albicans was evaluated by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide reduction assay. All selected culinary herbs and spices, except galangal, garlic, and turmeric, exhibited inhibitory effects on planktonic yeast cells. Cinnamon bark and sweet basil leaf essential oils exhibited potent fungicidal effect on planktonic and sessile fungus. Sessile MICs were 8-16 times higher than planktonic MICs. Consequently, both cinnamon bark and sweet basil leaf herbal oils seem to be highly effective anti-Candida choices. (J Oral Sci 58, 365-371, 2016).

  11. The research on biodistribution of 131I-iodosennoside A in normal mice and to evaluate myocardial activity

    International Nuclear Information System (INIS)

    Wang Junhu; Yin Zhiqi; Jiang Cuihua; Jiang Xiao; Li Yue; Zhang Jian; Sun Ziping; Ni Yicheng

    2013-01-01

    Purpose: The objective of this project is to evaluate biodistribution of [ 131 I]-Iodosennoside A in normal mice and explore the feasibility on the diagnosis of myocardial infarction. Methods: Iodogen method was used to radioiodinate sennoside A with 131 I. [ 131 I] Iodosennoside A was intravenously injected into mice. Three groups of mice were killed at 4 h, 24 h and 48 h post injection respectively and the radioactive uptake in major organs were calculated. Rats were subjected to left anterior descending (LAD) coronary artery ligation to induce acute myocardial infarction. Rat models of myocardial infarction were intravenously injected [ 131 I] iodosennoside A. 24 h after injection of [ 131 I] iodosennoside A, the regional distribution of radioiodinated sennoside A was determined by radioactivity counting technique. 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining and autoradiography were per- formed with 2 mm thick sections of hearts for postmortem verifications. Results: The study showed high uptake of [ 131 I] iodosennoside A in kidneys and fast blood clearance. At 24 h post injection, radioactivity concentration in infarcted myocardium was over 11.9 times higher than in normal myocardium. Preferential uptake of the [ 131 I] iodosennoside A in necrotic tissue was confirmed by perfect match of images from TTC staining and autoradiography. Conclusion: The result proved that [ 131 I] iodosennoside A has myocardial necrosis affinity and may serve as a marker on the diagnosis of myocardial infarction. (authors)

  12. Biocompatibility studies of polyacrylonitrile membranes modified with carboxylated polyetherimide

    Energy Technology Data Exchange (ETDEWEB)

    Senthilkumar, S.; Rajesh, S.; Jayalakshmi, A.; Mohan, D., E-mail: mohantarun@gmail.com

    2013-10-15

    Poly (ether-imide) (PEI) was carboxylated and used as the hydrophilic modification agent for the preparation of polyacrylonitrile (PAN) membranes. Membranes were prepared with different blend compositions of PAN and CPEI by diffusion induced precipitation. The modified membranes were characterized by thermo gravimetric analysis (TGA), mechanical analysis, scanning electron microscopy (SEM) and contact angle measurement to understand the influence of CPEI on the properties of the membranes. The biocompatibility studies exhibited reduced plasma protein adsorption, platelet adhesion and thrombus formation on the modified membrane surface. The complete blood count (CBC) results of CPEI incorporated membranes showed stable CBC values and significant decrease in the complement activation were also observed. In addition to good cytocompatibility, monocytes cultured on these modified membranes exhibited improved functional profiles in 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Thus it could be concluded that PAN/CPEI membranes with excellent biocompatibility can be useful for hemodialysis. Highlights: • Carboxylated PEI was prepared and utilized as hydrophilic modification agent. • CPEI incorporated into PAN to improved biocompatibility and cyto compatibility • Biocompatibility of membranes was correlated with morphology and hydrophilicity. • Antifouling studies of the PAN/CPEI membranes was studied by BSA as model foulant.

  13. Spirolactone and spirothiolactone rhodamine-pyrene probes for detection of Hg²⁺ with different sensing properties and its application in living cells.

    Science.gov (United States)

    Rui, Qing-Qing; Zhou, Yi; Fang, Yuan; Yao, Cheng

    2016-04-15

    Two new rhodamine B-based fluorescent probes PyRbS and PyRbO containing pyrene moiety were designed and synthesized. Both of the probes showed colorimetric and fluorometric sensing abilities for Hg(2+) with high selectivity over other metal ions. The binding analysis using Job's plot suggested 1:1 stoichiometry for the complexes formed for Hg(2+). Compared with PyRbO, the PyRbS showed higher selectivity and sensitivity due to the thiophilic property of Hg(2+) ion. The PyRbS exhibited the linear fluorescence quenching to Hg(2+) in the range of 0.3 to 4.8 μM (λ(ex)=365 nm) and 0.3 to 5.4 μM (λ(ex)=515 nm), and the detection limit was 0.72 μM. Moreover, ratiometric changes of PyRbS with Hg(2+) in absorption spectrum were observed, which could not be obtained in the combination of PyRbO with Hg(2+). In addition, the methyl thiazolyl tetrazolium (MTT) assay demonstrated that RbPyS had low cytotoxicity and was successfully used to monitor intracellular Hg(2+) levels in living cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Caffeine Increases Apolipoprotein A-1 and Paraoxonase-1 but not Paraoxonase-3 Protein Levels in Human-Derived Liver (HepG2) Cells.

    Science.gov (United States)

    Sayılan Özgün, Gülben; Özgün, Eray; Tabakçıoğlu, Kıymet; Süer Gökmen, Selma; Eskiocak, Sevgi; Çakır, Erol

    2017-12-01

    Apolipoprotein A-1, paraoxonase-1 and paraoxonase-3 are antioxidant and anti-atherosclerotic structural high-density lipoprotein proteins that are mainly synthesized by the liver. No study has ever been performed to specifically examine the effects of caffeine on paraoxonase enzymes and on liver apolipoprotein A-1 protein levels. To investigate the dose-dependent effects of caffeine on liver apolipoprotein A-1, paraoxonase-1 and paraoxonase-3 protein levels. In vitro experimental study. HepG2 cells were incubated with 0 (control), 10, 50 and 200 μM of caffeine for 24 hours. Cell viability was evaluated by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Apolipoprotein A-1, paraoxonase-1 and paraoxonase-3 protein levels were measured by western blotting. We observed a significant increase on apolipoprotein A-1 and paraoxonase-1 protein levels in the cells incubated with 50 µM of caffeine and a significant increase on paraoxonase-1 protein level in the cells incubated with 200 µM of caffeine. Our study showed that caffeine does not change paraoxonase-3 protein level, but the higher doses used in our study do cause an increase in both apolipoprotein A-1 and paraoxonase-1 protein levels in liver cells.

  15. Effect of Genistein and 17-β Estradiol on the Viability and Apoptosis of Human Hepatocellular Carcinoma HepG2 cell line

    Directory of Open Access Journals (Sweden)

    Masumeh Sanaei

    2017-01-01

    Full Text Available Background: One of the most lethal cancers is hepatocellular carcinoma (HCC. Genistein (GE is a choice compound for treatment of certain types of cancer. Phytoestrogens are plant derivatives that bear a structural similarity to 17-β estradiol (E2 and act in a similar manner. They are a group of lipophillic plant compounds with tumorigenic and antitumorigenic effects. E2 has stimulatory and inhibitory effects on cancer cell lines. This study was designed to investigate the antiproliferative and apoptotic effects of GE and E2 on the HCC HepG2 cell line. Materials and Methods: HepG2 cells were cultured and treated with various concentrations of GE and E2 and then 3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromideand flow cytometry assay were performed to determine cell viability and apoptosis. Results: GE and E2 induced apoptosis and inhibited cell growth significantly. Reduction of cell viability by 50% required 20 μM E2 for E2-treatment groups and 20 μMGE for GE-treatment groups. The percentage of the GE-treated apoptotic cells was reduced by about 35%, 42%, and 47% (P < 0.001 and that of E2-treated groups 34%, 39%, and 42% (P < 0.001 after 24, 48, and 72 h, respectively. Conclusions: Our experimental work clearly demonstrated that GE and E2 exhibited significant antiproliferative and apoptotic effects on human HCC HepG2 cells.

  16. Water-stable NaLuF4-based upconversion nanophosphors with long-term validity for multimodal lymphatic imaging.

    Science.gov (United States)

    Zhou, Jing; Zhu, Xingjun; Chen, Min; Sun, Yun; Li, Fuyou

    2012-09-01

    Multimodal imaging is rapidly becoming an important tool for biomedical applications because it can compensate for the deficiencies of individual imaging modalities. Herein, multifunctional NaLuF(4)-based upconversion nanoparticles (Lu-UCNPs) were synthesized though a facile one-step microemulsion method under ambient condition. The doping of lanthanide ions (Gd(3+), Yb(3+) and Er(3+)/Tm(3+)) endows the Lu-UCNPs with high T(1)-enhancement, bright upconversion luminescence (UCL) emissions, and excellent X-ray absorption coefficient. Moreover, the as-prepared Lu-UCNPs are stable in water for more than six months, due to the protection of sodium glutamate and diethylene triamine pentacetate acid (DTPA) coordinating ligands on the surface. Lu-UCNPs have been successfully applied to the trimodal CT/MR/UCL lymphatic imaging on the modal of small animals. It is worth noting that Lu-UCNPs could be used for imaging even after preserving for over six months. In vitro transmission electron microscope (TEM), methyl thiazolyl tetrazolium (MTT) assay and histological analysis demonstrated that Lu-UCNPs exhibited low toxicity on living systems. Therefore, Lu-UCNPs could be multimodal agents for CT/MR/UCL imaging, and the concept can be served as a platform technology for the next-generation of probes for multimodal imaging. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. Green synthesis of silver nanoparticles from aqueous leaf extract of Pomegranate (Punica granatum) and their anticancer activity on human cervical cancer cells

    Science.gov (United States)

    Sarkar, Sonia; Kotteeswaran, Venkatesan

    2018-06-01

    Plants contain different important phytochemicals that can be used as a potential treatment for various ailments including cancer. The green synthesis of silver nanoparticles from the extract of different plant parts has gained a wide range of engrossment among the researchers due to its unique optical and structural property. The aim of this study is green synthesis of silver nanoparticles from the aqueous leaf extract of pomegranate (Punica granatum) and to investigate its anticancer activity on human cervical cancer cells (HeLa). The synthesis of silver nanoparticle was depicted by the colour change from golden yellowish to dark brownish, UV-visible spectral analysis gave a characteristic surface plasmon absorption peak at . Further morphological characterization was done by Zeta potential where the size analysis was depicted to be 46.1 nm and zeta potential as . Fourier transform infrared spectroscopy (FTIR) inferred 3 intense sharp peaks at , , , confirmed the presence of flavonoids and polyphenols. The scanning electron microscopy (SEM) analysis with energy diffraction spectroscopy (EDS) confirmed the presence of silver nanoparticles with size ranged from to . X-ray diffraction (XRD) confirmed the crystallographic nature of silver. The cell proliferation activity of nanoparticles was tested by 3, ‑4, 5 dimethylthiazol-2,5 diphenyl tetrazolium bromide (MTT) assay where the inhibitory concentration () was found at inhibiting of HeLa cell line. The anticancer activity of nanoparticles was determined by lactate dehydrogenase (LDH) assay where showed of cytotoxicity. Furthermore, the anticancer property of nanoparticles was confirmed by the DNA fragmentation assay.

  18. Antifungal and Anti-Biofilm Activities of Acetone Lichen Extracts against Candida albicans

    Directory of Open Access Journals (Sweden)

    Marion Millot

    2017-04-01

    Full Text Available Candida albicans is a commensal coloniser of the human gastrointestinal tract and an opportunistic pathogen, especially thanks to its capacity to form biofilms. This lifestyle is frequently involved in infections and increases the yeast resistance to antimicrobials and immune defenses. In this context, 38 lichen acetone extracts have been prepared and evaluated for their activity against C. albicans planktonic and sessile cells. Minimum inhibitory concentrations of extracts (MICs were determined using the broth microdilution method. Anti-biofilm activity was evaluated using tetrazolium salt (XTT assay as the ability to inhibit the maturation phase (anti-maturation or to eradicate a preformed 24 h old biofilm (anti-biofilm. While none of the extracts were active against planktonic cells, biofilm maturation was limited by 11 of the tested extracts. Seven extracts displayed both anti-maturation and anti-biofilm activities (half maximal inhibitory concentrations IC50_mat and IC50_biof ≤ 100 µg/mL; Evernia prunastri and Ramalina fastigiata were the most promising lichens (IC50_mat < 4 µg/mL and IC50_biof < 10 µg/mL. Chemical profiles of the active extracts performed by thin layer chromatography (TLC and high performance liquid chromatography (HPLC have been analyzed. Depsides, which were present in large amounts in the most active extracts, could be involved in anti-biofilm activities. This work confirmed that lichens represent a reservoir of compounds with anti-biofilm potential.

  19. Fluoride injury symptoms in epiphytic lichens and mosses

    Energy Technology Data Exchange (ETDEWEB)

    Leblanc, F; Comeau, G; Rao, D N

    1971-01-01

    This study was conducted in Arvida, Quebec, where volatile fluorides and HF are released into the atmosphere from an aluminum factory. Lichen and moss-bearing bark discs, cut out from trees in an unpolluted area, were fixed in groups of six in rectangular wooden boards. All discs were photographed in color as well as in black and white. Fifteen sites, in addition to a control site were selected in different directions from the factory. At each site two boards were fixed with nails in a vertical position at a height of 8-10 m on a tree. One board was removed after 4 months exposure and the other after 12 months. The lichens and mosses exposed in control and polluted areas were compared with respect to their color, external morphology, plasmolysis in algal cells, loss of green color, and nature of reactions towards neutral red and 2,3,5-triphenyl-2h-tetrazolium chloride (ttc), absorption spectra of chlorophyll, and fluoride concentration. Results indicate that f-pollution affects moisture balance, causes chlorophyll damage, and produces other symptoms of injury which could lead to ultimate death of these organisms.

  20. Physiological seed quality of varieties of Ocimum produced under conditions of Valle del Cauca, Colombia

    Directory of Open Access Journals (Sweden)

    Leila Aceneth Durán Gaviria

    2016-01-01

    Full Text Available The genus Ocimum with over 150 species is characterized by high morphological variability and Chemotypes of great value in the industries of perfumery, cosmetics, food and pharmaceutical features. In order to evaluate the physiological quality of the seeds, in the Experimental Center and the Laboratory of Plant Physiology at the National University of Colombia at Palmira, germination and viability tests were performed with 2,3,5 triphenyl tetrazolium, TTZ. In trials, using a factorial design with five varieties of seeds, three treatments and three replications, the effect of imbibing the seeds for five minutes in Potassium nitrate 0.2% KNO3 and gibberellic acid GA3 250 ppm and 500 ppm respectively as first irrigation and a control (water was evaluated. The results showed that the seeds from the species O. selloi had the highest percentage (90% of viability in TTZ and germination (46.2%. The seeds soaked in 0.2% KNO3 showed the highest values of seed germination in three of the five varieties tested. The best responses were obtained with germination seeds O.selloi species (70% and O. micranthum (58%, followed by Ocimum sp. (26%. The results showed the presence of varying levels of latency, which was partially overcome by the application of potassium nitrate (KNO 3 0.2%.

  1. Investigation of Antileishmanial Effect of Alcoholic Extract and Essential Oil of Medicinal Plant Leaf Black Alfalfa (Medicago Lupulina, on The Number of Clinical Isolates of Leishmania Major Promastigotes in Vitro

    Directory of Open Access Journals (Sweden)

    E Gharirvand Eskandari

    2016-05-01

    Full Text Available Introduction: Leishmaniasis has created enormous global health problems. Side effects, drug resistance and the lack of effective vaccines had led to the new effective compounds effective of plants. The aim of this study was to introduce a traditional medicinal plant called Black alfalfa (Medicago Lupulina that can be used as a valuable resource against cutaneous leishmaniasis. Methods: In this experimental study, alcoholic extract was prepared by maceration and essential oil by distillation water method. Leishmania major promastigotes were cultured at 25 ± 2° C in N.N.N culturemedium, then in Schneider and next were cultured in RPMI- 1640. afterward, using MTT (Methyl Thiazole Tetrazolium, the IC50 (Inhibitory Concentrations 50% for extracts, essence and Glucantime were determined. The results using Tukey and t-test were analyzed and were presented by software SPSS16. MTT assay were repeated. 3 times for each sample. Results: IC50 for alfalfa leaf extract and essential oil of black alfalfa and Glucantime against L. major promastigotes was determined after 24, 48 and 72 hours, 240, 130 and 69 micrograms per ml, and 801, 340 and 190 micrograms per ml, also 26, 19 and 11 micrograms per ml , respectively. There was a significant differences between the IC50 plant extract and essential oil and Glucantime after 24, 48 and 72 hours. Conclusion: Alcoholic extracts and essential oil the plant had significant anti leishmaniasis effects in vitro. In this way, it can be considered as an anti-leishmaniasis among the herbs.

  2. Scavenging capacity of strawberry tree (Arbutus unedo L.) leaves on free radicals.

    Science.gov (United States)

    Oliveira, Ivo; Coelho, Valentim; Baltasar, Raquel; Pereira, José Alberto; Baptista, Paula

    2009-07-01

    Despite strawberry tree (Arbutus unedo L.) leaves had a long use in traditional medicine due to its antiseptic, diuretic, astringent and depurative properties, the potential of their antioxidant activity are still lacking. Our study goals to assess the antioxidant and free radical scavenging potential of water, ethanol, methanol and diethyl ether extracts of A. unedo leaves. Total phenols content was achieved spectrophotometrically using Folin-Ciocalteau reagent with gallic acid as standard. Antioxidant activity was evaluated using three different methods: reducing power of iron (III)/ferricyanide complex assay, scavenging effect on DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals and scavenging effect on superoxide radicals by using the PMS-NADH-nitroblue tetrazolium system. Ethanol extracts of A. unedo leaves were the highest in reducing power (IC(50) 232.7 microg/mL) and DPPH scavenging effect (IC(50) 63.2 microg/mL) followed by water extracts (with IC(50) of 287.7 and 73.7 microg/mL, respectively); whereas diethyl ether extracts were the lowest. In the scavenging on superoxide radical assay, methanol extracts obtained the best results (IC(50) 6.9 microg/mL). For all the methods tested the antioxidant activity was concentration dependent. In accordance with antioxidant activity, highest total phenols content were found in ethanol, followed by water, methanol and diethyl ether extract. The results indicated that A. unedo leaves are a potential source of natural antioxidants.

  3. Effects of water-current speed on hematological, biochemical and immune parameters in juvenile tinfoil barb, Barbonymus schwanenfeldii (Bleeker, 1854)

    Science.gov (United States)

    Zhu, Zhiming; Song, Bolan; Lin, Xiaotao; Xu, Zhongneng

    2016-01-01

    This study examines the effect of water-current speed on hematological, biochemical and immune parameters in juvenile tinfoil barb ( Barbonymus schwanenfeldii). Blood samples were taken on days 1, 23 and 45 from control fish and from two training groups maintained at current speeds of 0.06 bl/s (body length per second), 0.66 bl/s, and 1.92 bl/s, respectively. Significantly increased red-blood-cell counts and hematocrit were observed in the post-training groups on days 23 and 45. Significantly increased hemoglobin concentrations were also observed in the 1.92 bl/s group on days 23 and 45. In contrast, values of mean corpuscular volume were significantly lower in the 1.92 bl/s group than in the other groups on day 45. Nitroblue-tetrazolium-positive cells and lysozyme and superoxidase dismutase activities in the plasma increased significantly with increasing training intensity on days 23 and 45. Antibacterial activities were significantly increased in the trained groups compared with the control group on day 23; significantly elevated alkaline phosphatase activity was observed in the 1.92 bl/s groups on day 45. Therefore, training intensities of 0.66 and 1.92 bl/s enhanced the blood oxygen-carrying capability and plasma immune parameters of juvenile tinfoil barbs.

  4. Novel Hydrogel-Advanced Modified Clay Nanocomposites as Possible Vehicles for Drug Delivery and Controlled Release

    Directory of Open Access Journals (Sweden)

    Raluca Ianchis

    2017-12-01

    Full Text Available Present study refers to the synthesis of new advanced materials based on poly(methacrylic acid (PMAA with previously reported own advanced modified clays by edge covalent bonding. This will create the premises to obtain nanocomposite hydrogels with combined hydrophilic-hydrophobic behavior absolutely necessary for co-delivery of polar/nonpolar substances. For the synthesis, N,N’-methylenebisacrylamide was used as cross-linker and ammonium persulphate as initiator. As a consequence of the inclusion of clay into the polymer matrix and the intercalation of PMAA between the layers as well as the presence of hydrophobic interactions occurred between partners, the final hydrogel nanocomposites possessed greater swelling degrees, slower de-swelling process and enhanced mechanical properties depending on the clay type in comparison with pure hydrogel. In vitro MTS ([3-(4,5-dimethylthiazol-2-yl-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl-2H-tetrazolium, inner salt] colorimetric assay showed that direct exposure with PMMA-clay-based constructs did not affect cell viability and proliferation in time (24 and 48 h on either normal or adenocarcinoma cell lines.

  5. Novel Hydrogel-Advanced Modified Clay Nanocomposites as Possible Vehicles for Drug Delivery and Controlled Release.

    Science.gov (United States)

    Ianchis, Raluca; Ninciuleanu, Claudia M; Gifu, Ioana C; Alexandrescu, Elvira; Somoghi, Raluca; Gabor, Augusta R; Preda, Silviu; Nistor, Cristina L; Nitu, Sabina; Petcu, Cristian; Icriverzi, Madalina; Florian, Paula E; Roseanu, Anca M

    2017-12-13

    Present study refers to the synthesis of new advanced materials based on poly(methacrylic acid) (PMAA) with previously reported own advanced modified clays by edge covalent bonding. This will create the premises to obtain nanocomposite hydrogels with combined hydrophilic-hydrophobic behavior absolutely necessary for co-delivery of polar/nonpolar substances. For the synthesis, N , N '-methylenebisacrylamide was used as cross-linker and ammonium persulphate as initiator. As a consequence of the inclusion of clay into the polymer matrix and the intercalation of PMAA between the layers as well as the presence of hydrophobic interactions occurred between partners, the final hydrogel nanocomposites possessed greater swelling degrees, slower de-swelling process and enhanced mechanical properties depending on the clay type in comparison with pure hydrogel. In vitro MTS ([3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H -tetrazolium, inner salt]) colorimetric assay showed that direct exposure with PMMA-clay-based constructs did not affect cell viability and proliferation in time (24 and 48 h) on either normal or adenocarcinoma cell lines.

  6. 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.

  7. Cationic Gelatin Nanoparticles for Drug Delivery to the Ocular Surface: In Vitro and In Vivo Evaluation

    Directory of Open Access Journals (Sweden)

    Ching-Li Tseng

    2013-01-01

    Full Text Available To develop an effective ocular drug delivery carrier, we prepared two different charged gelatin nanoparticles (GPs and evaluated particle size, surface charge, and morphology. The in vitro biocompatibility of GPs was assessed using human corneal epithelium (HCE cells and in vivo safety by administering them as eye drops to New Zealand rabbits. The GPs prepared using type A gelatin were positively charged (GP(+, +33 mV; size, 180.6±45.7 nm. Water-soluble tetrazolium salt (WST-1 assay showed that both GPs were nontoxic to HCE cells. The fluorescence intensity of HCE cells cultured with cationic GPs conjugated with a fluorescent dye was higher than that of the anionic GP-treated HCE cells. In vivo examination showed no serious irritation to the rabbit eyes. Furthermore, corneal thickness and ocular pressure in the eyes of the treated rabbits were similar to those in the eyes of normal rabbits. Microscopic examination of corneal cryosections showed widely distributed fluorescent nanocarriers, from the anterior to the posterior part of the cornea of the GP(+ group, and higher fluorescence intensity in the GP(+ group was also observed. In conclusion, GPs as cationic colloidal carriers were efficiently adsorbed on the negatively charged cornea without irritating the eyes of the rabbits and can be retained in the cornea for a longer time. Thus, GPs(+ have a great potential as vehicles for ocular drug delivery.

  8. Antiproliferation and induction of cell death of Phaffia rhodozyma (Xanthophyllomyces dendrorhous) extract fermented by brewer malt waste on breast cancer cells.

    Science.gov (United States)

    Teo, Ivy Tuang Ngo; Chui, Chung Hin; Tang, Johnny Cheuk On; Lau, Fung Yi; Cheng, Gregory Yin Ming; Wong, Raymond Siu Ming; Kok, Stanton Hon Lung; Cheng, Chor Hing; Chan, Albert Sun Chi; Ho, Kwok Ping

    2005-11-01

    Astaxanthin has been shown to have antiproliferative activity on breast cancer and skin cancer cells. However, the high cost of production, isolation and purification of purified astaxanthin from natural sources or chemically synthetic methods limit its usage on cancer therapy. We show that astaxanthin could be produced by fermentating the Phaffia rhodozyma (Xanthophyllomyces dendrorhous) yeast cells with brewer malt waste using a 20 L B. Braun fermentor. The percentage composition of astaxanthin from the P. rhodozyma was >70% of total pigment as estimated by the high performance liquid chromatographic analysis. Furthermore, the antiproliferative activity of this P. rhodozyma cell extract (PRE) was demonstrated on breast cancer cell lines including the MCF-7 (estrogen receptor positive) and MDA-MB231 (estrogen receptor negative) by using the [3-(4,5-dimethylthiazol-2-yl)-5-(3-arboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium] (MTS) assay. No apoptotic cell death, but growth inhibitory effect was induced after 48 h of PRE incubation as suggested by morphological investigation. Anchorage-dependent clonogenicity assay showed that PRE could reduce the colony formation potential of both breast cancer cell lines. Cell death was observed from both breast cancer cell lines after incubation with PRE for 6 days. Taken together, our results showed that by using an economic method of brewer malt waste fermentation, we obtained P. rhodozyma with a high yield of astaxanthin and the corresponding PRE could have short-term growth inhibition and long-term cell death activity on breast cancer cells.

  9. Exploring the influence of culture conditions on kefir's anticancer properties.

    Science.gov (United States)

    Hatmal, Ma'mon M; Nuirat, Abeer; Zihlif, Malek A; Taha, Mutasem O

    2018-05-01

    Cancer is a major health problem in many parts of the world. Conventional anticancer treatments are painful, expensive, and unsafe. Therefore, demand is increasing for cancer treatments preferentially in the form of functional foods or nutritional supplements. Kefir, a traditional fermented milk dairy product, has significant antimutagenic and antitumor properties. This research addresses the hypothesis that kefir's anticancer properties are affected by fermentation conditions. Initially, kefir extracts prepared under standard conditions were screened against 7 cancer cell lines using the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay. Colon cancer and chronic myelogenous leukemia cells were found to be most susceptible to kefir extracts. Subsequently, a factorial design was implemented to assess the effects of 3 fermentation times (24, 48, and 72 h), 3 kefir-to-milk ratios (2, 5, and 10% wt/vol), and 3 fermentation temperatures (4, 25, and 40°C) on kefir's anticancer properties. Remarkably, exploration of the fermentation conditions allowed the anticancer properties of kefir to be enhanced by 5- to 8-fold against susceptible cell lines. Overall, these results demonstrate the possibility of optimizing the anticancer properties of kefir as a functional food in cancer therapy. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  10. Gemcitabine inhibits proliferation and induces apoptosis in human pancreatic cancer PANC-1 cells.

    Science.gov (United States)

    Yong-Xian, Gui; Xiao-Huan, Li; Fan, Zhang; Guo-Fang, Tian

    2016-10-01

    The aim of the study is to investigate the underlying molecular mechanisms by which gemcitabine (gem) inhibits proliferation and induces apoptosis in human pancreatic cancer PANC-1 cells in vitro. After PANC-1 cells had been treated by indicated concentration (0, 5, and 25 mg/L) of gem for 48 h, cell proliferation was evaluated by 3'-(4, 5 dimethyl-thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay; cell morphology was observed by transmission electron microscopy; Expression of c-IAP2 and Bcl-2 proteins was analyzed by Western blot; the activity of caspase-3 and -9 was detected by spectrophotometry. Gem significantly inhibited cell proliferation and could induce apoptosis of human pancreatic cancer PANC-1 cells, with a dose-dependent manner. Western blot analysis showed that gem significantly reduced c-IAP2 and Bcl-2 proteins expression level (P PANC-1 cells. Gem could induce apoptosis of human pancreatic cancer PANC-1 cells, probably through downregulating c-IAP2 and Bcl-2 expression levels, and at the same time activating caspase-3 and -9.

  11. 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.

  12. Tecoma sambucifolia: anti-inflammatory and antinociceptive activities, and 'in vitro' toxicity of extracts of the 'huarumo' of peruvian incas.

    Science.gov (United States)

    Alguacil, L F; Galán de Mera, A; Gómez, J; Llinares, F; Morales, L; Muñoz-Mingarro, M D; Pozuelo, J M; Vicente Orellana, J A

    2000-06-01

    Aqueous and alcoholic extracts of pods and flowers of Tecoma sambucifolia H.B.K. (Bignoniaceae) ('huarumo') were analysed to determine their anti-inflammatory activity (carrageenan-induced edema test), antinociceptive activity (acetic acid writhing test) and 'in vitro' toxicity in Chinese hamster ovary cells, human hepatome cells and human larynx epidermal carcinoma cells. The cytotoxic effects of both extracts were evaluated by two endpoint systems: neutral red uptake assay and tetrazolium assay. The results showed that all extracts have anti-inflammatory and antinociceptive activity, but the highest potency is that of the alcoholic extracts. There were significant differences in cytotoxicity between extracts and among the response of cells to them. The highest cytotoxicity was noted with the alcoholic extract, and the human hepatome cell line was the most sensitive, especially to the alcoholic extract of flowers. The aqueous pod extract appeared to have the best pharmaco-toxicological profile, since it provided a significant reduction of both pain and inflammation together with the lowest cytotoxicity.

  13. Cytotoxic activity of abietane diterpenoids from roots of Salvia sahendica by HPLC-based activity profiling

    Directory of Open Access Journals (Sweden)

    Fahimeh Moradi-Afrapoli

    Full Text Available ABSTRACT Screening of medicinal plants from Iranian flora against human cancer cell-lines have shown that an hexane extract from roots of Salvia sahendica Boiss. & Buhse, Lamiaceae, is active against human cervical cancer (HeLa and colorectal adenocarcinoma (Caco-2 cell-lines at the test concentration of 100 µg/ml (100% inhibition. Cytotoxicity of the extract was localized with the aid of HPLC-time-based activity profiling adapted to the tetrazolium colorimetric bioassay. Four abietane-type diterpenoids in active time-windows were identified as cytotoxic compounds namely: sahandone (1, sahandol (2, 12-deoxy-salvipisone (3 and sahandinone (4. Compound 1 showed the highest toxicity against HeLa cells (IC50 = 5.6 ± 0.1 µg/ml, which was comparable with betulinic acid (IC50 = 4.3 ± 1.2 µg/ml, the positive control. Compound 2 was active against the HeLa cells (IC50 = 8.9 ± 0.7 µg/ml but not the Caco-2 cell-line. Compounds 1, 3 and 4 exhibited moderate activity (IC50 = 22.9–41.4 µg/ml against the Caco-2 cells. This study reveals that the HeLa cells are more sensitive to all tested compounds than the Caco-2 cells. In silico molecular docking study showed a rigid binding of the compounds to tyrosine kinase pp60src, and proved their cytotoxic activity.

  14. Taraxinic acid, a hydrolysate of sesquiterpene lactone glycoside from the Taraxacum coreanum NAKAI, induces the differentiation of human acute promyelocytic leukemia HL-60 cells.

    Science.gov (United States)

    Choi, Jung-Hye; Shin, Kyung-Min; Kim, Na-Young; Hong, Jung-Pyo; Lee, Yong Sup; Kim, Hyoung Ja; Park, Hee-Juhn; Lee, Kyung-Tae

    2002-11-01

    The present work was performed to elucidate the active moiety of a sesquiterpene lactone, taraxinic acid-1'-O-beta-D-glucopyranoside (1). from Taraxacum coreanum NAKAI on the cytotoxicity of various cancer cells. Based on enzymatic hydrolysis and MTT assay, the active moiety should be attributed to the aglycone taraxinic acid (1a). rather than the glycoside (1). Taraxinic acid exhibited potent antiproliferative activity against human leukemia-derived HL-60. In addition, this compound was found to be a potent inducer of HL-60 cell differentiation as assessed by a nitroblue tetrazolium reduction test, esterase activity assay, phagocytic activity assay, morphology change, and expression of CD 14 and CD 66 b surface antigens. These results suggest that taraxinic acid induces the differentiation of human leukemia cells to monocyte/macrophage lineage. Moreover, the expression level of c-myc was down-regulated during taraxinic acid-dependent HL-60 cell differentiation, whereas p21(CIP1) and p27(KIP1) were up-regulated. Taken together, our results suggest that taraxinic acid may have potential as a therapeutic agent in human leukemia.

  15. 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

    Directory of Open Access Journals (Sweden)

    P. A. S. R. Santos

    2016-01-01

    Full Text Available 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.

  16. 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.

  17. Ultrastructural and metabolic changes in osteoblasts exposed to uranyl nitrate

    International Nuclear Information System (INIS)

    Tasat, D.R.; Orona, N.S.; Mandalunis, P.M.; Cabrini, R.L.; Ubios, A.M.

    2007-01-01

    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 μM) 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 μM 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 μM 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. 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) 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.

  19. Free radical scavenging activity and neuroprotective potentials of D138, one Cu(II)/Zn(II) Schiff-base complex derived from N,N'-bis(2-hydroxynaphthylmethylidene)-1,3-propanediamine.

    Science.gov (United States)

    Wang, Che; Cai, Zheng-Xu; You, Zhong-Lu; Guo, Hui-Shu; Shang, De-Jing; Wang, Xiao-Ling; Zhang, Liang; Ma, Li-Jie; Tan, Jun; Le, Wei-Dong; Li, Song

    2014-09-01

    There is increasing evidence that free radicals play an important role in neuronal damages induced by diabetes mellitus or cerebral ischemia insults. Antioxidants with free radical scavenging activities have been shown to be beneficial and neuroprotective for these pathological conditions. Here, we report free radical scavenging activity and neuroprotective potential of D138, one copper(II)/zinc(II) Schiff-base complex derived from N,N'-2(2-hydroxynaphthylmethylidene)-1,3-propanediamine. The data from three in vitro assays, 2,2-diphenyl-1-picrylhydrazyl assay, nitro blue tetrazolium assay and hydroxyl radical scavenging assay, indicated that D138 presented a potent free radical scavenging activity. The neuroprotective and antioxidative effects of D138 were further evaluated in vivo using bilateral common carotid artery occlusion (BCCAO) mouse model and streptozotocin (STZ) diabetic mouse model. Our results indicated that treatment of D138 significantly ameliorated the hippocampal neuronal damage and the oxidative stress levels in these animal models. Moreover, D138 also reversed the behavioral deficiencies induced by BCCAO or STZ, as assessed by Y-maze test and fear conditioning test. In conclusion, all these findings support that D138 exerts free radical scavenging and neuroprotective activities and has the potentials to be a potent therapeutic candidate for brain oxidative damage induced by cerebral ischemia or diabetes mellitus.

  20. Accumulation of polymorphonuclear leukocytes in reperfused ischemic canine myocardium: relation with tissue viability assessed by fluorine-18-2-deoxyglucose uptake

    International Nuclear Information System (INIS)

    Wijns, W.; Melin, J.A.; Leners, N.

    1988-01-01

    Polymorphonuclear leukocytes may participate in reperfusion injury. Whether leukocytes affect viable or only irreversibly injured tissue is not known. Therefore, we assessed the accumulation of 111In-labeled leukocytes in tissue samples characterized as either ischemic but viable or necrotic by metabolic, histochemical, and ultrastructural criteria. Six open-chest dogs received left anterior descending coronary occlusion for 2 hr followed by 4 hr reperfusion. Myocardial blood flow was determined by microspheres and autologous 111In-labeled leukocytes were injected intravenously. Fluorine-18-2-deoxyglucose, a tracer of exogenous glucose utilization, was injected 3 hr after reperfusion. The dogs were killed 4 hr after reperfusion. The risk and the necrotic regions were assessed following in vivo dye injection and postmortem tetrazolium staining. Myocardial samples were obtained in the ischemic but viable, necrotic and normal zones, and counted for 111In and 18F activity. Compared to normal, leukocytes were entrapped in necrotic regions (111In activity: 207 +/- 73%) where glucose uptake was decreased (26 +/- 15%). A persistent glucose uptake, marker of viability, was mainly seen in risk region (135 +/- 85%) where leukocytes accumulation was moderate in comparison to normal zone (146 +/- 44%). Thus, the glucose uptake observed in viable tissue is mainly related to myocytes metabolism and not to leukocytes metabolism

  1. The Impact of the Roast Levels of Coffee Extracts on their Potential Anticancer Activities.

    Science.gov (United States)

    Mojica, Benigno E; Fong, Lisa E; Biju, Denny; Muharram, Alfeah; Davis, Isabel M; Vela, Klarisse O; Rios, Diana; Osorio-Camacena, Elena; Kaur, Baljit; Rojas, Sebastian M; Forester, Sarah C

    2018-04-01

    Coffee is one of the most widely consumed beverages in the world and contains numerous phytochemicals that are beneficial to consumer health. The phytochemical profile of coffee, however, can be affected by the roast level. In this study, we compared the effect of roasting level on the growth inhibitory activity of HT-29 (colon) and SCC-25 (oral) cancer cell lines. The different roasting stages selected for this study were green, cinnamon/blonde, city/medium, full city/medium-dark, and full city plus/dark. Cancer cells were treated with various concentrations of coffee extracts for 72 hr. Cell viability was quantified using the thiazolyl blue tetrazolium bromide assay. It was found that the lighter roast extracts, Cinnamon in particular, reduced cell growth more than darker roast extracts. The Cinnamon extract had the greatest amount of total phenolic content and antioxidant activity. Relative levels of gallic, caffeic, and chlorogenic acid in the extracts were also compared. The Cinnamon coffee extract had the highest levels of gallic and caffeic acids, which have both been widely-regarded as bioactive phytochemicals. In conclusion, the consumption of lighter roasted coffee, may contribute to the prevention of certain types of cancer such as oral and colon. Chemical compounds in coffee may reduce the risk for certain types of cancers. These compounds may be particularly abundant in lighter roasted coffee. Therefore, lighter roasted coffee could contribute to the prevention of cancer through a healthy diet. © 2018 Institute of Food Technologists®.

  2. Study on the enhanced cellular uptake effect of daunorubicin on leukemia cells mediated via functionalized nickel nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Guo Dadong; Wu Chunhui; Hu Hongli; Wang Xuemei [State Key Lab of Bioelectronics (Chien-Shiung Wu Lab), Southeast University, Nanjing 210096 (China); Li Xiaomao [Department of Physics, University of Saarland, D-66041 Saarbruecken (Germany); Chen Baoan, E-mail: xuewang@seu.edu.c [Zhongda Hospital, School of Clinical Medical, Southeast University, Nanjing 210096 (China)

    2009-04-15

    The success of cancer chemotherapy is largely dependent on the efficient anticancer drug accumulation in target tumor tissues and cells so as to inhibit the proliferation of the cancer cells. Recently, some biocompatible nanomaterials have been utilized as drug target delivery systems and have shown the great potential to effectively afford the sustained drug delivery for the target cancer cells. In this study, we have explored the possibility for the bio-application of the functionalized nickel (Ni) nanoparticles and the efficiency of the functionalized Ni nanoparticles on drug permeability, and cellular uptake of leukemia K562 cells in vitro has been probed via atomic force microscopy, inverted fluorescence microscopy and confocal microscopy, electrochemical study and MTT (3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl-tetrazolium bromide) assay. It is observed that the presence of relevant Ni nanoparticles could induce the membrane structure change of target cells and efficiently improve the permeability of the cell membrane so that the combination of these Ni nanoparticles with anticancer drug daunorubicin could have a synergistic effect on the efficient cytotoxicity suppression in leukemia cancer cells. These observations indicate the great potential of Ni nanoparticles in the future biomedical application including target cancer diagnosis and chemotherapy.

  3. Cytotoxic effect of microbial biosurfactants against human embryonic kidney cancerous cell: HEK-293 and their possible role in apoptosis.

    Science.gov (United States)

    Pradhan, Arun Kumar; Pradhan, Nilotpala; Mohapatra, Purusottam; Kundu, Chanakya Nath; Panda, Prasanna Kumar; Mishra, Barada Kanta

    2014-11-01

    Two different microbial biosurfactants S9BS and CHBS were isolated from Lysinibacillus fusiformis S9 and Bacillus tequilensis CH. Cytotoxicity effect of these biosurfactants on human embryonic kidney cancerous cell (HEK-293) were studied with the help of 3-(4,5-dimethylthiazol-2yl-)-2, 5-diphenyl tetrazolium bromide (MTT) assay and morphological changes were observed under inverted microscope. The biosurfactants exhibited positive cytotoxic effect on HEK-293 cell line. It was found that LC50 of S9BS and CHBS were 75 and 100 μg ml(-1), respectively. Further cell cycle and apoptosis analysis of biosurfactant-treated HEK-293 cell line were done by FACS. In this study, cytotoxic effect of glycolipid biosurfactant against HEK-293 cell lines is reported for the first time. Mechanism towards increased membrane permeability of biosurfactant-treated cancer cell may be the incorporation of its lipid moiety into the plasma membrane leading to formation of pores and membrane disruption. Hence, these microbial biosurfactants can prove to be significant biomolecule for cancer treatment.

  4. Bortezomib interactions with chemotherapy agents in acute leukemia in vitro.

    Science.gov (United States)

    Horton, Terzah M; Gannavarapu, Anurhadha; Blaney, Susan M; D'Argenio, David Z; Plon, Sharon E; Berg, Stacey L

    2006-07-01

    Although there is effective chemotherapy for many patients with leukemia, 20% of children and up to 65% of adults relapse. Novel therapies are needed to treat these patients. Leukemia cells are very sensitive to the proteasome inhibitor bortezomib (VELCADE(R), PS-341), which enhances the in vitro cytotoxic effects of dexamethasone and doxorubicin in multiple myeloma. To determine if bortezomib enhances the cytotoxicity of agents used in leukemia, we employed an in vitro tetrazolium-based colorimetric assay (MTT) to evaluate the cytotoxic effects of bortezomib alone and in combination with dexamethasone, vincristine, doxorubicin, cytarabine, asparaginase, geldanamycin, trichostatin A, and the bcl-2 inhibitor HA14.1. We demonstrated that primary leukemia lymphoblasts and leukemia cell lines are sensitive to bortezomib, with an average IC(50) of 12 nM. Qualitative and quantitative bortezomib-drug interactions were evaluated using the universal response surface approach (URSA). Bortezomib was synergistic with dexamethasone in dexamethasone-sensitive leukemia cells, and additive with vincristine, asparaginase, cytarabine, and doxorubicin. The anti-leukemic activity of bortezomib was also additive with geldanamycin and HA14.1, and additive or synergistic with trichostatin A. These results were compared to analysis using the median-dose effect method, which generated complex drug interactions due to differences in dose-response curve sigmoidicities. These data suggest bortezomib could potentiate the cytotoxic effects of combination chemotherapy in patients with leukemia.

  5. Antiproliferative effects of cinobufacini on human hepatocellular carcinoma HepG2 cells detected by atomic force microscopy

    Science.gov (United States)

    Wu, Qing; Lin, Wei-Dong; Liao, Guan-Qun; Zhang, Li-Guo; Wen, Shun-Qian; Lin, Jia-Ying

    2015-01-01

    AIM: To investigate the antiproliferative activity of cinobufacini on human hepatocellular carcinoma HepG2 cells and the possible mechanism of its action. METHODS: HepG2 cells were treated with different concentrations of cinobufacini. Cell viability was measured by methylthiazolyl tetrazolium (MTT) assay. Cell cycle distribution was analyzed by flow cytometry (FCM). Cytoskeletal and nuclear alterations were observed by fluorescein isothiocyanate-phalloidin and DAPI staining under a laser scanning confocal microscope. Changes in morphology and ultrastructure of cells were detected by atomic force microscopy (AFM) at the nanoscale level. RESULTS: MTT assay indicated that cinobufacini significantly inhibited the viability of HepG2 cells in a dose-dependent manner. With the concentration of cinobufacini increasing from 0 to 0.10 mg/mL, the cell viability decreased from 74.9% ± 2.7% to 49.41% ± 2.2% and 39.24% ± 2.1% (P deep pores in the cell membrane, with larger particles and a rougher cell surface. CONCLUSION: Cinobufacini inhibits the viability of HepG2 cells via cytoskeletal destruction and cell membrane toxicity. PMID:25624718

  6. Impact of the Fenton process in meat digestion as assessed using an in vitro gastro-intestinal model.

    Science.gov (United States)

    Oueslati, Khaled; de La Pomélie, Diane; Santé-Lhoutellier, Véronique; Gatellier, Philippe

    2016-10-15

    The production of oxygen free radicals catalysed by non-haem iron was investigated in an in vitro mimetic model of the digestive tract using specific chemical traps. Superoxide radicals (O2(∗-)) and their protonated form (hydroperoxyl radicals, HO2(∗)) were detected by the reduction of nitroblue tetrazolium into formazan, and hydroxyl radicals (OH(∗)) were detected by the hydroxylation of terephthalate. Under gastric conditions, O2(∗-)/HO2(∗) were detected in higher quantity than OH(∗). Increasing the pH from 3.5 to 6.5 poorly affected the kinetics of free radical production. The oxidations generated by these free radicals were estimated on myofibrils prepared from pork rectus femoris muscle. Myofibrillar lipid and protein oxidation increased with time and oxidant concentration, with a negative impact on the digestibility of myofibrillar proteins. Plant food antioxidants considerably decreased free radical production and lipid oxidation but not protein oxidation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. 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.

  8. Modification of bone graft by blending with lecithin to improve hydrophilicity and biocompatibility

    International Nuclear Information System (INIS)

    Wang, Y; Cui, F Z; Jiao, Y P; Hu, K; Fan, D D

    2008-01-01

    Lecithin was blended to improve the hydrophilicity and biocompatibility of bone graft containing poly(l-lactic acid) (PLLA). Solution blending and freeze drying were used to fabricate symmetrical scaffolds containing different percentages of lecithin (lecithin: PLLA = 0, 5, 10 wt%). Scanning electron microscopy showed that the scaffolds maintained the three-dimensional porous structure. A water uptake experiment proved the significant improvement of hydrophilicity of the blend scaffold. With the addition of lecithin, the compressive strength and compressive modulus decreased. When the weight ratio of lecithin to PLLA was up to 10%, the compressive strength was still more than the lower limit of natural cancellous bone. To test the biocompatibility of the scaffolds, cell culture in vitro and subcutaneous implantation in vivo were performed. MC3T3-E1 preosteoblastic cells were cultured on the scaffolds for 7 days. Methylthiazol tetrazolium assay and laser scanning confocal microscopy were used to exhibit proliferation and morphology of the cells. The subcutaneous implantation in rats tested inflammatory response to the scaffolds. The results proved the better biocompatibility and milder inflammatory reactions of the blend scaffold (lecithin: PLLA = 5%) compared with the scaffold without lecithin. The modified scaffold containing lecithin is promising for bone tissue engineering

  9. Overexpression of YB1 C-terminal domain inhibits proliferation, angiogenesis and tumorigenicity in a SK-BR-3 breast cancer xenograft mouse model.

    Science.gov (United States)

    Shi, Jian-Hong; Cui, Nai-Peng; Wang, Shuo; Zhao, Ming-Zhi; Wang, Bing; Wang, Ya-Nan; Chen, Bao-Ping

    2016-01-01

    Y-box-binding protein 1 (YB1) is a multifunctional transcription factor with vital roles in proliferation, differentiation and apoptosis. In this study, we have examined the role of its C-terminal domain (YB1 CTD) in proliferation, angiogenesis and tumorigenicity in breast cancer. Breast cancer cell line SK-BR-3 was infected with GFP-tagged YB1 CTD adenovirus expression vector. An 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) proliferation assay showed that YB1 CTD decreased SK-BR-3 cell proliferation, and down-regulated cyclin B1 and up-regulated p21 levels in SK-BR-3 cells. YB1 CTD overexpression changed the cytoskeletal organization and slightly inhibited the migration of SK-BR-3 cells. YB1 CTD also inhibited secreted VEGF expression in SK-BR-3 cells, which decreased SK-BR-3-induced EA.hy926 endothelial cell angiogenesis in vitro. YB1 CTD overexpression attenuated the ability of SK-BR-3 cells to form tumours in nude mice, and decreased in vivo VEGF levels and angiogenesis in the xenografts in SK-BR-3 tumour-bearing mice. Taken together, our findings demonstrate the vital role of YB1 CTD overexpression in inhibiting proliferation, angiogenesis and tumorigenicity of breast cancer cell line SK-BR-3.

  10. Evaluation of genotoxicity and cytotoxicity of water samples from the Sinos River Basin, southern Brazil

    Directory of Open Access Journals (Sweden)

    E Bianchi

    Full Text Available Some water bodies in the Sinos River Basin (SRB have been suffering the effects of pollution by residential, industrial and agroindustrial wastewater. The presence of cytotoxic and genotoxic compounds could compromise the water quality and the balance of these ecosystems. In this context, the research aimed to evaluate the genotoxicity and cytotoxicity of the water at four sites along the SRB (in the cities of Santo Antônio da Patrulha, Parobé, Campo Bom and Esteio, using bioassays in fish and cell culture. Samples of surface water were collected and evaluated in vitro using the Astyanax jacuhiensis fish species (micronucleus test and comet assay and the Vero lineage of cells (comet assay and cytotoxicity tests, neutral red - NR and tetrazolium MTT. The micronucleus test in fish showed no significant differences between the sampling sites, and neither did the comet assay and the MTT and NR tests in Vero cells. The comet assay showed an increase in genetic damage in the fish exposed to water samples collected in the middle and lower sections of the basin (Parobé, Campo Bom and Esteio when compared to the upper section of the basin (Santo Antônio da Patrulha. The results indicate contamination by genotoxic substances starting in the middle section of the SRB.

  11. In Vitro Cytotoxic Effects of Cuscuta chinensis Whole Extract on Human Acute Lymphoblastic Leukemia Cell Line

    Directory of Open Access Journals (Sweden)

    Fatemeh Zeraati

    2010-12-01

    Full Text Available Background: One of the major paths for drug development isthe study of bioactivities of natural products. Therefore, theaim of this study was to compare the cytotoxic effects ofaqueous extract of whole Cuscuta chinensis Lam., which is atraditional medicinal herb commonly used in Iran and otheroriental countries, on the human caucasian acute lymphoblasticleukemia (CCRF-CEM and another human lymphocyte,Jurkat (JM cell lines.Methods: In vitro cytotoxic screening with various concentrations(0, 0.1, 1, 10, 25 and 50 μg/ml of the extract wasperformed using microscope and methyl tetrazolium bromidetest (MTT.Results: The minimum effective concentration of the plantextract was 1 μg/ml, and increasing the dose to 10 μg/mlinduced increasingly stronger effects. The inhibitory concentration50% (IC50 of the extract against CCRF wasabout 3 μg/ml in 24 hours and 2.5 μg/ml in 48 hrs. In contrast,the extract did not have cytotoxic effect for the JMcells at these doses.Conclusion: The findings of the present study suggest that C.chinensis is toxic against CCRF-CEM and JM tumor cells.Whether or not such effects can be employed for the treatmentof such tumors must await future studies.Iran J Med Sci 2010; 35(4: 310-314.

  12. In Vitro Pollen Viability and Pollen Germination in Cherry Laurel (Prunus laurocerasus L.

    Directory of Open Access Journals (Sweden)

    Melekber Sulusoglu

    2014-01-01

    Full Text Available Pollen quality is important for growers and breeders. This study was carried out to determine in vitro pollen viability and pollen germination in seven genotypes of cherry laurel (Prunus laurocerasus L.. Two pollen viability tests, TTC (2,3,5-triphenyl tetrazolium chloride and IKI (iodine potassium iodide, were used. Pollen traits of genotypes were studied using an in vitro medium containing 0%, 5%, 10%, 15%, and 20% sucrose to determine the best sucrose concentrations for germination. In the second step, the germinated pollen was counted 1, 4, 6, 10, 12, 24, and 48 hours later until there was no further germination. The viability rates were different according to genotypes and tests used. The IKI and TTC staining tests and pollen germination had low correlation (r2 = 0.0614 and r2 = 0.0015, resp.. Painted pollen rate was higher and pollen was well-stained with IKI test and pollen viability estimated with TTC staining test was better than that estimated with the IKI staining test. 15% sucrose gave the best germination rates in most of the genotypes. Pollen germination rates were recorded periodically from one hour to 48 hours in 15% sucrose and the results showed that pollen germination rates increased after 6 hours of being placed in culture media.

  13. In vitro pollen viability and pollen germination in cherry laurel (Prunus laurocerasus L.).

    Science.gov (United States)

    Sulusoglu, Melekber; Cavusoglu, Aysun

    2014-01-01

    Pollen quality is important for growers and breeders. This study was carried out to determine in vitro pollen viability and pollen germination in seven genotypes of cherry laurel (Prunus laurocerasus L.). Two pollen viability tests, TTC (2,3,5-triphenyl tetrazolium chloride) and IKI (iodine potassium iodide), were used. Pollen traits of genotypes were studied using an in vitro medium containing 0%, 5%, 10%, 15%, and 20% sucrose to determine the best sucrose concentrations for germination. In the second step, the germinated pollen was counted 1, 4, 6, 10, 12, 24, and 48 hours later until there was no further germination. The viability rates were different according to genotypes and tests used. The IKI and TTC staining tests and pollen germination had low correlation (r(2) = 0.0614 and r(2) = 0.0015, resp.). Painted pollen rate was higher and pollen was well-stained with IKI test and pollen viability estimated with TTC staining test was better than that estimated with the IKI staining test. 15% sucrose gave the best germination rates in most of the genotypes. Pollen germination rates were recorded periodically from one hour to 48 hours in 15% sucrose and the results showed that pollen germination rates increased after 6 hours of being placed in culture media.

  14. Standardization of metachromatic staining method of myofibrillar ATPase activity of myosin to skeletal striated muscle of mules and donkeys

    Directory of Open Access Journals (Sweden)

    Flora H.F. D'Angelis

    2014-09-01

    Full Text Available This study aims at standardizing the pre-incubation and incubation pH and temperature used in the metachromatic staining method of myofibrillar ATPase activity of myosin (mATPase used for asses and mules. Twenty four donkeys and 10 mules, seven females and three males, were used in the study. From each animal, fragments from the Gluteus medius muscle were collected and percutaneous muscle biopsy was performed using a 6.0-mm Bergström-type needle. In addition to the metachromatic staining method of mATPase, the technique of nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR was also performed to confirm the histochemical data. The histochemical result of mATPase for acidic pre-incubation (pH=4.50 and alkaline incubation (pH=10.50, at a temperature of 37ºC, yielded the best differentiation of fibers stained with toluidine blue. Muscle fibers were identified according to the following colors: type I (oxidative, light blue, type IIA (oxidative-glycolytic, intermediate blue and type IIX (glycolytic, dark blue. There are no reports in the literature regarding the characterization and distribution of different types of muscle fibers used by donkeys and mules when performing traction work, cargo transportation, endurance sports (horseback riding and marching competitions. Therefore, this study is the first report on the standardization of the mATPase technique for donkeys and mules.

  15. Preparation of Graphene Quantum Dots and Their Application in Cell Imaging

    Directory of Open Access Journals (Sweden)

    Jie Zhang

    2016-01-01

    Full Text Available Objective. This study aims to increase the fluorescence quantum yield by improving the conditions of preparing graphene quantum dots (GQDs through the solvothermal route and observe the GQDs performance in imaging oral squamous cells. Methodology. The following experimental conditions of GQDs preparation through the solvothermal route were improved: graphene oxide (GO/N-N dimethyl formamide (DMF ratio, filling percentage, and reaction time. A fluorescence spectrophotometer was used to measure photoluminescence, and the peak values were compared. Methylthiazolyldiphenyl-tetrazolium (MTT bromide was used to detect the cytotoxicity of GQDs, which was compared with that of cadmium telluride quantum dots (CdTe QDs. GQDs were cultured with tongue cancer cells. After the coculture, a laser scanning confocal microscope (LSCM was used to observe cell imaging. Results. The optimal conditions of GQD preparation through the solvothermal route included the following: 10 mg/mL GO/DMF ratio, 80% filling percentage, 12 h reaction time, and 17.4% fluorescence quantum yield. As the cell concentration increased, the GQD and CdTe QD groups exhibited a decreasing cell survival rate, with the decrease in the CdTe QD group being more significant. The LSCM observations showed bright green fluorescence images. Conclusion. The improved experimental conditions increased the fluorescence quantum yield of GQDs. In this study, the prepared GQDs exhibited low cytotoxicity level and satisfactory cell imaging performance.

  16. Radiation-induced reduction of ditetrazolium salt in aqueous solutions

    International Nuclear Information System (INIS)

    Sadeghi, Ali; Chaychian, Mahnaz; Al-Sheikhly, Mohamad; McLaughlin, W.L.

    2002-01-01

    Color formation in aqueous solutions of the ditetrazolium salt blue tetrazolium (BT 2+ ) in the absence or presence of oxygen is a complex radiation chemical reaction. The final stable product is the poorly soluble diformazan violet to blue pigment having a broad spectral absorption band (λ max =552 nm). The reaction of BT 2+ with the hydrated electron proceeds by rapid reduction of BT 2+ followed by protonation at the nitrogen closest to the unsubstituted phenyl group, via the two intermediate tetrazolinyl radicals shared by the ditetrazole ring nitrogens. The effect of solution pH, N 2 O saturation, and the presence of the reducing agent dextrose are examined. The system serves as a radiochromic sensor and a dosimeter of ionizing radiations. Solutions of 5 mmol l -1 BT 2+ at pH 7.3 serve as dosimeters over an absorbed-dose range of approximately 0.2-6 kGy (dearated, with a range of 1-8 mmol l -1 dextrose) and of about 1-15 kGy (aerated, with 0.1 mol l -1 sodium formate and 5 mmol l -1 dextrose)

  17. Electrochemical & osteoblast adhesion study of engineered TiO2 nanotubular surfaces on titanium alloys

    International Nuclear Information System (INIS)

    Rahman, Zia Ur; Haider, Waseem; Pompa, Luis; Deen, K.M.

    2016-01-01

    TiO 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 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 2 nanotubes were grafted on cpTi, Ti6Al4V and Ti6Al4V-ELI via anodization. • MC3T3 cells interact differently with nanotubes of different titanium alloys. • TiO 2 nanotubes have a positive impact on the osteoblast cell viability.

  18. Microelectrode Array-evaluation of Neurotoxic Effects of Magnesium as an Implantable Biomaterial.

    Science.gov (United States)

    Huang, Ting; Wang, Zhonghai; Wei, Lina; Kindy, Mark; Zheng, Yufeng; Xi, Tingfei; Gao, Bruce Z

    2016-01-01

    Magnesium (Mg)-based biomaterials have shown great potential in clinical applications. However, the cytotoxic effects of excessive Mg 2+ and the corrosion products from Mg-based biomaterials, particularly their effects on neurons, have been little studied. Although viability tests are most commonly used, a functional evaluation is critically needed. Here, both methyl thiazolyl tetrazolium (MTT) and lactate dehydrogenase (LDH) assays were used to test the effect of Mg 2+ and Mg-extract solution on neuronal viability. Microelectrode arrays (MEAs), which provide long-term, real-time recording of extracellular electrophysiological signals of in vitro neuronal networks, were used to test for toxic effects. The minimum effective concentrations (EC min ) of Mg 2+ from the MTT and LDH assays were 3 mmol/L and 100 mmol/L, respectively, while the EC min obtained from the MEA assay was 0.1 mmol/L. MEA data revealed significant loss of neuronal network activity when the culture was exposed to 25% Mg-extract solution, a concentration that did not affect neuronal viability. For evaluating the biocompatibility of Mg-based biomaterials with neurons, MEA electrophysiological testing is a more precise method than basic cell-viability testing.

  19. Determination of glycated hemoglobin in patients with advanced liver disease

    Science.gov (United States)

    Lahousen, Theresa; Hegenbarth, Karin; Ille, Rottraut; Lipp, Rainer W.; Krause, Robert; Little, Randie R.; Schnedl, Wolfgang J.

    2004-01-01

    AIM: To evaluate the glycated hemoglobin (HbA 1c) determination methods and to determine fructosamine in patients with chronic hepatitis, compensated cirrhosis and in patients with chronic hepatitis treated with ribavirin. METHODS: HbA1c values were determined in 15 patients with compensated liver cirrhosis and in 20 patients with chronic hepatitis using the ion-exchange high performance liquid chromatography and the immunoassay methods. Fructosamine was determined using nitroblue tetrazolium. RESULTS: Forty percent of patients with liver cirrhosis had HbA1c results below the non-diabetic reference range by at least one HbA1c method, while fructosamine results were either within the reference range or elevated. Twenty percent of patients with chronic hepatitis (hepatic fibrosis) had HbA1c results below the non -diabetic reference range by at least one HbA1c method. In patients with chronic hepatitis treated with ribavirin, 50% of HbA1c results were below the non-diabetic reference using at least one of the HbA1c methods. CONCLUSION: Only evaluated in context with all liver function parameters as well as a red blood count including reticulocytes, HbA 1c results should be used in patients with advanced liver disease. HbA 1c and fructosamine measurements should be used with caution when evaluating long-term glucose control in patients with hepatic cirrhosis or in patients with chronic hepatitis and ribavirin treatment. PMID:15259084

  20. Modulation of mitochondrial activity in HaCaT keratinocytes by the cell penetrating peptide Z-Gly-RGD(DPhe)-mitoparan.

    Science.gov (United States)

    Richardson, Adam; Muir, Lewis; Mousdell, Sasha; Sexton, Darren; Jones, Sarah; Howl, John; Ross, Kehinde

    2018-01-30

    Biologically active cell penetrating peptides (CPPs) are an emerging class of therapeutic agent. The wasp venom peptide mastoparan is an established CPP that modulates mitochondrial activity and triggers caspase-dependent apoptosis in cancer cells, as does the mastoparan analogue mitoparan (mitP). Mitochondrial depolarisation and activation of the caspase cascade also underpins the action of dithranol, a topical agent for treatment of psoriasis. The effects of a potent mitP analogue on mitochondrial activity were therefore examined to assess its potential as a novel approach for targeting mitochondria for the treatment of psoriasis. In HaCaT keratinocytes treated with the mitP analogue Z-Gly-RGD(DPhe)-mitP for 24 h, a dose-dependent loss of mitochondrial activity was observed using the methyl-thiazolyl-tetrazolium (MTT) assay. At 10 μmol L -1 , MTT activity was less than 30% that observed in untreated cells. Staining with the cationic dye JC-1 suggested that Z-Gly-RGD(DPhe)-mitP also dissipated the mitochondrial membrane potential, with a threefold increase in mitochondrial depolarisation levels. However, caspase activity appeared to be reduced by 24 h exposure to Z-Gly-RGD(DPhe)-mitP treatment. Furthermore, Z-Gly-RGD(DPhe)-mitP treatment had little effect on overall cell viability. Our findings suggest Z-Gly-RGD(DPhe)-mitP promotes the loss of mitochondrial activity but does not appear to evoke apoptosis in HaCaT keratinocytes.

  1. Effects of Iron-Oxide Nanoparticle Surface Chemistry on Uptake Kinetics and Cytotoxicity in CHO-K1 Cells

    Directory of Open Access Journals (Sweden)

    Camille C. Hanot

    2015-12-01

    Full Text Available Superparamagnetic iron-oxide nanoparticles (SPIONs show great promise for multiple applications in biomedicine. While a number of studies have examined their safety profile, the toxicity of these particles on reproductive organs remains uncertain. The goal of this study was to evaluate the cytotoxicity of starch-coated, aminated, and PEGylated SPIONs on a cell line derived from Chinese Hamster ovaries (CHO-K1 cells. We evaluated the effect of particle diameter (50 and 100 nm and polyethylene glycol (PEG chain length (2k, 5k and 20k Da on the cytotoxicity of SPIONs by investigating cell viability using the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT and sulforhodamine B (SRB assays. The kinetics and extent of SPION uptake by CHO-K1 cells was also studied, as well as the resulting generation of intracellular reactive oxygen species (ROS. Cell toxicity profiles of SPIONs correlated strongly with their cellular uptake kinetics, which was strongly dependent on surface properties of the particles. PEGylation caused a decrease in both uptake and cytotoxicity compared to aminated SPIONs. Interestingly, 2k Da PEG-modifed SPIONs displayed the lowest cellular uptake and cytotoxicity among all studied particles. These results emphasize the importance of surface coatings when engineering nanoparticles for biomedical applications.

  2. Nanotoxicity of pure silica mediated through oxidant generation rather than glutathione depletion in human lung epithelial cells.

    Science.gov (United States)

    Akhtar, Mohd Javed; Ahamed, Maqusood; Kumar, Sudhir; Siddiqui, Huma; Patil, Govil; Ashquin, Mohd; Ahmad, Iqbal

    2010-10-09

    Though, oxidative stress has been implicated in silica nanoparticles induced toxicity both in vitro and in vivo, but no similarities exist regarding dose-response relationship. This discrepancy may, partly, be due to associated impurities of trace metals that may present in varying amounts. Here, cytotoxicity and oxidative stress parameters of two sizes (10 nm and 80 nm) of pure silica nanoparticles was determined in human lung epithelial cells (A549 cells). Both sizes of silica nanoparticles induced dose-dependent cytotoxicity as measured by MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide] and lactate dehydrogenase (LDH) assays. Silica nanoparticles were also found to induce oxidative stress in dose-dependent manner indicated by induction of reactive oxygen species (ROS) generation, and membrane lipid peroxidation (LPO). However, both sizes of silica nanoparticles had little effect on intracellular glutathione (GSH) level and the activities of glutathione metabolizing enzymes; glutathione reductase (GR) and glutathione peroxidase (GPx). Buthionine-[S,R]-sulfoximine (BSO) plus silica nanoparticles did not result in significant GSH depletion than that caused by BSO alone nor N-acetyl cysteine (NAC) afforded significant protection from ROS and LPO induced by silica nanoparticles. The rather unaltered level of GSH is also supported by finding no appreciable alteration in the level of GR and GPx. Our data suggest that the silica nanoparticles exert toxicity in A549 cells through the oxidant generation (ROS and LPO) rather than the depletion of GSH. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  3. Toona Sinensis Extracts Induced Cell Cycle Arrest and Apoptosis in the Human Lung Large Cell Carcinoma

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    Cheng-Yuan Wang

    2010-02-01

    Full Text Available Toona sinensis extracts have been shown to exhibit anti-cancer effects in human ovarian cancer cell lines, human promyelocytic leukemia cells and human lung adenocarcinoma. Its safety has also been confirmed in animal studies. However, its anti-cancer properties in human lung large cell carcinoma have not been studied. Here, we used a powder obtained by freeze-drying the super-natant of centrifuged crude extract from Toona sinensis leaves (TSL-1 to treat the human lung carcinoma cell line H661. Cell viability was evaluated by the 3-(4-,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide assay. Flow cytometry analysis revealed that TSL-1 blocked H661 cell cycle progression. Western blot analysis showed decreased expression of cell cycle proteins that promote cell cycle progression, including cyclin-dependent kinase 4 and cyclin D1, and increased the expression of proteins that inhibit cell cycle progression, including p27. Furthermore, flow cytometry analysis showed that TSL-1 induced H661 cell apoptosis. Western blot analysis showed that TSL-1 reduced the expression of the anti-apoptotic protein B-cell lymphoma 2, and degraded the DNA repair protein, poly(ADP-ribose polymerase. TSL-1 shows potential as a novel therapeutic agent or for use as an adjuvant for treating human lung large cell carcinoma.

  4. Effect of Zebularine loaded MePEG-PCL nanoparticles on viability, attachment of in vitro cultured lens epithelial cells

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    Si-Wei Liu

    2015-01-01

    Full Text Available AIM: To investigate the effect of zebularine(Zebloaded Poly(ethylene glycol-block-poly(ε-caprolactonemethyl ether(MePEG-PCLnanoparticles(NPson the viability, attachment, and apoptosis of in vitro cultured lens epithelial cells(LECs. METHODS: In vitro cultured infant human lens tissue HLE B-3 immortalized cells were distributed randomly divided into six groups. Each group was administered with free Zeb 50μmol/L(ZebF1 group, 100μmol/L(ZebF2 group, Zeb -loaded MePEG-PCL NPs 50μmol/L(ZebNP1 group, Zeb -loaded MePEG-PCL NPs 100μmol/L(ZebNP2 group, MePEG-PCL empty NPs(NPs groupor blank medium(group Crespectively. A tetrazolium dye assay(MTTtest and modified MTT test were performed to determine cell viability and cell attachment. DNA ladder was used to detect the cell apoptosis. RESULTS: Determined by MTT colorimetric method: Cell proliferation rate of LECs were suppressed by all Zeb administration groups in a concentration-time dependent manner(PPP ZebNP1>ZebF2(PCONCLUSION: Zeb loaded MePEG-PCL NPs had better effect on suppressing the viability and attachment of in vitro cultured LECs than the free Zeb groups, as well as enhancing the apoptosis.

  5. Antiproliferative and Pro-Apoptotic Effect of Novel Nitro-Substituted Hydroxynaphthanilides on Human Cancer Cell Lines

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    Tereza Kauerova

    2016-07-01

    Full Text Available Ring-substituted hydroxynaphthanilides are considered as cyclic analogues of salicylanilides, compounds possessing a wide range of pharmacological activities, including promising anticancer properties. The aim of this study was to evaluate the potential anticancer effect of novel nitro-substituted hydroxynaphthanilides with a special focus on structure-activity relationships. The antiproliferative effect was assessed by Water Soluble Tetrazolium Salts-1 (WST-1 assay, and cytotoxicity was evaluated via dye exclusion test. Flow cytometry was used for cell cycle analysis and detection of apoptosis using Annexin V-FITC/PI assay. Protein expression was estimated by Western blotting. Our data indicate that the potential to cause the antiproliferative effect increases with the shift of the nitro substituent from the ortho- to the para-position. The most potent compounds, 3-hydroxy-N-(3-nitrophenylnaphthalene-2-carboxamide (2, and 2-hydroxy-N-(4-nitrophenyl-naphthalene-1-carboxamide (6 showed antiproliferative activity against THP-1 and MCF-7 cancer cells without affecting the proliferation of 3T3-L1 non-tumour cells. Compounds 2 and 6 induced the accumulation of THP-1 and MCF-7 cells in G1 phase associated with the downregulation of cyclin E1 protein levels, while the levels of cyclin B1 were not affected. Moreover, compound 2 was found to exert the pro-apoptotic effect on the THP-1 cells. These results suggest that hydroxynaphthanilides might represent a potential model structure for the development of novel anticancer agents.

  6. In vitro investigating of anticancer activity of focuxanthin from marine brown seaweed species

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    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.

  7. Effect of clinostat rotation on differentiation of embryonic bone in vitro

    Science.gov (United States)

    Al-Ajmi, N.; Braidman, I. P.; Moore, D.

    We have investigated the effect of changes in the gravity vector on osteoblast behaviour, using the clinostat set at 8 rpm. Two sources of osteoblasts were used: secondary cultures of fetal rat bone cells, and the rat osteosarcoma line 17/2.8 (ROS). Cell number was determined by incubation with 3-(4,dimethyl-2yl)-2,3 diphenyl) tetrazolium bromide (MTT) and measurement of optical density at 570 nm (OD). Alkaline phosphatase activity was detected by standard cytochemical methods. Dividing cells were localised by labelling dividing nuclei with Bromodeoxyuridine (BrdU), detected by immunofluorescence. Cell culture was initiated at densities between 1-4x10^4 cells ml^-1. Growth rates in all cultures during the first 48 hours exposure to clinostat rotation were less than in stationary controls. After 3 days, ROS cell numbers were 35% lower, and calvarial cells 39% lower than their respective controls. Alkaline phosphatase activity in calvarial control cultures was uniformly present in characteristically polygonal cells, but after culture in the clinostat the enzyme was present sporadically, and the cells were cuboid. There was also no BrdU uptake in nuclei, but it was present in cell cytoplasms. We conclude that the clinostat decreases cell numbers and cell division. Both cell shape and the distribution of alkaline phosphatase activity in calvarial cell cultures were also affected. This implies that changes in the gravity vector can affect osteoblasts directly, without interaction with other cell types.

  8. Cytotoxic effects of the newly-developed chemotherapeutic agents 17-AAG in combination with oxaliplatin and capecitabine in colorectal cancer cell lines.

    Science.gov (United States)

    Mohammadian, Mahshid; Zeynali, Shima; Azarbaijani, Anahita Fathi; Khadem Ansari, Mohammad Hassan; Kheradmand, Fatemeh

    2017-12-01

    The use of heat shock protein 90 inhibitors like 17-allylamino-17-demethoxy-geldanamycin (17-AAG) has been recently introduced as an attractive anticancer therapy. It has been shown that 17-AAG may potentiate the inhibitory effects of some classical anticolorectal cancer (CRC) agents. In this study, two panels of colorectal carcinoma cell lines were used to evaluate the effects of 17-AAG in combination with capecitabine and oxaliplatin as double and triple combination therapies on the proliferation of CRC cell lines. HT-29 and all HCT-116 cell lines were seeded in culture media in the presence of different doses of the mentioned drugs in single, double, and triple combinations. Water-soluble tetrazolium-1 (WST-1) assay was used to investigate cell proliferation 24 h after treatments. Then, dose-response curves were plotted using WST-1outputs, and IC 50 values were determined. For double and triple combinations respectively 0.5 × IC 50 and 0.25 × IC 50 were used. Data was analyzed with the software CompuSyn. Drug interactions were analyzed using Chou-Talalay method to calculate the combination index (CI).The data revealed that 17-AAG shows a potent synergistic interaction (CI 1) in HT-29 and a synergistic effect (CI AAG with oxaliplatin or capecitabine might be effective against HCT-116 and HT-29 cell lines. However, in triple combinations, positive results were seen only against HCT-116. Further investigation is suggested to confirm the effectiveness of these combinations in clinical trials.

  9. Novel polypropylene biocomposites reinforced with carbon nanotubes and hydroxyapatite nanorods for bone replacements

    Energy Technology Data Exchange (ETDEWEB)

    Liao, Cheng Zhu; Li, Kai [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon (Hong Kong); Wong, Hoi Man; Tong, Wing Yin; Yeung, Kelvin Wai Kwok [Department of Orthopedics and Traumatology, The University of Hong Kong (Hong Kong); Tjong, Sie Chin, E-mail: aptjong@cityu.edu.hk [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon (Hong Kong)

    2013-04-01

    Multi-walled carbon nanotubes (MWNTs) of 0.1 and 0.3 wt.% and hydoxyapatite nanorods (nHAs) of 8–20 wt.% were incorporated into polypropylene (PP) to form biocomposites using melt-compounding and injection molding techniques. The structural, mechanical, thermal and in vitro cell responses of the PP/MWNT–nHA hybrids were investigated. Tensile and impact tests demonstrated that the MWNT additions are beneficial in enhancing the stiffness, tensile strength and impact toughness of the PP/nHA nanocomposites. According to thermal analysis, the nHA and MWNT fillers were found to be very effective to improve dimensional and thermal stability of PP. The results of osteoblast cell cultivation and dimethyl thiazolyl diphenyl tetrazolium (MTT) tests showed that the PP/MWNT–nHA nanocomposites are biocompatible. Such novel PP/MWNT–nHA hybrids are considered to be potential biomaterials for making orthopedic bone implants. - Highlights: ► Multiwalled carbon nanotubes (MWNTs) and hydroxyapatite nanorods (nHA) are used as hybrid fillers to reinforce polypropylene. ► MWNT additions are beneficial in enhancing tensile strength and stiffness of PP/nHA composites. ► Hybridizing MWNT with nHA fillers enhance thermal and dimensional stability of PP significantly. ► Hybridizing MWNT with nHA greatly enhance osteoblast adhesion and proliferation. ► PP/MWNT–nHA composites show attractive applications as load-bearing materials in orthopedics.

  10. 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.

  11. Comparative assessment of antibiotic susceptibility of coagulase-negative staphylococci in biofilm versus planktonic culture as assessed by bacterial enumeration or rapid XTT colorimetry.

    Science.gov (United States)

    Cerca, Nuno; Martins, Silvia; Cerca, Filipe; Jefferson, Kimberly K; Pier, Gerald B; Oliveira, Rosário; Azeredo, Joana

    2005-08-01

    To quantitatively compare the antibiotic susceptibility of biofilms formed by the coagulase-negative staphylococci (CoNS) Staphylococcus epidermidis and Staphylococcus haemolyticus with the susceptibility of planktonic cultures. Several CoNS strains were grown planktonically or as biofilms to determine the effect of the mode of growth on the level of susceptibility to antibiotics with different mechanisms of action. The utility of a new, rapid colorimetric method that is based on the reduction of a tetrazolium salt (XTT) to measure cell viability was tested by comparison with standard bacterial enumeration techniques. A 6 h kinetic study was performed using dicloxacillin, cefazolin, vancomycin, tetracycline and rifampicin at the peak serum concentration of each antibiotic. In planktonic cells, inhibitors of cell wall synthesis were highly effective over a 3 h period. Biofilms were much less susceptible than planktonic cultures to all antibiotics tested, particularly inhibitors of cell wall synthesis. The susceptibility to inhibitors of protein and RNA synthesis was affected by the biofilm phenotype to a lesser degree. Standard bacterial enumeration techniques and the XTT method produced equivalent results both in biofilms and planktonic assays. This study provides a more accurate comparison between the antibiotic susceptibilities of planktonic versus biofilm populations, because the cell densities in the two populations were similar and because we measured the concentration required to inhibit bacterial metabolism rather than to eradicate the entire bacterial population. While the biofilm phenotype is highly resistant to antibiotics that target cell wall synthesis, it is fairly susceptible to antibiotics that target RNA and protein synthesis.

  12. Electrospun polyurethane membranes for Tissue Engineering applications

    Energy Technology Data Exchange (ETDEWEB)

    Gabriel, Laís P., E-mail: lagabriel@gmail.com [National Institute of Biofabrication, Campinas (Brazil); Department of Chemical Engineering, University of Campinas, Campinas (Brazil); Rodrigues, Ana Amélia [National Institute of Biofabrication, Campinas (Brazil); Department of Medical Sciences, University of Campinas, Campinas (Brazil); Macedo, Milton; Jardini, André L.; Maciel Filho, Rubens [National Institute of Biofabrication, Campinas (Brazil); Department of Chemical Engineering, University of Campinas, Campinas (Brazil)

    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 72 h 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.

  13. Influence of Hard Segments on the Thermal, Phase-Separated Morphology, Mechanical, and Biological Properties of Polycarbonate Urethanes

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    Rong Zhu

    2017-03-01

    Full Text Available Abstract: In this study, we have fabricated a series of polycarbonate polyurethanes using a two-step bulk reaction by the melting pre-polymer solution-casting method in order to synthesize biomedical polyurethane elastomers with good mechanical behavior and biostability. The polyurethanes were prepared using dibutyltin dilaurate as the catalyst, poly(1,6-hexanediolcarbonate microdiols (PCDL as the soft segment, and the chain extender 1,4-butanediol (BDO and aliphatic 1,6-hexamethylene diisocyanate (HDI as the hard segments. The chemical structures and physical properties of the obtained films were characterized by attenuated total reflectance Fourier transform infrared (ATR-FTIR spectroscopy, gel permeation chromatography (GPC, differential scanning calorimeter (DSC, and mechanical property tests. The surface properties and degrees of microphase separation were further analyzed by water droplet contact angle measurements (CA and atomic force microscopy (AFM. The materials exhibited a moderate toxic effect on the tetrazolium (MTT assay and good hemocompatibility through hemolytic tests, indicating a good biocompatibility of the fabricated membranes. The materials could be considered as potential and beneficial suitable materials for tissue engineering, especially in the fields of artificial blood-contacting implants or other biomedical applications.

  14. A novel bedside diagnostic test for methanol poisoning using dry chemistry for formate.

    Science.gov (United States)

    Hovda, Knut Erik; Gadeholt, Gaut; Evtodienko, Vladimir; Jacobsen, Dag

    2015-11-01

    The standard diagnostic approach to methanol poisoning is chromatographic measurement of methanol on centrally placed stationary equipment. Methanol poisoning in places where such equipment is unavailable is thus often not diagnosed. Methanol is metabolized to a toxic metabolite, formate; the presence of this compound indicates methanol poisoning. We have developed an enzymatic test for formate and modified it into a portable dry chemistry system that could be used anywhere. The method consists of two enzymatic steps: Formation of NADH from NAD by formate dehydrogenase, and subsequent use of NADH as a reductant of a tetrazolium into a formazan dye that can be quantified photometrically or visually. The photometer gave a good correlation of R(2) = 0.9893 in serum and R(2) = 0.9949 in whole blood, showing an instrumental detection limit of less than 1 mM (4.5 mg/dL). The visual readings showed a correlation of R(2) = 0.8966. Users experienced some difficulty in separating the negative control from the low positives. We have documented the feasibility of an affordable formate strip test for bedside diagnosis of methanol poisoning and for screening of metabolic acidosis of unknown origin. Visual reading is possible, but a reader will improve reliability at lower levels of formate. Future studies are necessary to study the sensitivity and specificity towards other causes of metabolic acidosis and other acids present in human blood.

  15. 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.

  16. 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.

  17. Influence of indicators for thiotriazolin phagocytic activity of leukocytes in the blood in the later period of development of stomach ulcers against pneumonia

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    L. O. Furdychko

    2017-07-01

    Full Text Available Important for the pathogenesis of pneumonia and gastric ulcers should state phagocytic activity of leukocytes (PHAL in the blood. In fact the state of nonspecific resistance factors to some extent depends on the disease, the development of complications, prognosis and therapy. Materials and methods. The study was conducted on 49 male guinea pigs. The experimental pneumonia caused by the method Shlyapnykova V. N., Solodova T. L., gastric ulcer simulated method Komarov V. I. Nonspecific resistance of animals we evaluated, examining the phagocytic activity of leukocytes (PHAL, nitroblue tetrazolium test (NST- test. Phagocytic activity of leukocytes (PHAL in blood were studied in terms phagocytic index (PHI and the phagocytic number (PHN and determined by the method Menshikov V. V. NST - test method Vyksmana M. E., Mayanskoho A. H. The figures research results processed by statistical method Student. Results and discussion. For the experiment, we selected two models of disease: experimental pneumonia (EP and peptic ulcer (PU. State PHAL determined by the level of phagocytic number (PHN, phagocytic index (PHI, NST-test levels in the ЕP+ PU. Found that in the 10-th and 18-th day of development of pathological process both in the stomach and the lungs of guinea pigs, there was reduction of PHI respectively 15,8% (P

  18. Both Creatine and Its Product Phosphocreatine Reduce Oxidative Stress and Afford Neuroprotection in an In Vitro Parkinson’s Model

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    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.

  19. Efficient and safe internalization of magnetic iron oxide nanoparticles: two fundamental requirements for biomedical applications.

    Science.gov (United States)

    Calero, Macarena; Gutiérrez, Lucía; Salas, Gorka; Luengo, Yurena; Lázaro, Ana; Acedo, Pilar; Morales, M Puerto; Miranda, Rodolfo; Villanueva, Angeles

    2014-05-01

    We have performed a series of in vitro tests proposed for the reliable assessment of safety associated with nanoparticles-cell interaction. A thorough analysis of toxicity of three different coating iron oxide nanoparticles on HeLa cells has been carried out including, methyl thiazol tetrazolium bromide (MTT) and Trypan blue exclusion tests, cell morphology observation by optical and Scanning Electron Microscopy (SEM), study of cytoskeletal components, analysis of cell cycle and the presence of reactive oxygen species (ROS). We have quantified magnetic nanoparticle internalization, determined possible indirect cell damages and related it to the nanoparticle coating. The results confirm a very low toxicity of the analyzed iron oxide nanoparticles into HeLa cells by multiple assays and pave the way for a more successful cancer diagnostic and treatment without secondary effects. In this paper, three different iron oxide nanoparticles are studied and compared from the standpoint of safety and toxicity in HeLa cells, demonstrating low toxicity for each preparation, and paving the way to potential future clinical applications. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Lysosome destruction and lipoperoxide formation due to active oxygen generated from haematoporphyrin and UV irradiation

    International Nuclear Information System (INIS)

    Torinuki, W.; Miura, T.; Seiji, M.

    1980-01-01

    The lysosomal enzymes, acid-phosphates and β-glucuronidase, were released from rat liver lysosome when exposed to 400 nm irradiation in the presence of haematoporphyrin and the release was prevented by adding vitamin E, diazabicyclo-octane, bovine serum albumin, superoxide dismutase or D-mannitol to the reaction mixture. Monochromatic irradiation with wavelengths from 380 to 410 nm caused no significant differences in the release of lysosomal enzymes, but 420 nm irradiation caused three-fifths of that of 400 nm irradiation. The malondialdeyhde level in rat liver homogenate increased after 400 nm irradiation in the presence of haematoporphyrin Reduction of nitroblue-tetrazolium was not observed when haematoporphyrin was excited by 400 nm; it was considered that superoxide anion radical (0 2 - ) was not primarily generated. The following mechanism was assumed; that porphyrin which had been excited by 400 nm, converted ground-state molecular oxygen ( 3 0 2 ) to excited singlet oxygen ( 1 0 2 ), which formed lipid peroxides in lysosomal membrane resulting in destruction of the membrane; skin changes would occur from these released lysosomal enzymes. (author)

  1. Cytotoxicity and Genotoxicity of Cypermethrin in Hepatocarcinoma Cells: A Dose- and Time-Dependent Study.

    Science.gov (United States)

    AlKahtane, Abdullah A; Alarifi, Saud; Al-Qahtani, Ahmed A; Ali, Daoud; Alomar, Suliman Y; Aleissia, Mohammed S; Alkahtani, Saad

    2018-01-01

    Most of the agricultural workers are potentially exposed to pesticides through different routes. Inhalation exposures may result in numerous diseases that can adversely affect an individual's health and capacity to perform at work. The aim of this study was to determine the cytotoxic potential of cypermethrin pesticide on cultured human hepatocarcinoma (HepG2) cells. The HepG2 cells were exposed to cypermethrin (0, 5, 15, 40 ng/mL) for 24 and 48 hours. We observed that cypermethrin caused cell death of HepG2 cells using 3-(4, 5-dimethylthiozolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase tests. Furthermore, cypermethrin reduced HepG2 cells viability in a time and dose dependent basis, that was probably mediated through the induction of reactive oxygen species (ROS) and apoptosis. An increase in ROS generation with a concomitant increase in expression of the proapoptotic protein Bcl-2 and cytochrome c and decrease in the antiapoptosis protein Bax suggested that a mitochondria-mediated pathway was involved in cypermethrin-induced apoptosis. These findings provide insights into the underlying mechanisms involved in cytotoxicity of cypermethrin in HepG2 cells.

  2. Bacteria hold their breath upon surface contact as shown in a strain of Escherichia coli, using dispersed surfaces and flow cytometry analysis.

    Directory of Open Access Journals (Sweden)

    Jing Geng

    Full Text Available Bacteria are ubiquitously distributed throughout our planet, mainly in the form of adherent communities in which cells exhibit specific traits. The mechanisms underpinning the physiological shift in surface-attached bacteria are complex, multifactorial and still partially unclear. Here we address the question of the existence of early surface sensing through implementation of a functional response to initial surface contact. For this purpose, we developed a new experimental approach enabling simultaneous monitoring of free-floating, aggregated and adherent cells via the use of dispersed surfaces as adhesive substrates and flow cytometry analysis. With this system, we analyzed, in parallel, the constitutively expressed GFP content of the cells and production of a respiration probe--a fluorescent reduced tetrazolium ion. In an Escherichia coli strain constitutively expressing curli, a major E. coli adhesin, we found that single cell surface contact induced a decrease in the cell respiration level compared to free-floating single cells present in the same sample. Moreover, we show here that cell surface contact with an artificial surface and with another cell caused reduction in respiration. We confirm the existence of a bacterial cell "sense of touch" ensuring early signalling of surface contact formation through respiration down modulation.

  3. In vitro testing of drug combinations employing nilotinib and alkylating agents with regard to pretransplant conditioning treatment of advanced-phase chronic myeloid leukemia.

    Science.gov (United States)

    Radujkovic, Aleksandar; Luft, Thomas; Dreger, Peter; Ho, Anthony D; Jens Zeller, W; Fruehauf, Stefan; Topaly, Julian

    2014-08-01

    The prognosis of patients with advanced-phase chronic myeloid leukemia (CML) remains dismal despite the availability of targeted therapies and allogeneic stem cell transplantation (allo-SCT). Increasing the antileukemic efficacy of the pretransplant conditioning regimen may be a strategy to increase remission rates and duration. We therefore investigated the antiproliferative effects of nilotinib in combination with drugs that are usually used for conditioning: the alkylating agents mafosfamide, treosulfan, and busulfan. Drug combinations were tested in vitro in different imatinib-sensitive and imatinib-resistant BCR-ABL-positive cell lines. A tetrazolium-based MTT assay was used for the assessment and quantification of growth inhibition after exposure to alkylating agents alone or to combinations with nilotinib. Drug interaction was analyzed using the median-effect method of Chou and Talalay, and combination index (CI) values were calculated according to the classic isobologram equation. Treatment of imatinib-sensitive, BCR-ABL-positive K562 and LAMA84 cells with nilotinib in combination with mafosfamide, treosulfan, or busulfan resulted in synergistic (CI 1) effects, respectively. In imatinib-resistant K562-R and LAMA84-R cells, all applied drug combinations were synergistic (CI conditioning regimens for allo-SCT in advanced-phase CML.

  4. Effect of Evodiamine on Inducing Apoptosis of Human Gastric Cancer Cell Line SGC-7901 in Vitro

    Directory of Open Access Journals (Sweden)

    LIU Shao-ping

    2014-09-01

    Full Text Available Objective: To explore the proliferation inhibition and apoptosis-inducing effect of evodiamine in human gastric cancer SGC-7901 cells. Methods: After 48 or 24 h exposure to different concentrations of evodiamine, cell proliferation was analyzed using tetrazolium blue (MTT assay while apoptosis and cell-cycle phase distribution using flow cytometry. Results: In 0.01~30.00 μg/mL range of concentrations, evodiamine inhibited the proliferation of SGC-7901 cells in dose-dependent manner, and the overall mean IC50 was (3.79±0.16 μg/mL; the apoptosis rate was increased from 3.4% to 7.0%, 13.8% and 36.3% at concentrations of 0, 0.5, 1.5 and 30 μg/mL of evodiamine, respectively; the percentage of cells accumulated in G2/M phase was increased from 17.26% to 98.92% in the cells treated with evodiamine for 24 h in 0.01~30.00 μg/mL range of concentrations. Conclusion: Evodiamine can inhibit the proliferation, induce apoptosis in human gastric cancer cell line SGC-7901 in vitro and arrest the cell cycle at the G2/M phase.

  5. Schisandrin B protects PC12 cells by decreasing the expression of amyloid precursor protein and vacuolar protein sorting 35★

    Science.gov (United States)

    Yan, Mingmin; Mao, Shanping; Dong, Huimin; Liu, Baohui; Zhang, Qian; Pan, Gaofeng; Fu, Zhiping

    2012-01-01

    PC12 cell injury was induced using 20 μM amyloid β-protein 25–35 to establish a model of Alzheimer's disease. The cells were then treated with 5, 10, and 25 μM Schisandrin B. Methylthiazolyldiphenyl-tetrazolium bromide assays and Hoechst 33342 staining results showed that with increasing Schisandrin B concentration, the survival rate of PC12 cells injured by amyloid β-protein 25–35 gradually increased and the rate of apoptosis gradually decreased. Reverse transcription-PCR, immunocytochemical staining and western blot results showed that with increasing Schisandrin B concentration, the mRNA and protein expression of vacuolar protein sorting 35 and amyloid precursor protein were gradually decreased. Vacuolar protein sorting 35 and amyloid precursor protein showed a consistent trend for change. These findings suggest that 5, 10, and 25 μM Schisandrin B antagonizes the cellular injury induced by amyloid β-protein 25–35 in a dose-dependent manner. This may be caused by decreasing the expression of vacuolar protein sorting 35 and amyloid precursor protein. PMID:25745458

  6. Aspidosperma pyrifolium Has Anti-Inflammatory Properties: An Experimental Study in Mice with Peritonitis Induced by Tityus serrulatus Venom or Carrageenan.

    Science.gov (United States)

    Souza Lima, Maíra Conceição Jerônimo de; Oliveira Bitencourt, Mariana Angélica; Furtado, Allanny Alves; Torres-Rêgo, Manoela; Siqueira, Emerson Michell da Silva; Oliveira, Ruth Medeiros; Oliveira Rocha, Hugo Alexandre; Ferreira Rocha, Keyla Borges; Silva-Júnior, Arnóbio Antônio da; Zucolotto, Silvana Maria; Fernandes-Pedrosa, Matheus de Freitas

    2017-11-11

    Scorpions of the genus Tityus are responsible for the majority of envenomation in Brazil, the Tityus serrulatus species being the most common and dangerous in South America. In this approach, we have investigated the ability of the aqueous extract from the leaves of Aspidosperma pyrifolium in reducing carrageenan-induced inflammation and the inflammation induced by T. serrulatus envenomation in mice. We also evaluated the cytotoxic effects of this extract, using the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl-2H-tetrazolium (MTT) assay and the results revealed that the extract is safe. Analysis by High Performance Liquid Chromatography coupled with Diode Array Detector (HPLC-DAD) and Liquid Chromatography Coupled with Mass Spectrometry with Diode Array Detection (LC-DAD-MS) showed one major chemical component, the flavonoid rutin and phenolics compounds. For in vivo studies in carrageenan-induced peritonitis model, mice received extracts, dexamethasone, rutin or saline, before administration of carrageenan. For venom-induced inflammation model, animals received T. serrulatus venom and were, simultaneously, treated with extracts, antivenom, rutin or saline. The extract and rutin showed a reduction in the cell migration into the peritoneal cavity, and in the same way the envenomated animals also showed reduction of edema, inflammatory cell infiltration and vasodilation in lungs. This is an original study revealing the potential action of A. pyrifolium against inflammation caused by Tityus serrulatus venom and carrageenan, revealing that this extract and its bioactive molecules, specifically rutin, may present potential anti-inflammatory application.

  7. Enhancement of bioavailability by formulating rhEPO ionic complex with lysine into PEG-PLA micelle

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Yanan; Sun, Fengying; Wang, Dan; Zhang, Renyu [Jilin University, College of Life Science (China); Dou, Changlin; Liu, Wanhui; Sun, Kaoxiang, E-mail: sunkx@ytu.edu.cn [Yantai University, School of Pharmacy (China); Li, Youxin, E-mail: liyouxin@jlu.edu.cn [Jilin University, College of Life Science (China)

    2013-10-15

    A composite micelle of ionic complex encapsulated into poly(ethylene glycol)-poly(d,l-lactide) (PEG-PLA) di-block copolymeric micelles was used for protein drug delivery to improve its pharmacokinetic performance. In this study, recombinant human erythropoietin (rhEPO, as a model protein) was formulated with lysine into composite micelles at a diameter of 71.5 nm with narrow polydispersity indices (PDIs < 0.3). Only a trace amount of protein was in aggregate form. The zeta potential of the spherical micelles was ranging from -0.54 to 1.39 mv, and encapsulation efficiency is high (80 %). The stability of rhEPO was improved significantly in composite micelles in vitro. Pharmacokinetic studies in rats showed significant, enhanced plasma retention of the composite micelles in comparison with native rhEPO. Areas under curve (AUCs) of the rhEPO released from the composite micelles were 4.5- and 2.3-folds higher than those of the native rhEPO and rhEPO-loaded PEG-PLA micelle, respectively. In addition, the composite micelles exhibited good biocompatibility using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay with human embryonic kidney (HEK293T) cells. All these features are preferable for utilizing the composite micelles as a novel protein delivery system.

  8. Enhancement of bioavailability by formulating rhEPO ionic complex with lysine into PEG-PLA micelle

    Science.gov (United States)

    Shi, Yanan; Sun, Fengying; Wang, Dan; Zhang, Renyu; Dou, Changlin; Liu, Wanhui; Sun, Kaoxiang; Li, Youxin

    2013-10-01

    A composite micelle of ionic complex encapsulated into poly(ethylene glycol)-poly( d, l-lactide) (PEG-PLA) di-block copolymeric micelles was used for protein drug delivery to improve its pharmacokinetic performance. In this study, recombinant human erythropoietin (rhEPO, as a model protein) was formulated with lysine into composite micelles at a diameter of 71.5 nm with narrow polydispersity indices (PDIs protein was in aggregate form. The zeta potential of the spherical micelles was ranging from -0.54 to 1.39 mv, and encapsulation efficiency is high (80 %). The stability of rhEPO was improved significantly in composite micelles in vitro. Pharmacokinetic studies in rats showed significant, enhanced plasma retention of the composite micelles in comparison with native rhEPO. Areas under curve (AUCs) of the rhEPO released from the composite micelles were 4.5- and 2.3-folds higher than those of the native rhEPO and rhEPO-loaded PEG-PLA micelle, respectively. In addition, the composite micelles exhibited good biocompatibility using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay with human embryonic kidney (HEK293T) cells. All these features are preferable for utilizing the composite micelles as a novel protein delivery system.

  9. Tissue-transglutaminase contributes to neutrophil granulocyte differentiation and functions.

    Science.gov (United States)

    Balajthy, Zoltán; Csomós, Krisztián; Vámosi, György; Szántó, Attila; Lanotte, Michel; Fésüs, László

    2006-09-15

    Promyelocytic NB4 leukemia cells undergo differentiation to granulocytes following retinoic acid treatment. Here we report that tissue transglutaminase (TG2), a protein cross-linking enzyme, was induced, then partially translocated into the nucleus, and became strongly associated with the chromatin during the differentiation process. The transglutaminase-catalyzed cross-link content of both the cytosolic and the nuclear protein fractions increased while NB4 cells underwent cellular maturation. Inhibition of cross-linking activity of TG2 by monodansylcadaverin in these cells led to diminished nitroblue tetrazolium (NBT) positivity, production of less superoxide anion, and decreased expression of GP91PHOX, the membrane-associated subunit of NADPH oxidase. Neutrophils isolated from TG2(-/-) mice showed diminished NBT reduction capacity, reduced superoxide anion formation, and down-regulation of the gp91phox subunit of NADPH oxidase, compared with wild-type cells. It was also observed that TG2(-/-) mice exhibited increased neutrophil phagocytic activity, but had attenuated neutrophil chemotaxis and impaired neutrophil extravasation with higher neutrophil counts in their circulation during yeast extract-induced peritonitis. These results clearly suggest that TG2 may modulate the expression of genes related to neutrophil functions and is involved in several intracellular and extracellular functions of extravasating neutrophil.

  10. Screening of antiproliferative effect of aqueous extracts of plant foods consumed in México on the breast cancer cell line MCF-7.

    Science.gov (United States)

    García-Solís, Pablo; Yahia, Elhadi M; Morales-Tlalpan, Verónica; Díaz-Muñoz, Mauricio

    2009-01-01

    We evaluated the antiproliferative effect of aqueous extracts of 14 plant foods consumed in Mexico on the breast cancer cell line MCF-7. The plant foods used were avocado, black sapote, guava, mango, prickly pear cactus stems (called nopal in Mexico, cooked and raw), papaya, pineapple, four different cultivars of prickly pear fruit, grapes and tomato. β-Carotene, total phenolics and gallic acid contents and the antioxidant capacity, measured by the ferric reducing/antioxidant power and the 2,2-diphenyl-1,1-picrylhydrazyl radical scavenging assays, were analyzed in each aqueous extract. Only the papaya extract had a significant antiproliferative effect measured with the methylthiazolydiphenyl-tetrazolium bromide assay. We did not notice a relationship between the total phenolic content and the antioxidant capacity with antiproliferative effect. It is suggested that each extract of plant food has a unique combination of the quantity and quality of phytochemicals that could determine its biological activity. Besides, papaya represents a very interesting fruit to explore its antineoplastic activities.

  11. Tetrandrine, a Compound Common in Chinese Traditional Medicine, Preferentially Kills Breast Cancer Tumor Initiating Cells (TICs) In Vitro

    International Nuclear Information System (INIS)

    Xu, Wei; Debeb, Bisrat G.; Lacerda, Lara; Li, Jessica; Woodward, Wendy A.

    2011-01-01

    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 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 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

  12. On methods for the detection of reactive oxygen species generation by human spermatozoa: analysis of the cellular responses to catechol oestrogen, lipid aldehyde, menadione and arachidonic acid.

    Science.gov (United States)

    Aitken, R J; Smith, T B; Lord, T; Kuczera, L; Koppers, A J; Naumovski, N; Connaughton, H; Baker, M A; De Iuliis, G N

    2013-03-01

    Oxidative stress is known to have a major impact on human sperm function and, as a result, there is a need to develop sensitive methods for measuring reactive oxygen species (ROS) generation by these cells. A variety of techniques have been developed for this purpose including chemiluminescence (luminol and lucigenin), flow cytometry (MitoSOX Red, dihydroethidium, 4,5-diaminofluorescein diacetate and 2',7'-dichlorodihydrofluorescein diacetate) and spectrophotometry (nitroblue tetrazolium). The relative sensitivity of these assays and their comparative ability to detect ROS generated in different subcellular compartments of human spermatozoa, have not previously been investigated. To address this issue, we have compared the performance of these assays when ROS generation was triggered with a variety of reagents including 2-hydroxyestradiol, menadione, 4-hydroxynonenal and arachidonic acid. The results revealed that menadione predominantly induced release of ROS into the extracellular space where these metabolites could be readily detected by luminol-peroxidase and, to a lesser extent, 2',7'-dichlorodihydrofluorescein. However, such sensitivity to extracellular ROS meant that these assays were particularly vulnerable to interference by leucocytes. The remaining reagents predominantly elicited ROS generation by the sperm mitochondria and could be optimally detected by MitoSOX Red and DHE. Examination of spontaneous ROS generation by defective human spermatozoa revealed that MitoSOX Red was the most effective indicator of oxidative stress, thereby emphasizing the general importance of mitochondrial dysregulation in the aetiology of defective sperm function. © 2013 American Society of Andrology and European Academy of Andrology.

  13. Gamma ray sterilization of delta inulin adjuvant particles (Advax™) makes minor, partly reversible structural changes without affecting adjuvant activity

    Science.gov (United States)

    Cooper, P. D.; Barclay, T. G.; Ginic-Markovic, M.; Petrovsky, N.

    2014-01-01

    We earlier identified a developmental series of seven isoforms/polymorphs of microparticulate inulin by comparing non-covalent bonding strengths. Their pharmaceutical utility lies in modulation of cellular immunity, exploited as vaccine adjuvants (Advax™) especially for delta inulin (DI). As such particles cannot be sterilized by filtration we explore the effect of 60Co gamma radiation (GR) on inulin isoforms, particularly DI. Its adjuvant activity and overt physical properties were unaffected by normal GR sterilizing doses (up to 25 kGy). Heating irradiated isoform suspensions near their critical dissolution temperature revealed increased solubility deduced to reflect a single lethal event in one component of a multi-component structure. Local oxidative effects of GR on DI were not found. The observed DI loss was almost halved by re-annealing at the critical temperature: surviving inulin chains apparently reassemble into smaller amounts of the original type of structure. Colorimetric tetrazolium assay revealed increases in reducing activity after GR of raw inulin powder, which yielded DI with normal physical properties but only 25% normal recovery yet 4× normal reducing ability, implying final retention of some GR-changed inulin chains. These findings suggest minimal inulin chain cleavage and confirm that GR may be a viable strategy for terminal sterilization of microparticulate inulin adjuvants. PMID:24342245

  14. Biological Activities of Toninia candida and Usnea barbata Together with Their Norstictic Acid and Usnic Acid Constituents

    Directory of Open Access Journals (Sweden)

    Nedeljko Manojlović

    2012-11-01

    Full Text Available The aim of this study was to investigate the chemical composition of acetone extracts of the lichens Toninia candida and Usnea barbata and in vitro antioxidant, antimicrobial, and anticancer activities of these extracts together with some of their major metabolites. The chemical composition of T. candida and U. barbata extracts was determined using HPLC-UV analysis. The major phenolic compounds in these extracts were norstictic acid (T. candida and usnic acid (U. barbata. Antioxidant activity was evaluated by free radical scavenging, superoxide anion radical scavenging, reducing power and determination of total phenolic compounds. Results of the study proved that norstictic acid had the largest antioxidant activity. The total content of phenols in the extracts was determined as the pyrocatechol equivalent. The antimicrobial activity was estimated by determination of the minimal inhibitory concentration using the broth microdilution method. The most active was usnic acid with minimum inhibitory concentration values ranging from 0.0008 to 0.5 mg/mL. Anticancer activity was tested against FemX (human melanoma and LS174 (human colon carcinoma cell lines using the microculture tetrazolium test. Usnic acid was found to have the strongest anticancer activity towards both cell lines with IC50 values of 12.72 and 15.66 μg/mL.

  15. Microculture virus titration--a simple colourimetric assay for influenza virus titration.

    Science.gov (United States)

    Levi, R; Beeor-Tzahar, T; Arnon, R

    1995-03-01

    Influenza antigens can be detected by several well established methods. However, when it is important to determine the titre of infective virions, a bioassay should be employed. The standard and the most widely used tests for influenza infectivity are titration carried out in embryonated hen eggs, or the plaque assay employing tissue culture techniques. A simple colourimetric assay for influenza virus detection and titration is described. Samples of allantoic fluid or mice lung homogenates were used to infect MDCK cultures in microplate wells. After an incubation period, the tetrazolium (MTT) colourimetric assay was used to determine cell viability, and when compared to untreated culture control enabled the detection and titration of several influenza strains. When samples were assayed simultaneously in embryonated eggs and by the MCVT method, good correlation in determined titres was obtained. The availability of an additional method for influenza titration allows more flexibility in the choice of titration method according to the specific needs of the study. Furthermore, this method lends itself to full automatization. Similar procedures should also be applicable to titration of other cytopathic viruses.

  16. Cytotoxic glucosphingolipid from Celtis Africana.

    Science.gov (United States)

    Perveen, Shagufta; Al-Taweel, Areej Mohammad; Fawzy, Ghada Ahmed; El-Shafae, Azza Muhammed; Khan, Afsar; Proksch, Peter

    2015-05-01

    Literature survey proved the use of the powdered sun-dried bark and roots of Celtis africana for the treatment of cancer in South Africa. The aim of this study was to do further isolation work on the ethyl acetate fraction and to investigate the cytotoxic activities of the various fractions and isolated compound. Cytotoxicity of petroleum ether, chloroform, ethyl acetate, n-butanol fractions and compound 1 were tested on mouse lymphoma cell line L5178Y using the microculture tetrazolium assay. One new glucosphingolipid 1 was isolated from the aerial parts of C. africana. The structure of the new compound was determined by extensive analysis by one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy and mass spectrometry. The ethyl acetate fraction and compound 1 showed strong cytotoxic activity with an EC50 value of 8.3 μg/mL and 7.8 μg/mL, respectively, compared with Kahalalide F positive control (6.3 μg/mL). This is the first report of the occurrence of a cytotoxic glucosphingolipid in family Ulmaceae.

  17. 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.

  18. Autophagy in RAW264.7 Cells Treated with Surface-Functionalized Graphene Oxides

    Directory of Open Access Journals (Sweden)

    Chang Seok Park

    2015-01-01

    Full Text Available This study investigated cytotoxicity, particularly autophagy, in RAW264.7 cells exposed to graphene oxide (GO and its derivatives (dodecylamine-GO (DA-GO, reduced GO (rGO, and sodium dodecyl sulfate-rGO (SDS-rGO. Appearance of amine stretching bands, out-of-plane C-H stretching vibrations, and S=O stretching bands in infrared spectra indicated the formation of DA-GO, rGO, and SDS-rGO, respectively. Light microscopy and microculture tetrazolium assay showed that all the GO types exerted cytotoxic effects on RAW264.7 cells in a concentration-dependent manner. Higher concentrations of the GO types downregulated the expression of PU.1, a unique transcription factor in monocytes and macrophages, and decreased the conversion of LC3A/B-I to LC3A/B-II, suggesting that PU.1 was associated with autophagy in RAW264.7 cells. These results suggested that surface-functionalized GOs exerted cytotoxic effects in a concentration-dependent manner by changing the expression of critical genes and inducing autophagy in macrophages.

  19. Synergetic Effects of PARP Inhibitor AZD2281 and Cisplatin in Oral Squamous Cell Carcinoma in Vitro and in Vivo

    Directory of Open Access Journals (Sweden)

    Masaaki Yasukawa

    2016-02-01

    Full Text Available Cisplatin is a commonly used chemotherapeutic drug for treatment of oral carcinoma, and combinatorial effects are expected to exert greater therapeutic efficacy compared with monotherapy. Poly(ADP-ribosylation is reported to be involved in a variety of cellular processes, such as DNA repair, cell death, telomere regulation, and genomic stability. Based on these properties, poly(ADP-ribose polymerase (PARP inhibitors are used for treatment of cancers, such as BRCA1/2 mutated breast and ovarian cancers, or certain solid cancers in combination with anti-cancer drugs. However, the effects on oral cancer have not been fully evaluated. In this study, we examined the effects of PARP inhibitor on the survival of human oral cancer cells in vitro and xenografted tumors derived from human oral cancer cells in vivo. In vitro effects were assessed by microculture tetrazolium and survival assays. The PARP inhibitor AZD2281 (olaparib showed synergetic effects with cisplatin in a dose-dependent manner. Combinatorial treatment with cisplatin and AZD2281 significantly inhibited xenografted tumor growth compared with single treatment of cisplatin or AZD2281. Histopathological analysis revealed that cisplatin and AZD2281 increased TUNEL-positive cells and decreased Ki67- and CD31-positive cells. These results suggest that PARP inhibitors have the potential to improve therapeutic strategies for oral cancer.

  20. Characterization of chemical compounds for dosimetry of the radiation in industrial processes

    International Nuclear Information System (INIS)

    Galante, Ana Maria Sisti

    1999-01-01

    Different chemical compounds have been studied to optimize dosimetric systems in irradiation processes. In this study 2,3,5 Triphenyl -2H- Tetrazolium Chloride, Brilliant Cresyl Blue, Bromocresol Green and Potassium Nitrate were investigated for their merits or faults, for 60 Co gamma field, in order to verify if can be considered as dosimeters. Fricke solution was used as reference dosimeter to determine absorption dose rates at the gamma facilities.Only Bromocresol Green and Potassium Nitrate are recommended for dosimetry purposes since the main characteristics were achieved. The other two compounds could be used in dosimetry with changes in their formulation. Bromocresol Green and potassium Nitrate are reproducible and radiation sensitive for absorbed doses from 300 Gy to 150 kGy Bromocresol Green was used in liquid form and Potassium Nitrate was prepared in solid pellets form. Spectrophotometry in the visible region was used as the main detection technique, which allows relating optical absorption, before and after irradiation, with the absorbed dose. The maximum absorption wavelength for each compound was observed at 450-460nm for bromocresol Green and 546nm for Potassium Nitrate. Dose calibration curves are linear for both compounds in all dose intervals. When irradiated with accelerated electrons, with energies between o,9 MeV and 1,5MeV, optical absorption intensification, of about 2,6 times, was observed when comparing results for Potassium Nitrate, with those for gamma rays. All the evaluations are presented in this work. (author)

  1. 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.

  2. In vitro evaluation of alginate encapsulated adipose-tissue stromal cells for use as injectable bone graft substitute

    International Nuclear Information System (INIS)

    Abbah, S.A.; Lu, W.W.; Chan, D.; Cheung, K.M.C.; Liu, W.G.; Zhao, F.; Li, Z.Y.; Leong, J.C.Y.; Luk, K.D.K.

    2006-01-01

    This study aims to investigate the survival and osteogenic behavior of murine-derived adipose-tissue stromal cells (ATSCs) encapsulated in alginate microcapsules thereby instigating further studies in this cell delivery strategy for in vivo osteogenesis. Cell viability was quantified using a tetrazolium-based assay and osteogenic differentiation was evaluated by both alkaline-phosphatase (ALP) histochemistry and osteocalcin mRNA analysis. Following microencapsulation, cell numbers increased from 3.9 x 10 3 on day 1 to 7.8 x 10 3 on day 7 and maintained excellent viability in the course of 21-day culture. ALP was 6.9, 5.5, and 3.2 times higher than monolayer cultures on days 7, 14, and 21, respectively. In addition, osteocalcin mRNA was detectable in encapsulated cultures earlier (day 14) than monolayer cultures. We conclude that alginate microcapsules can act as three-dimensional matrix for ATSC proliferation and has potential for use as injectable, biodegradable scaffold in bone tissue engineering

  3. Optimizing Tuberculosis Testing for Basic Laboratories

    Science.gov (United States)

    Ramos, Eric; Schumacher, Samuel G.; Siedner, Mark; Herrera, Beatriz; Quino, Willi; Alvarado, Jessica; Montoya, Rosario; Grandjean, Louis; Martin, Laura; Sherman, Jonathan M.; Gilman, Robert H.; Evans, Carlton A.

    2010-01-01

    Optimal tuberculosis testing usually involves sputum centrifugation followed by broth culture. However, centrifuges are biohazardous and scarce in the resource-limited settings where most tuberculosis occurs. To optimize tuberculosis testing for these settings, centrifugation of 111 decontaminated sputum samples was compared with syringe-aspiration through polycarbonate membrane-filters that were then cultured in broth. To reduce the workload of repeated microscopic screening of broth cultures for tuberculosis growth, the colorimetric redox indicator 2,3-diphenyl-5-(2-thienyl) tetrazolium chloride was added to the broth, which enabled naked-eye detection of culture positivity. This combination of filtration and colorimetric growth-detection gave similar results to sputum centrifugation followed by culture microscopy regarding mean colony counts (43 versus 48; P = 0.6), contamination rates (0.9% versus 1.8%; P = 0.3), and sensitivity (94% versus 95%; P = 0.7), suggesting equivalency of the two methods. By obviating centrifugation and repeated microscopic screening of cultures, this approach may constitute a more appropriate technology for rapid and sensitive tuberculosis diagnosis in basic laboratories. PMID:20889887

  4. Involvement of Bax and Bcl-2 in Induction of Apoptosis by Essential Oils of Three Lebanese Salvia Species in Human Prostate Cancer Cells

    Directory of Open Access Journals (Sweden)

    Alessandra Russo

    2018-01-01

    Full Text Available Prostate cancer is one of the most common forms of cancer in men, and research to find more effective and less toxic drugs has become necessary. In the frame of our ongoing program on traditionally used Salvia species from the Mediterranean Area, here we report the biological activities of Salvia aurea, S. judaica and S. viscosa essential oils against human prostate cancer cells (DU-145. The cell viability was measured by 3(4,5-dimethyl-thiazol-2-yl2,5-diphenyl-tetrazolium bromide (MTT test and lactate dehydrogenase (LDH release was used to quantify necrosis cell death. Genomic DNA, caspase-3 activity, expression of cleaved caspase-9, B-cell lymphoma 2 (Bcl-2 and Bcl-2 associated X (Bax proteins were analyzed in order to study the apoptotic process. The role of reactive oxygen species in cell death was also investigated. We found that the three essential oils, containing caryophyllene oxide as a main constituent, are capable of reducing the growth of human prostate cancer cells, activating an apoptotic process and increasing reactive oxygen species generation. These results suggest it could be profitable to further investigate the effects of these essential oils for their possible use as anticancer agents in prostate cancer, alone or in combination with chemotherapy agents.

  5. Assessment of cytotoxicity of Portulaca oleracea Linn. against human colon adenocarcinoma and vero cell line

    Science.gov (United States)

    Mali, Prashant Y.

    2015-01-01

    Background: Portulaca oleracea Linn. (Portulacaceae) is commonly known as purslane in English. In traditional system it is used to cure diarrhea, dysentery, leprosy, ulcers, asthma, and piles, reduce small tumors and inflammations. Aim: To assess cytotoxic potential of chloroform extract of P. oleracea whole plant against human colon adenocarcinoma (HCT-15) and normal (Vero) cell line. Materials and Methods: Characterization of chloroform extract of P. oleracea by Fourier transform infrared (FTIR) spectroscopy was performed. Cytotoxicity (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay was used for assessment of cytotoxic potential of chloroform extract of P. oleracea. The concentrations of 1000–0.05 μg/ml were used in the experiment. Doxorubicin was considered as standard reference drug. Results: FTIR spectrum showed the peak at 1019.52 and 1396.21 center. The 50% cell growth inhibition (IC50) of chloroform extract of P. oleracea and doxorubicin was 1132.02 μg/ml and 460.13 μg/ml against human colon adenocarcinoma and 767.60 μg/ml and 2392.71 μg/ml against Vero cell line, respectively. Conclusion: Chloroform extract of P. oleracea whole plant was less efficient or does not have cytotoxic activity against human colon adenocarcinoma cell line. It was not safe to normal Vero cell line. But, there is a need to isolate, identify, and confirm the phytoconstituents present in extract by sophisticated analytical techniques. PMID:27833374

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

    Science.gov (United States)

    Foley, Rhonda C; Kidd, Brendan N; Hane, James K; Anderson, Jonathan P; Singh, Karam B

    2016-01-01

    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.

  7. 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.

  8. Preliminary evaluation of in vitro cytotoxicity and in vivo antitumor activity of Xanthium strumarium in transplantable tumors in mice.

    Science.gov (United States)

    Aranjani, Jesil Mathew; Manuel, Atulya; Mallikarjuna Rao, Chamallamudi; Udupa, Nayanabhirama; Rao, Josyula Venkata; Joy, Ann Mary; Gandhi, Prajay; Radhakrishnan, Ethiraj Kannat

    2013-01-01

    In the present study, active fractions of the methanolic extract of Xanthium strumarium (XS) showing potent cytotoxicity were determined using microculture tetrazolium (MTT) and sulforhodamine B (SRB) assays in selected cancer cell lines. The active fractions viz., chloroform soluble fraction of root (CEXSR), hexane soluble fraction of leaf (HEXSL), hexane soluble fraction of fruits (HEXSF) and chloroform soluble fraction of fruits (CEXSF) of XS were tested in transplantable animal tumor models for their antitumor potential. Dalton's ascitic lymphoma (DLA) cells were used to induce solid and liquid (ascites) tumor in mice. The tumor bearing animals were treated with active fractions at two dose levels (100 and 200 mg/kg). The antitumor activities of the active fractions in tumor bearing animals were monitored with parameters such as body weight and increase in life-span as well as biochemical and hematological modalities (in the case of liquid tumor). Tumor incidence and tumor volume were the parameters monitored in the case of the solid tumor model. The results were analyzed by one-way ANOVA followed by Tukey's post hoc test. The extracts were found to increase the life-span of tumor bearing animals and restore the altered hematological and biochemical parameters significantly.

  9. Nucleic acid hybridization assays employing dA-tailed capture probes. II. Advanced multiple capture methods

    International Nuclear Information System (INIS)

    Hunsaker, W.R.; Badri, H.; Lombardo, M.; Collins, M.L.

    1989-01-01

    A fourth capture is added to the reversible target capture procedure. This results in an improved radioisotopic detection limit of 7.3 x 10(-21) mol of target. In addition, the standard triple capture method is converted into a nonradioactive format with a detection limit of under 1 amol of target. The principal advantage of nonradioactive detection is that the entire assay can be performed in about 1 h. Nucleic acids are released from cells in the presence of the (capture probe) which contains a 3'-poly(dA) sequence and the (labeled probe) which contains a detectable nonradioactive moiety such as biotin. After a brief hybridization in solution, the target is captured on oligo(dT) magnetic particles. The target is further purified from sample impurities and excess labeled probe by recapture either once or twice more on fresh magnetic particles. The highly purified target is then concentrated to 200 nl by recapture onto a poly(dT) nitrocellulose filter and rapidly detected with streptavidin-alkaline phosphatase using bromochloroindolyl phosphate and nitroblue tetrazolium. Using this procedure, as little as 0.25 amol of a target plasmid has been detected nonradioactively in crude samples in just 1 h without prior purification of the DNA and RNA. Finally, a new procedure called background capture is introduced to complement the background-reducing power of RTC

  10. In vitro cytotoxic, genotoxic and antioxidant/oxidant effects of guaiazulene on human lymphocytes

    Directory of Open Access Journals (Sweden)

    Başak Toğar

    2015-02-01

    Full Text Available The aim of this study was to evaluate for the cytotoxicity, genotoxicity and antioxidant/oxidant activity of GYZ on human peripheral blood lymphocytes (PBLs. Guaiazulene (GYZ was added into culture tubes at various concentrations (0-400 µg/mL-1. Cytotoxicity against the human lymphocytes cultures was examined by lactate dehydrogenase (LDH release assay. The proliferative response was estimated by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT assay. Antioxidant/oxidant activity was evaluated by measuring the total oxidant status (TOS and total antioxidant capacity (TAC levels. Micronucleus (MN and chromosomal aberration (CA tests were used in genotoxicity studies. The results showed that GYZ caused cytotoxicity in the PBLs at high concentrations, but TOS level were not affected, while the level of TAC was significantly increased. GYZ also did not induce chromosomal aberrations when compared to that of the control group. Results this study clearly revealed that GYZ was not genotoxic and also increased the capacity of the antioxidant in the culture of human PBL cells. This report is first report on the impact of GYZ on human PBL cells.

  11. Biocompatibility of crystalline opal nanoparticles.

    Science.gov (United States)

    Hernández-Ortiz, Marlen; Acosta-Torres, Laura S; Hernández-Padrón, Genoveva; Mendieta, Alicia I; Bernal, Rodolfo; Cruz-Vázquez, Catalina; Castaño, Victor M

    2012-10-22

    Silica nanoparticles are being developed as a host of biomedical and biotechnological applications. For this reason, there are more studies about biocompatibility of silica with amorphous and crystalline structure. Except hydrated silica (opal), despite is presents directly and indirectly in humans. Two sizes of crystalline opal nanoparticles were investigated in this work under criteria of toxicology. In particular, cytotoxic and genotoxic effects caused by opal nanoparticles (80 and 120 nm) were evaluated in cultured mouse cells via a set of bioassays, methylthiazolyldiphenyl-tetrazolium-bromide (MTT) and 5-bromo-2'-deoxyuridine (BrdU). 3T3-NIH cells were incubated for 24 and 72 h in contact with nanocrystalline opal particles, not presented significant statistically difference in the results of cytotoxicity. Genotoxicity tests of crystalline opal nanoparticles were performed by the BrdU assay on the same cultured cells for 24 h incubation. The reduction of BrdU-incorporated cells indicates that nanocrystalline opal exposure did not caused unrepairable damage DNA. There is no relationship between that particles size and MTT reduction, as well as BrdU incorporation, such that the opal particles did not induce cytotoxic effect and genotoxicity in cultured mouse cells.

  12. Protection of MES23.5 dopaminergic cells by obestatin is mediated by proliferative rather than anti-apoptotic action.

    Science.gov (United States)

    Shen, Xiao-Li; Jia, Feng-Ju; Song, Ning; Xie, Jun-Xia; Jiang, Hong

    2014-02-01

    Obestatin is an endogenous peptide sharing a precursor with ghrelin. This study aims to investigate whether and how obestatin protects MES23.5 dopaminergic cells against 1-methyl-4-phenylpyridinium (MPP(+))-induced neurotoxicity. MES23.5 cells were pretreated with obestatin (10(-13)-10(-6) mol/L) for 20 min prior to incubation with 200 μmol/L MPP(+) for 12 or 24 h, or treated with obestatin alone (10(-13) to 10(-6) mol/L) for 0, 6, 12, and 24 h. The methyl thiazolyl tetrazolium (MTT) assay was used to measure cell viability. Flow cytometry was used to measure the caspase-3 activity and the mitochondrial transmembrane potential. Proliferating cell nuclear antigen (PCNA) protein levels were determined by Western blotting. Obestatin (10(-13) to 10(-7) mol/L) pretreatment blocked or even reversed the MPP(+)-induced reduction of viability in MES23.5 cells, but had no effect on MPP(+)-induced mitochondrial transmembrane potential collapse and caspase-3 activation. When applied alone, obestatin increased viability. Elevated PCNA levels occurred with 10(-7), 10(-9), 10(-11) and 10(-13) mol/L obestatin treatment for 12 h. The results suggest that the protective effects of obestatin against MPP(+) in MES23.5 cells are due to its proliferation-promoting rather than anti-apoptotic effects.

  13. Inhibitory effect of farnesol on biofilm formation by Candida tropicalis

    Directory of Open Access Journals (Sweden)

    E Zibafar

    2009-03-01

    Full Text Available ABSTRACT Background: Candidiasis associated with indwelling medical devices is especially problematic since they can act as substrates for biofilm growth which are highly resistant to antifungal drugs. Farnesol is a quorum-sensing molecule that inhibits filamentation and biofilm formation in Candida albicans. Since in recent years Candida tropicalis have been reported as an important and common non-albicans Candida species with high drug resistance pattern, the inhibitory effect of farnesol on biofilm formation by Candida tropicalis was evaluated. Methods: Five Candida tropicalis strains were treated with different concentration of farnesol (0, 30 and 300 µM after 0, 1 and 4 hrs of adherence and then they were maintained under biofilm formation condition in polystyrene, 96-well microtiter plates at 37°C for 48 hrs. Biofilm formation was measured by a semiquantitative colorimetric technique based on reduction assay of 2,3- bis  -2H-tetrazolium- 5- carboxanilide (XTT. Results: The results indicated that the initial adherence time had no effect on biofilm formation and low concentration of farnesol (30 µM could not inhibit biofilm formation. However the presence of non-adherent cells increased biofilm formation significantly and the high concentration of farnesol (300 µM could inhibit biofilm formation. Conclusion: Results of this study showed that the high concentration of farnesol could inhibit biofilm formation and may be used as an adjuvant in prevention and in therapeutic strategies with antifungal drugs.

  14. Hypericin and pulsed laser therapy of squamous cell cancer in vitro.

    Science.gov (United States)

    Bublik, Michael; Head, Christian; Benharash, Peyman; Paiva, Marcos; Eshraghi, Adrian; Kim, Taiho; Saxton, Romaine

    2006-06-01

    This in vitro study compares continuous wave and pulsed laser light at longer wavelengths for activation of the phototoxic drug hypericin in human cancer cells. Two-photon pulsed laser light now allows high-resolution fluorescent imaging of cancer cells and should provide deeper tissue penetration with near infrared light for improved detection as well as phototoxicity in human tumors. Cultured Seoul National University (SNU)-1 tumor cells from a squamous cell carcinoma (SCC) were incubated with hypericin before photoirradiation at four laser wavelengths. Phototoxicity of hypericin sensitized SCC cells was measured by dimethyl thiazoldiphenyl (MTT) tetrazolium bromide cell viability assays and by confocal fluorescence microscopy via 532-nm and infrared two-photon pulsed laser light. Phototoxic response increased linearly with hypericin dose of 0.1-2 microM, light exposure time of 5-120 sec, and pulsed dye laser wavelengths of 514-593 nm. Light energy delivery for 50% cell phototoxicity (LD50) response was 9 joules at 514 nm, 3 joules at 550 nm, and less than 1 joule at the 593 nm hypericin light absorption maxima. Fluorescence confocal microscopy revealed membrane and perinuclear localization of hypericin in the SNU cells with membrane damage seen after excitation with visible 532 nm continuous wave light or two-photon 700-950 nm picosecond pulsed laser irradiation. Hypericin may be a powerful tumor targetting drug when combined with pulsed laser light in patients with recurrent head and neck SCC.

  15. Isolation and biological activities of decanal, linalool, valencene, and octanal from sweet orange oil.

    Science.gov (United States)

    Liu, Kehai; Chen, Qiulin; Liu, Yanjun; Zhou, Xiaoyan; Wang, Xichang

    2012-11-01

    Product 1 (82.25% valencene), product 2 (73.36% decanal), product 3 (78.12% octanal), and product 4 (90.61% linalool) were isolated from sweet orange oil by combined usage of molecular distillation and column chromatography. The antioxidant activity of sweet orange oil and these products was investigated using 2,2-diphenyl-1-picrylhydrazyl and reducing power assays. In this test, product 1 (82.25% valencene), product 2 (73.36% decanal), and product 4 (90.61% linalool) had antioxidant activity, but lower than sweet orange oil. The antimicrobial activity was investigated in order to evaluate their efficacy against 5 microorganisms. The results showed that sweet orange oil, product 2 (73.36% decanal), product 3 (78.12% octanal), and product 4 (90.61% linalool) had inhibitory and bactericidal effect on the test microorganisms (except Penicillium citrinum). Valencene did not show any inhibitory effect. Saccharomyces cerivisiae was more susceptible, especially to the crude sweet orange oil (minimal inhibitory concentration 6.25 μL/mL). The cytotoxicity was evaluated on Hela cells using the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. All test samples showed significant cytotoxicity on the cell lines with IC(50) values much less than 20 μg/mL. © 2012 Institute of Food Technologists®

  16. 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.

  17. Wound healing properties of PVA/starch/chitosan hydrogel membranes with nano Zinc oxide as antibacterial wound dressing material.

    Science.gov (United States)

    Baghaie, Shaghayegh; Khorasani, Mohammad T; Zarrabi, Ali; Moshtaghian, Jamal

    2017-12-01

    In this work, hydrogel membranes were developed based on poly vinyl alcohol (PVA), starch (St), and chitosan (Cs) hydrogels with nano Zinc oxide (nZnO). PVA/St/Cs/nZnO hydrogel membranes were prepared by freezing-thawing cycles, and the aqueous PVA/St solutions were prepared by dissolving PVA in distilled water. After the dissolution of PVA, starch was mixed, and the mixture was stirred. Then, chitosan powder was added into acetic acid, and the mixture was stirred to form a chitosan solution. Subsequently, Cs, St and PVA solutions were blended together to form a homogeneous PVA/St/Cs ternary blend solution. Measurement of Equilibrium Swelling Ratio (ESR), Water Vapor Transmission Test (WVTR), mechanical properties, scanning electron microscopy (SEM), MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] assay, antibacterial studies, in vivo wound healing effect and histopathology of the hydrogel membranes were then performed. The examination revealed that the hydrogel membranes were more effective as a wound dressing in the early stages of wound healing and that the gel could be used in topic applications requiring a large spectrum of antibacterial activity; namely, as a bandage for wound dressing.

  18. Real-time ultrasound imaging of irreversible electroporation in a porcine liver model adequately characterizes the zone of cellular necrosis.

    Science.gov (United States)

    Schmidt, Carl R; Shires, Peter; Mootoo, Mary

    2012-02-01

      Irreversible electroporation (IRE) is a largely non-thermal method for the ablation of solid tumours. The ability of ultrasound (US) to measure the size of the IRE ablation zone was studied in a porcine liver model.   Three normal pig livers were treated in vivo with a total of 22 ablations using IRE. Ultrasound was used within minutes after ablation and just prior to liver harvest at either 6 h or 24 h after the procedure. The area of cellular necrosis was measured after staining with nitroblue tetrazolium and the percentage of cell death determined by histomorphometry.   Visible changes in the hepatic parenchyma were apparent by US after all 22 ablations using IRE. The mean maximum diameter of the ablation zone measured by US during the procedure was 20.1 ± 2.7 mm. This compared with a mean cellular necrosis zone maximum diameter of 20.3 ± 2.9 mm as measured histologically. The mean percentage of dead cells within the ablation zone was 77% at 6 h and 98% at 24 h after ablation.   Ultrasound is a useful modality for measuring the ablation zone within minutes of applying IRE to normal liver tissue. The area of parenchymal change measured by US correlates with the area of cellular necrosis. © 2011 International Hepato-Pancreato-Biliary Association.

  19. Design, Synthesis and Antitumor Evaluation of Novel Pyrazolopyrimidines and Pyrazoloquinazolines

    Directory of Open Access Journals (Sweden)

    Mohamed El-Naggar

    2018-05-01

    Full Text Available A series of N-aryl-7-aryl-pyrazolo[1,5-a]pyrimidines 18a–u and N-aryl-pyrazolo[1,5-a]quinazolines 25a–c were designed and synthesized via the reaction of 5-aminopyrazoles 11a–c with enaminones 12a–g or 19, respectively. The new compounds were screened for their in vitro antitumor activity toward liver (HepG-2 and breast (MCF-7 human cancer cells using 3-[4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2H-tetrazolium bromide MTT assay. From the results, it was found that all compounds showed dose-dependent cytotoxic activities against both HepG-2 and MCF-7 cells. Two compounds 18o and 18a were selected for further investigations. Cell cycle analysis of liver (HepG-2 cells treated with 18o and breast (MCF-7 cells treated with 18a showed cell cycle arrest at G2/M phase and pro-apoptotic activity as indicated by annexin V-FITC staining.

  20. Modified Polymeric Nanoparticles Exert In Vitro Antimicrobial Activity Against Oral Bacteria.

    Science.gov (United States)

    Toledano-Osorio, Manuel; Babu, Jegdish P; Osorio, Raquel; Medina-Castillo, Antonio L; García-Godoy, Franklin; Toledano, Manuel

    2018-06-14

    Polymeric nanoparticles were modified to exert antimicrobial activity against oral bacteria. Nanoparticles were loaded with calcium, zinc and doxycycline. Ions and doxycycline release were measured by inductively coupled plasma optical emission spectrometer and high performance liquid chromatography. Porphyromonas gingivalis , Lactobacillus lactis , Streptoccocus mutans , gordonii and sobrinus were grown and the number of bacteria was determined by optical density. Nanoparticles were suspended in phosphate-buffered saline (PBS) at 10, 1 and 0.1 mg/mL and incubated with 1.0 mL of each bacterial suspension for 3, 12, and 24 h. The bacterial viability was assessed by determining their ability to cleave the tetrazolium salt to a formazan dye. Data were analyzed by ANOVA and Scheffe’s F ( p Nanoparticles (60% to 99% reduction) followed by Ca-Nanoparticles or Zn-Nanoparticles (30% to 70% reduction) and finally the non-doped nanoparticles (7% to 35% reduction). P. gingivalis , S. mutans and L. lactis were the most susceptible bacteria, being S. gordonii and S. sobrinus the most resistant to the tested nanoparticles.

  1. METHODS FOR OVERCOMING SEED DORMANCY AND THE INITIAL GROWTH OF Ziziphus joazeiro Mart. IN DIFFERENT SOILS

    Directory of Open Access Journals (Sweden)

    JOSENARA DAIANE DE SOUZA COSTA

    2016-01-01

    Full Text Available Considering the potential and importance of biodiversity of the Caatinga biome, studies on endemic species are relevant to conservation of its natural resources. The objectives of this work was to evaluate seed viability and physical methods for overcoming seed dormancy in juazeiro (Ziziphus joazeiro Mart. and assess the influence of different types of soil on the initial growth of juazeiro plants. The experiments were conducted in three stages. In the first stage, the viability of seeds from different lots was evaluated using the tetrazolium test. The second stage was conducted in a plant nursery, evaluating the application of physical processes (scarification with sandpaper and hammer in overcoming seed dormancy, and the initial growth of the plants. In the third step, the chlorophyll a and b contents, gas exchange and dry matter accumulation were evaluated in young juazeiro plants in three types of soil (Oxisol, Entisol and Vertisol. The results indicate that the viability of juazeiro seeds is greatly influenced by differences between the progenitor plants and by storage conditions, which enable a longevity of two years when stored at 22°C and 16% of relative humidity. The scarification methods with sandpaper and hammer are not efficient to reduce the time and uniform seed germination. The juazeiro plants have high growth potential in different edaphic environments, adapting well to soils of different textures and chemical compositions.

  2. 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.

  3. 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.

  4. Compounds used to produce cloned animals are genotoxic and mutagenic in mammalian assays in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    R.J. Oliveira

    2014-04-01

    Full Text Available 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.

  5. Candida albicans survival and biofilm formation under starvation conditions.

    Science.gov (United States)

    Ning, Y; Hu, X; Ling, J; Du, Y; Liu, J; Liu, H; Peng, Z

    2013-01-01

    To investigate the survival and biofilm formation capacity of Candida albicans in starvation and under anaerobic conditions. Candida albicans growth and survival were monitored in vitro for up to 8 months. Fungal suspensions from late exponential, stationary and starvation phases were incubated on human dentine, polystyrene and glass slides. Scanning electron microscopy (SEM) was used to observe the process of biofilm formation. 2,3-bis(2-Methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide inner salt (XTT) reduction assay was performed to quantify the biofilm formation capability, and confocal laser scanning microscopy (CLSM) was used to study and make semi-quantitative comparisons of the ultrastructure of biofilms formed on human dentine. 'XTT bioactivity' and 'COMSTAT results' were analysed by two-way analysis of variance (ANOVA) and one-way ANOVA, respectively. Candida albicans survived for over six months. SEM demonstrated that starving C. albicans produced mature biofilms on different substrata. C. albicans of the same growth phase incubated on human dentine displayed significantly higher biofilm formation capability than on polystyrene or glass slides (P roughness coefficient and surface/volume ratio (P < 0.05). Candida albicans cells can survive and form biofilms in anaerobic and nutrient-limited conditions and may pose a treatment challenge. © 2012 International Endodontic Journal.

  6. Self-Assembled Lipid Nanoparticles for Oral Delivery of Heparin-Coated Iron Oxide Nanoparticles for Theranostic Purposes.

    Science.gov (United States)

    Truzzi, Eleonora; Bongio, Chiara; Sacchetti, Francesca; Maretti, Eleonora; Montanari, Monica; Iannuccelli, Valentina; Vismara, Elena; Leo, Eliana

    2017-06-09

    Recently, solid lipid nanoparticles (SLNs) have attracted increasing attention owing to their potential as an oral delivery system, promoting intestinal absorption in the lymphatic circulation which plays a role in disseminating metastatic cancer cells and infectious agents throughout the body. SLN features can be exploited for the oral delivery of theranostics. Therefore, the aim of this work was to design and characterise self-assembled lipid nanoparticles (SALNs) to encapsulate and stabilise iron oxide nanoparticles non-covalently coated with heparin (Fe@hepa) as a model of a theranostic tool. SALNs were characterised for physico-chemical properties (particle size, surface charge, encapsulation efficiency, in vitro stability, and heparin leakage), as well as in vitro cytotoxicity by methyl thiazole tetrazolium (MTT) assay and cell internalisation in CaCo-2, a cell line model used as an indirect indication of intestinal lymphatic absorption. SALNs of about 180 nm, which are stable in suspension and have a high encapsulation efficiency (>90%) were obtained. SALNs were able to stabilise the heparin coating of Fe@hepa, which are typically unstable in physiological environments. Moreover, SALNs-Fe@hepa showed no cytotoxicity, although their ability to be internalised into CaCo-2 cells was highlighted by confocal microscopy analysis. Therefore, the results indicated that SALNs can be considered as a promising tool to orally deliver theranostic Fe@hepa into the lymphatic circulation, although further in vivo studies are needed to comprehend further potential applications.

  7. 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

  8. Phenolic composition of selected herbal infusions and their anti-inflammatory effect on a colonic model in vitro in HT-29 cells

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    Elda Herrera-Carrera

    2015-12-01

    Full Text Available Some herbal infusions used in folk medicine in Mexico to treat gastrointestinal disorders were evaluated. Antioxidant activity and phenolic compounds were analyzed on the lyophilized aqueous crude extracts (LACE of arnica (Aster gymnocephalus, chamomile (Chamaemelum nobile, cumin (Cominum cyminum, desert resurrection plant (DRP (Selaginella lepidophylla, laurel (Listea glaucescens, marjoram (Origanum majorana, mint (Mentha spicata, salvilla (Buddleia scordioides and yerbaniz (Tagetes lucida. Total phenolic content ranged from 8.0 to 70.7 μg GAE/mg for DRP and laurel respectively. Major phenolic compounds were identified by gas chromatography–mass spectrometry and high-performance liquid chromatography. The IC50 determined by the degradation of the deoxy-d-ribose ranged from 2,452.53 to 5,097.11 μg/mL. The cytoprotective effect of the LACE alone and on indomethacin-induced oxidative stress in HT-29 cells was tested. The tetrazolium dye MTT assay was performed in concentrations of 0.125–10 mg/mL allowing choosing the lowest concentration for this experimentation. Inflammation markers were measured by Western blotting. None of the extracts inhibited COX-1 by themselves; however, it was observed that extracts have a modulation effect over COX-2, TNFα, NFκB, and IL-8. By the decrease in the expression of pro-inflammatory cytokines, it follows that salvilla, chamomile, and laurel show promising anti-inflammatory effects.

  9. Sinularin Selectively Kills Breast Cancer Cells Showing G2/M Arrest, Apoptosis, and Oxidative DNA Damage

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    Hurng-Wern Huang

    2018-04-01

    Full Text Available The natural compound sinularin, isolated from marine soft corals, is antiproliferative against several cancers, but its possible selective killing effect has rarely been investigated. This study investigates the selective killing potential and mechanisms of sinularin-treated breast cancer cells. In 3-(4,5-dimethylthiazol-2-yl-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl-2H- tetrazolium, inner salt (MTS assay, sinularin dose-responsively decreased the cell viability of two breast cancer (SKBR3 and MDA-MB-231 cells, but showed less effect on breast normal (M10 cells after a 24 h treatment. According to 7-aminoactinomycin D (7AAD flow cytometry, sinularin dose-responsively induced the G2/M cycle arrest of SKBR3 cells. Sinularin dose-responsively induced apoptosis on SKBR3 cells in terms of a flow cytometry-based annexin V/7AAD assay and pancaspase activity, as well as Western blotting for cleaved forms of poly(ADP-ribose polymerase (PARP, caspases 3, 8, and 9. These caspases and PARP activations were suppressed by N-acetylcysteine (NAC pretreatment. Moreover, sinularin dose-responsively induced oxidative stress and DNA damage according to flow cytometry analyses of reactive oxygen species (ROS, mitochondrial membrane potential (MitoMP, mitochondrial superoxide, and 8-oxo-2′-deoxyguanosine (8-oxodG. In conclusion, sinularin induces selective killing, G2/M arrest, apoptosis, and oxidative DNA damage of breast cancer cells.

  10. Investigation on cell biocompatible behaviors of polyaniline film fabricated via electroless surface polymerization

    International Nuclear Information System (INIS)

    Liu Sheng; Wang Jinqing; Zhang Dong; Zhang Puliang; Ou Junfei; Liu Bin; Yang Shengrong

    2010-01-01

    Considering for the potential application in tissue engineering, polyaniline (PANi) film was fabricated via a two-step route: a self-assembled monolayer of C 6 H 5 NHC 3 H 6 Si(OMe) 3 was firstly formed on the single-crystal Si substrate; the conducting PANi film was then prepared through electroless surface polymerization of the aniline molecules on the aniline monolayer-bearing silane surface in an acidic aqueous solution. The formation of PANi film on Si surface was confirmed by characterizations of X-ray photoelectron spectroscope (XPS) and specular reflectance Fourier transform infrared (SR-FTIR) spectrum, etc. At last, the proliferation behaviors of PC-12 cells on the PANi film surface were studied by the [3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) colorimetric assays, acridine orange fluorometric staining, and scanning electron microscope (SEM) observation, etc. The results demonstrate that the as-prepared PANi film provides high ability for cell proliferation, exhibiting promising potentials as surface coating to cultivate neuronal cells for applications in the tissue engineering.

  11. Highly fluorescent CdTe quantum dots with reduced cytotoxicity-A Robust biomarker

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    Jandi Kim

    2015-03-01

    Full Text Available l-Cysteine (Cys capped CdTe quantum dots (CdTe@Cys QDs were successfully synthesized in an aqueous medium. The synthesized CdTe@Cys samples were analyzed using Fourier transform infrared (FT-IR spectroscopy, fluorescence (FL spectroscopy, transmission electron microscopy (TEM, confocal microscopy and subsequently subjected to the antibacterial test. Systematic investigations were carried out for the determination of optimal conditions namely the ratios of Cd:Te, CdTe:Cys, pH value and the chemical stability of CdTe@Cys. Moreover, the reactivation of FL intensity in the CdTe@Cys sample was done easily by the addendum of Cys. The introduction of additional cysteine to the CdTe@Cys QDs sample showed an enhancement in terms of the FL intensity and stability along with the reduced antibacterial activity. This was further confirmed through Thiazolyl blue tetrazolium bromide (MTT assays. Both the result of the bio-stability tests namely the antibacterial test and MTT assay displayed similarities between the externally added Cys and cytotoxicity of the bacteria and human HeLa cancer cell lines. Confocal microscopic images were captured for the CdTe@Cys conjugated Escherichia coli.

  12. Conjugated Linoleic Acid Stimulates Apoptosis in RH and Tehran Strains of Toxoplasma gondii, in Vitro.

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    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.

  13. Potential protection of green tea polyphenols against 1800 MHz electromagnetic radiation-induced injury on rat cortical neurons.

    Science.gov (United States)

    Liu, Mei-Li; Wen, Jian-Qiang; Fan, Yu-Bo

    2011-10-01

    Radiofrequency electromagnetic fields (EMF) are harmful to public health, but the certain anti-irradiation mechanism is not clear yet. The present study was performed to investigate the possible protective effects of green tea polyphenols against electromagnetic radiation-induced injury in the cultured rat cortical neurons. In this study, green tea polyphenols were used in the cultured cortical neurons exposed to 1800 MHz EMFs by the mobile phone. We found that the mobile phone irradiation for 24 h induced marked neuronal cell death in the MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazolium bromide) and TUNEL (TdT mediated biotin-dUTP nicked-end labeling) assay, and protective effects of green tea polyphenols on the injured cortical neurons were demonstrated by testing the content of Bcl-2 Assaciated X protein (Bax) in the immunoprecipitation assay and Western blot assay. In our study results, the mobile phone irradiation-induced increases in the content of active Bax were inhibited significantly by green tea polyphenols, while the contents of total Bax had no marked changes after the treatment of green tea polyphenols. Our results suggested a neuroprotective effect of green tea polyphenols against the mobile phone irradiation-induced injury on the cultured rat cortical neurons.

  14. Antitumor effects evaluation of a novel porphyrin derivative in photodynamic therapy.

    Science.gov (United States)

    Li, Jian-Wei; Wu, Zhong-Ming; Magetic, Davor; Zhang, Li-Jun; Chen, Zhi-Long

    2015-12-01

    In this paper, the antitumor activity of a novel porphyrin-based photosensitizer 5,10,15,20-tetrakis[(5-diethylamino)pentyl] porphyrin (TDPP) was reported in vitro and in vivo. The photophysical and cellular properties of TDPP were investigated. The singlet oxygen generation quantum yield of TDPP was detected; it showed a high singlet oxygen quantum yield of 0.52. The intracellular distribution of photosensitizer was detected with laser scanning confocal microscopy. The efficiency of TDPP-photodynamic therapy (PDT) in vitro was analyzed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and in situ trypan blue exclusion test. Treated with a 630-nm laser, TDPP can kill cultured human esophageal cancer cell line (Eca-109) cells and reduce the growth of Eca-109 xenograft tumors significantly in BABL/c nude mice. And histopathological study was also used to confirm the antitumor effect. It has the perspective to be developed as a new antitumor drug in photodynamic therapy and deserves further investigation.

  15. Cytotoxicity of peracetic acid: evaluation of effects on metabolism, structure and cell death.

    Science.gov (United States)

    Viola, K S; Rodrigues, E M; Tanomaru-Filho, M; Carlos, I Z; Ramos, S G; Guerreiro-Tanomaru, J M; Faria, G

    2017-01-30

    To evaluate the cytotoxicity and the mechanism of cell aggression of peracetic acid (PA) in comparison with sodium hypochlorite (NaOCl). L929 fibroblasts were exposed to 1% PA and 2.5% NaOCl, at several dilutions for 10 min. The following parameters were evaluated: cell metabolism by methylthiazol tetrazolium assay, external morphology by scanning electron microscopy, ultrastructure by transmission electron microscopy, the cytoskeleton by means of actin and α-tubulin labelling, and the type of cell death by flow cytometry (apoptosis/necrosis). The data were analysed by two-way anova and the Bonferroni post-test (α = 0.05). The PA group had lower cell viability and a higher percentage of necrotic cells than the NaOCl group (P < 0.05). Both solutions diminished cell metabolism, led to destructuring of the cytoskeleton, created changes in the external morphology, resulted in the accumulation of proteins in the rough endoplasmic reticulum and induced cell death predominantly by necrosis. However, these changes were observed in lower doses of PA when compared with NaOCl. Although they had the same mechanism of cytotoxicity, 1% PA had greater cytotoxic potential than 2.5% NaOCl. © 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  16. Disinfection of Streptococcus mutans biofilm by a non-thermal atmospheric plasma brush

    Science.gov (United States)

    Hong, Qing; Dong, Xiaoqing; Chen, Meng; Xu, Yuanxi; Sun, Hongmin; Hong, Liang; Wang, Yong; Yu, Qingsong

    2016-07-01

    This study investigated the argon plasma treatment effect on disinfecting dental biofilm by using an atmospheric pressure plasma brush. Streptococcus mutans biofilms were developed for 3 days on the surfaces of hydroxyapatite (HA) discs, which were used to simulate human tooth enamel. After plasma treatment, cell viability in the S. mutans biofilms was characterized by using 3-(4,5-dimethylazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and confocal laser scanning microscopy (CLSM). Compared with the untreated control group, about 90% bacterial reduction in the biofilms was observed after 1 min plasma treatment. Scanning electron microscopy (SEM) examination indicated severe cell damages occurred on the top surface of the plasma treated biofilms. Confocal laser scanning microscopy (CLSM) showed that plasma treatment was effective as deep as 20 µm into the biofilms. When combined with antibiotic treatment using 0.2% chlorhexidine digluconate solution, the plasma treatment became more effective and over 96% bacterial reduction was observed with 1 min plasma treatment.

  17. 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.

  18. Assessment of bacterial endospore viability with fluorescent dyes.

    Science.gov (United States)

    Laflamme, C; Lavigne, S; Ho, J; Duchaine, C

    2004-01-01

    To validate three fluorescence viability assays designed primarily for vegetative cells on pure Bacillus endospores. Purified fresh and gamma-irradiated Bacillus endospores (Bacillus cereus, B. coagulans and two strains of B. subtilis) were used. The viability assays were: 5-cyano-2,3-diotolyl tetrazolium chloride (CTC) to test respiratory activity and early germination, DiBAC4(3) and Live/Dead BacLight to measure membrane energization and permeabilization, respectively. Gamma irradiation treatment completely eliminated spore culturability and was used as negative control. The untreated spores showed respiratory activity after 1 h of incubation and this was characteristic of almost 100% of spores after 24 h. The membrane potential assessment gave no answer about spore viability. A lower proportion of untreated spores had permeabilized membrane compared with gamma-irradiated spores using Live/Dead BacLight (P plate count. This study shows that fluorescence tests could be applied to assess viability in potentially pathogenic Bacillus spore preparations within 1 h.

  19. Nicotine promotes proliferation and collagen synthesis of chondrocytes isolated from normal human and osteoarthritis patients.

    Science.gov (United States)

    Ying, Xiaozhou; Cheng, Shaowen; Shen, Yue; Cheng, Xiaojie; An Rompis, Ferdinand; Wang, Wei; Lin, Zhongqin; Chen, Qingyu; Zhang, Wei; Kou, Dongquan; Peng, Lei; Tian, Xin Qiao; Lu, Chuan Zhu

    2012-01-01

    The aims of the study were to show the direct effect of nicotine with different concentrations (0, 25, 50, and 100 ng/ml) on chondrocytes isolated from normal human and osteoarthritis patients, respectively. Microscopic observation was performed during the culture with an inverted microscope. Methyl thiazolyl tetrazolium (MTT) assay method was adopted to observe the influence of nicotine on the proliferation of chondrocytes, and real-time PCR and ELISA were used to assay the mRNA and protein expression of type II collagen and aggrecan, respectively. We discovered that the OA chondrocytes were similar to fibroblasts in shape and grow slower than normal chondrocytes. The proliferation of the two kinds of chondrocytes was increased in a concentration-dependent manner and in a time-dependent manner (P<0.05). Also, we found that the mRNA level of type II collagen were upregulated under 25-100 ng/ml nicotine doses both in the two kinds of chondrocytes compared with control. The expression of protein levels of type II collagen were synthesized in line with the increase in mRNA. No effect was observed on aggrecan synthesis with any nicotine dose. We concluded that nicotine has the same effect on both chondrocytes, obtained either from osteoarthritis patients or from normal human, and the positive effect of smoking in OA may relate to the alteration in metabolism of chondrocytes.

  20. Porphyromonas gingivalis decreases osteoblast proliferation through IL-6-RANKL/OPG and MMP-9/TIMPs pathways

    Directory of Open Access Journals (Sweden)

    Le Xuan

    2009-01-01

    Full Text Available Background: Porphyromonas gingivalis, an important periodontal pathogen, is closely associated with inflammatory alveolar bone resorption. This bacterium exerts its pathogenic effect indirectly through multiple virulence factors, such as lipopolysaccharides, fimbriae, and proteases. Another possible pathogenic path may be through a direct interaction with the host′s soft and hard tissues (e.g., alveolar bone, which could lead to periodontitis. Aims and Objectives: The aim of the present study was to investigate the direct effect of live and heat-inactivated P gingivalis on bone resorption, using an in vitro osteoblast culture model. Results: Optical microscopy and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide MTT assay revealed that live P gingivalis induced osteoblast detachment and reduced their proliferation. This effect was specific to live bacteria and was dependent on their concentration. Live P gingivalis increased IL-6 mRNA expression and protein production and downregulated RANKL and OPG mRNA expression. The effect of live P gingivalis on bone resorption was strengthened by an increase in MMP-9 expression and its activity. This increase was accompanied by an increase in TIMP-1 and TIMP-2 mRNA expression and protein production by osteoblasts infected with live P gingivalis. Conclusion: Overall, the results suggest that direct contact of P gingivalis with osteoblasts induces bone resorption through an inflammatory pathway that involves IL-6, RANKL/OPG, and MMP-9/TIMPs.

  1. The Flavin-Containing Reductase Domain of Cytochrome P450 BM3 Acts as a Surrogate for Mammalian NADPH-P450 Reductase.

    Science.gov (United States)

    Park, Seon-Ha; Kang, Ji-Yeon; Kim, Dong-Hyun; Ahn, Taeho; Yun, Chul-Ho

    2012-11-01

    Cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium is a self-sufficient monooxygenase that consists of a heme domain and FAD/FMN-containing reductase domain (BMR). In this report, the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) by BMR was evaluated as a method for monitoring BMR activity. The electron transfer proceeds from NADPH to BMR and then to BMR substrates, MTT and CTC. MTT and CTC are monotetrazolium salts that form formazans upon reduction. The reduction of MTT and CTC followed classical Michaelis-Menten kinetics (kcat =4120 min(-1), Km =77 μM for MTT and kcat =6580 min(-1), Km =51 μM for CTC). Our continuous assay using MTT and CTC allows the simple, rapid measurement of BMR activity. The BMR was able to metabolize mitomycin C and doxorubicin, which are anticancer drug substrates for CPR, producing the same metabolites as those produced by CPR. Moreover, the BMR was able to interact with CYP1A2 and transfer electrons to promote the oxidation reactions of substrates by CYP1A2 and CYP2E1 in humans. The results of this study suggest the possibility of the utilization of BMR as a surrogate for mammalian CPR.

  2. Water extract of Semecarpus parvifolia Thw. leaves inhibits cell proliferation and induces apoptosis on HEp-2 cells.

    Science.gov (United States)

    Soysa, Preethi; Jayarthne, Panchima; Ranathunga, Imali

    2018-03-05

    Semecarpus parvifolia Thw is used as an ingredient of poly herbal decoctions to treat cancer in traditional medicine. The present study aims to investigate the antiproliferative activity on HEp 2 cells by the water extract of S. parvifolia leaves and to evaluate potential mechanisms. The plant extract was exposed to S. parvifolia for 24 hours and antiproliferative activity was quantified by Sulforhodamine B (SRB), 3-(4, 5-dimethythiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) and Lactate dehydrogenase (LDH) assays. Morphological changes were observed after staining cells with ethidium bromide/acridine orange (EB/AO) and Giemsa dye. Comet assay was performed to evaluate the DNA damage. The toxicity of the plant extract was determined by brine shrimp lethality assay. S. parvifolia leaves reduced the cell proliferation in a dose and time dependent manner. A two fold increase in NO level was observed at higher concentrations. Morphological changes characteristic to apoptosis were observed in light microscopy, Giemsa and EB/AO stained cells. Fragmented DNA further confirmed its capacity to induce apoptosis. No lethality was observed with brine shrimps. The results suggest that Semecarpus parvifolia Thw induces apoptosis in HEp-2 cells through a NO dependent pathway.

  3. Structure-based pharmacophore design and virtual screening for novel potential inhibitors of epidermal growth factor receptor as an approach to breast cancer chemotherapy.

    Science.gov (United States)

    Mahernia, Shabnam; Hassanzadeh, Malihe; Sharifi, Niusha; Mehravi, Bita; Paytam, Fariba; Adib, Mehdi; Amanlou, Massoud

    2018-02-01

    Cancer cells are described with features of uncontrolled growth, invasion and metastasis. The epidermal growth factor receptor subfamily of tyrosine kinases (EGFR-TK) plays a crucial regulatory role in the control of cellular proliferation and progression of various cancers. Therefore, its inhibition might lead to the discovery of a new generation of anticancer drugs. In the present study, structure-based pharmacophore modeling, molecular docking and molecular dynamics simulations were applied to identify potential hits, which exhibited good inhibition on the proliferation of MCF-7 breast cancer cell line and favorable binding interactions on EGFR-TK. Selected compounds were examined for their anticancer activity against the Michigan Cancer Foundation-7 (MCF-7) breast cancer cell line which overexpresses EGFR using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay. Compounds 1 and 2, with an isoindoline-1-one core, induced significant inhibition of breast cancer cells proliferation with IC[Formula: see text] values 327 and 370 nM, respectively.

  4. 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.

  5. Increased Loading, Efficacy and Sustained Release of Silibinin, a Poorly Soluble Drug Using Hydrophobically-Modified Chitosan Nanoparticles for Enhanced Delivery of Anticancer Drug Delivery Systems

    Directory of Open Access Journals (Sweden)

    Cha Yee Kuen

    2017-11-01

    Full Text Available Conventional delivery of anticancer drugs is less effective due to pharmacological drawbacks such as lack of aqueous solubility and poor cellular accumulation. This study reports the increased drug loading, therapeutic delivery, and cellular accumulation of silibinin (SLB, a poorly water-soluble phenolic compound using a hydrophobically-modified chitosan nanoparticle (pCNP system. In this study, chitosan nanoparticles were hydrophobically-modified to confer a palmitoyl group as confirmed by 2,4,6-Trinitrobenzenesulfonic acid (TNBS assay. Physicochemical features of the nanoparticles were studied using the TNBS assay, and Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR analyses. The FTIR profile and electron microscopy correlated the successful formation of pCNP and pCNP-SLB as nano-sized particles, while Dynamic Light Scattering (DLS and Field Emission-Scanning Electron Microscopy (FESEM results exhibited an expansion in size between pCNP and pCNP-SLB to accommodate the drug within its particle core. To evaluate the cytotoxicity of the nanoparticles, a Methylthiazolyldiphenyl-tetrazolium bromide (MTT cytotoxicity assay was subsequently performed using the A549 lung cancer cell line. Cytotoxicity assays exhibited an enhanced efficacy of SLB when delivered by CNP and pCNP. Interestingly, controlled release delivery of SLB was achieved using the pCNP-SLB system, conferring higher cytotoxic effects and lower IC50 values in 72-h treatments compared to CNP-SLB, which was attributed to the hydrophobic modification of the CNP system.

  6. Novel T-cell epitopes of ovalbumin in BALB/c mouse: Potential for peptide-immunotherapy

    International Nuclear Information System (INIS)

    Yang, Marie; Mine, Yoshinori

    2009-01-01

    The identification of food allergen T-cell epitopes provides a platform for the development of novel immunotherapies. Despite extensive knowledge of the physicochemical properties of hen ovalbumin (OVA), a major egg allergen, the complete T-cell epitope map of OVA has surprisingly not been defined in the commonly used BALB/c mouse model. In this study, spleen cells obtained from OVA-sensitized mice were incubated in the presence of 12-mer overlapping synthetic peptides, constructed using the SPOTS synthesis method. Proliferative activity was assessed by 72-h in vitro assays with use of the tetrazolium salt WST-1 and led to identification of four mitogenic sequences, i.e., A39R50, S147R158, K263E274, and A329E340. ELISA analyses of interferon (IFN)-γ and interleukin (IL)-4 productions in cell culture supernatants upon stimulation with increasing concentrations of peptides confirmed their immunogenicity. Knowledge of the complete T-cell epitope map of OVA opens the way to a number of experimental investigations, including the exploration of peptide-based immunotherapy.

  7. A smart multifunctional nanocomposite for intracellular targeted drug delivery and self-release

    International Nuclear Information System (INIS)

    Wang Chan; Tao Shengyang; Hu Tao; Yang Jingbang; Meng Changgong; Lv Piping; Wei Wei

    2011-01-01

    A multifunctional 'all-in-one' nanocomposite is fabricated using a colloid, template and surface-modification method. This material encompasses magnetic induced target delivery, cell uptake promotion and controlled drug release in one system. The nanocomposite is characterized by scanning electron microscopy, transmission electron microscopy, x-ray diffraction, N 2 adsorption and vibrating sample magnetometry. The prepared material has a diameter of 350-400 nm, a high surface area of 420.29 m 2 g -1 , a pore size of 1.91 nm and a saturation magnetization of 32 emu g -1 . Doxorubicin (DOX) is loaded in mesopores and acid-sensitive blockers are introduced onto the orifices of the mesopores by a Schiff base linker to implement pH-dependent self-release. Folate was also introduced to improve DOX targeted delivery and endocytosis. The linkers remained intact to block pores with ferrocene valves and inhibit the diffusion of DOX at neutral pH. However, in lysosomes of cancer cells, which have a weak acidic pH, hydrolysis of the Schiff base group removes the nanovalves and allows the trapped DOX to be released. These processes are demonstrated by UV-visible absorption spectra, confocal fluorescence microscopy images and methyl thiazolyl tetrazolium assays in vitro, which suggest that the smart nanocomposite successfully integrates targeted drug delivery with internal stimulus induced self-release and is a potentially useful material for nanobiomedicine.

  8. 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.

  9. Apoptotic activity and gene responses in Drosophila melanogaster S2 cells, induced by azadirachtin A.

    Science.gov (United States)

    Xu, Lin; Li, Sheng; Ran, Xueqin; Liu, Chang; Lin, Rutao; Wang, Jiafu

    2016-09-01

    Azadirachtin has been used as an antifeedant and growth disruption agent for many insect species. Previous investigations have reported the apoptotic effects of azadirachtin on some insect cells, but the molecular mechanisms are still not clear. This study investigated the underlying molecular mechanisms for the apoptotic effects induced by azadirachtin on Drosophila melanogaster S2 cells in vitro. The results of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay demonstrated that azadirachtin exhibited significant cytotoxicity to S2 cells in a time- and dose-dependent manner. The changes in cellular morphology and the DNA fragmentation demonstrated that azadirachtin induced remarkable apoptosis of S2 cells. Expression levels of 276 genes were found to be significantly changed in S2 cells after exposure to azadirachtin, as detected by Drosophila genome array. Among these genes, calmodulin (CaM) was the most highly upregulated gene. Azadirachtin was further demonstrated to trigger intracellular Ca(2+) release in S2 cells. The genes related to the apoptosis pathway, determined from chip data, were validated by the real-time quantitative polymerase chain reaction method. The results showed that azadirachtin-mediated intracellular Ca(2+) release was the primary event that triggered apoptosis in Drosophila S2 cells through both pathways of the Ca(2+) -CaM and EcR/Usp signalling cascade. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

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

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    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.

  11. Agmatine Protects Against 6-OHDA-Induced Apoptosis, and ERK and Akt/GSK Disruption in SH-SY5Y Cells.

    Science.gov (United States)

    Amiri, Esmat; Ghasemi, Rasoul; Moosavi, Maryam

    2016-08-01

    6-Hydroxydopamine (6-OHDA), a metabolite of dopamine is known to induce dopaminergic cell toxicity which makes that a suitable agent inducing an experimental model of Parkinson's disease (PD). Agmatine has been shown to protect against some cellular and animal PD models. This study was aimed to assess whether agmatine prevents 6-OHDA-induced SH-SY5Y cell death and if yes, then how it affects Akt/glycogen synthesis kinase-3β (GSK-3β) and extracellular signal-regulated kinases (ERK) signals. The cells were treated with different drugs, and their viability was examined via MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay and morphological observation. Western blot studies were done to assess cleaved caspase-3, Akt/GSK-3β, and ERK proteins. 6-OHDA-induced cell death and caspase-3 cleavage, while agmatine prevented those changes. 6-OHDA also decreased the amount of phosphorylated Akt (pAkt)/Akt while increased GSK-3β activity which was prevented by agmatine. Additionally, this toxin increased pERK/ERK ratio which was averted again by agmatine. The PI3/Akt inhibitor, LY294002, impeded the changes induced by agmatine, while ERK inhibitor (PD98059) did not disturb the effects of agmatine, and by itself, it preserved the cells against 6-OHDA toxicity. This study revealed that agmatine is protective in 6-OHDA model of PD and affects Akt/GSK-3β and ERK pathways.

  12. Targeting EGFR-overexpressing tumor cells using Cetuximab-immunomicelles loaded with doxorubicin and superparamagnetic iron oxide

    International Nuclear Information System (INIS)

    Liao Chengde; Sun Qiquan; Liang, Biling; Shen Jun; Shuai Xintao

    2011-01-01

    Epidermal growth factor receptor (EGFR), a cellular transmembrane receptor, plays a key role in cell proliferation and is linked to a poor prognosis in various human cancers. In this study, we constructed Cetuximab-immunomicelles in which the anti-EGFR monoclonal antibody was linked to poly(ethylene glycol)-block-poly(ε-caprolactone) (PEG–PCL) nanomicelles that were loaded with doxorubicin (DOX) and superparamagnetic iron oxide (SPIO). The specific interactions between EGFR-overexpressing tumor cells (A431) and immunomicelles were observed using confocal laser scanning microscopy (CLSM) and flow cytometry. Furthermore, the capacity of transporting SPIO into tumor cells using these immunomicelles was evaluated with a 1.5 T clinical magnetic resonance imaging (MRI) scanner. It was found that the acquired MRI T2 signal intensity of A431 cells that were treated with the SPIO-loaded and antibody-functionalized micelles decreased significantly. Using the thiazolyl blue tetrazolium bromide (MTT) assay, we also demonstrated that the immunomicelles inhibited cell proliferation more effectively than their nontargeting counterparts. Our results suggest that Cetuximab-immunomicelles are a useful delivery vehicle for DOX and SPIO to EGFR-overexpressing tumor cells in vitro and that Cetuximab-immunomicelles can serve as a MRI-visible and targeted drug delivery agent for better tumor imaging and therapy.

  13. The anti-biofouling behavior of high voltage pulse electric field (HPEF) mediated by carbon fiber composite coating in seawater.

    Science.gov (United States)

    Feng, Tiantian; Wu, Jinyi; Chai, Ke; Yang, Pengpeng

    2018-04-25

    One of the most important research areas in the marine industry is to investigate new and effective anti-biofouling technologies. In this study, high voltage pulse electric field (HPEF) mediated by carbon fiber (CF) composite coating was utilized to prevent the fouling of bacteria, microalgae and barnacle larvae in seawater. The plate count, 2, 3, 5-triphenyl-tetrazolium chloride (TTC) reduction assay and neutral red (NR) staining and larval motility detection showed that the inactivation rates were at the highest levels, which reached 99.1%, 99.9%, 99.7%, 98.7% and 85% respectively for Pseudomonas sp., Vibrio sp., iron bacteria, Navicula sp. and the second stage nauplii of Balanus reticulatus, under the HPEF with 19 kV pulse amplitude, 23.15 kHz frequency and 0.5 duty cycle. The field-emission scanning electron microscopy (FE-SEM) of Navicula sp. revealed that the HPEF brought about the cell lysis and the cell organic matter release on the coating, which could be the mechanism of the inactivation by the HPEF. Additionally, the FE-SEM and Raman spectroscopy indicated that the HPEF hardly damaged the coating. Copyright © 2018 Elsevier B.V. All rights reserved.

  14. Inhibition of human polymorphonuclear leukocyte function by components of human colostrum and mature milk.

    Science.gov (United States)

    Pickering, L K; Cleary, T G; Caprioli, R M

    1983-04-01

    To compare the effect of human colostrum (days 1 to 3 postpartum) and mature milk (days 170 +/- 24 postpartum) on the function of polymorphonuclear leukocytes (PMNL), Ficoll-Hypaque-separated PMNL from the blood of 60 healthy volunteers were incubated with whole colostrum, colostral lipid, and colostral aqueous phase from 30 mothers, or with mature whole milk and its separated components from 30 mothers, and tested for resting and zymosan-stimulated oxidative metabolism, functional activity, and the presence of Fc receptors. Stimulated oxygen consumption, quantitative nitroblue tetrazolium dye reduction, [1-(14)C]glucose utilization, and Fc receptors were significantly (P cells or cells exposed to the aqueous phase of colostrum. In contrast, PMNL exposed to whole mature milk or to its lipid or aqueous phase caused no significant decrease in any of these parameters when compared to nonexposed cells. In assays of phagocytosis, colostral PMNL or blood PMNL exposed to colostral lipid had a significant (P < 0.001) decrease in their ability to ingest [methyl-(3)H]thymidine-labeled Staphylococcus aureus when compared to non-lipid-exposed PMNL. Blood PMNL exposed to lipid from mature milk had no decrease in ability to ingest S. aureus. Analysis of total lipid and total and individual fatty acid content revealed a uniform increase in all components in mature milk when compared to colostrum. Lipid or lipid-soluble material present in human colostrum but not mature milk causes inhibition of phagocytosis and respiratory burst-related activities of PMNL.

  15. Acetal-Linked Paclitaxel Polymeric Prodrug Based on Functionalized mPEG-PCL Diblock Polymer for pH-Triggered Drug Delivery

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    Yinglei Zhai

    2017-12-01

    Full Text Available The differences in micro-environment between cancer cells and the normal ones offer the possibility to develop stimuli-responsive drug-delivery systems for overcoming the drawbacks in the clinical use of anticancer drugs, such as paclitaxel, doxorubicin, and etc. Hence, we developed a novel endosomal pH-sensitive paclitaxel (PTX prodrug micelles based on functionalized poly(ethylene glycol-poly(ε-caprolactone (mPEG-PCL diblock polymer with an acid-cleavable acetal (Ace linkage (mPEG-PCL-Ace-PTX. The mPEG-PCL-Ace-PTX5 with a high drug content of 23.5 wt % was self-assembled in phosphate buffer (pH 7.4, 10 mM into nanosized micelles with an average diameter of 68.5 nm. The in vitro release studies demonstrated that mPEG-PCL-Ace-PTX5 micelles was highly pH-sensitive, in which 16.8%, 32.8%, and 48.2% of parent free PTX was released from mPEG-PCL-Ace-PTX5 micelles in 48 h at pH 7.4, 6.0, and 5.0, respectively. Thiazolyl Blue Tetrazolium Bromide (MTT assays suggested that the pH-sensitive PTX prodrug micelles displayed higher therapeutic efficacy against MCF-7 cells compared with free PTX. Therefore, the PTX prodrug micelles with acetal bond may offer a promising strategy for cancer therapy.

  16. FRAKSINASI PROTEIN KAPANG LAUT Xylaria psidii KT30 DAN SITOTOKSISITASNYA TERHADAP SEL HeLa [Fractionation of Proteins of Marine Fungus Xylaria psidii KT30 and their Cytotoxicity against HeLa Cells

    Directory of Open Access Journals (Sweden)

    Mita Gebriella Inthe

    2014-06-01

    Full Text Available Cervical cancer is the most common cause of death for Indonesian women after human breast cancer. One of the efforts of cancer treatment is the utilization of natural compounds. One of the microorganisms having the potential as anticancer agent is endophytic fungi. Endophytic fungi from the marine habitat can be isolated from sea weeds, sea grasses, sponges, and mangroves. Xylaria psidii KT30, a marine fungus used in this study was isolated from red seaweed Kappaphycus alvarezii. Xylaria psidii KT30 was cultivated in potato dextrose broth medium for nine days at room temperature 27-29°C in shaking condition. This study aimed to obtain protein fractions from X. psidii KT30 and determine their toxicity againt Chang and HeLa cells. The fractionation process was conducted using DEAE Sephadex A-50 column chromatography and the toxicity was determined by Brine Shrimp Lethality Test (BSLT. The metabolites excreted in the culture broth was extracted using 90% of ammonium sulphate. The extract was then tested for their toxicity against HeLa and Chang cells by Microculture Tetrazolium Technique (MTT assay.The results revealed that LC50 of the protein extract of X. psidii KT30 was 104.95 ppm and IC50 was 69.9 ppm. Based on the National Cancer Institute (NCI, this value showed moderate cytotoxicity against HeLa cells.

  17. Isolation and Partial Characterization of Bioactive Fucoxanthin from Himanthalia elongata Brown Seaweed: A TLC-Based Approach

    Directory of Open Access Journals (Sweden)

    Gaurav Rajauria

    2013-01-01

    Full Text Available 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 Rf (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.

  18. [Preparation of sodium alginate-nanohydroxyapatite composite material for bone repair and its biocompatibility].

    Science.gov (United States)

    Wang, Yanmei; He, Jiacai; Li, Quanli; Shen, Jijia

    2014-02-01

    To prepare sodium alginate-nanohydroxyapatite composite material and to explore its feasibility as a bone repair material. Sodium alginate-nanohydroxyapatite composite material was prepared using chemical cross-linking and freeze-drying technology. The composite was characterized by X-ray diffraction (XRD) and scanning electron microscope (SEM) and its porosity was measured by liquid displacement method. The fifth passage of bone marrow stromal stem cells (BMSCs) were incubated on the composite material and then growth was observed by inverted microscope and SEM. BMSCs were cultured with liquid extracts of the material, methyl thiazolyl tetrazolium (MTT) assay was used to calculate the relative growth rate (RGR) on 1, 3, 5 d and to evaluate the cytotoxicity. Fresh dog blood was added into the liquid extracts to conduct hemolysis test, the spectrophotometer was used to determine the optical density (OD) and to calculate the hemolysis rate. Sodium alginate-nanohydroxyapatite composite material displayed porosity, the porous pore rate was (88.6 +/- 4.5)%. BMSCs showed full stretching and vigorous growth under inverted microscope and SEM. BMSCs cultured with liquid extracts of the material had good activities. The toxicity of composite material was graded as 1. Hemolysis test results showed that the hemolysis rate of the composite material was 1.28%, thus meeting the requirement of medical biomaterials. The composite material fabricated in this study has high porosity and good biocompatibility.

  19. High-Throughput Screening of Coenzyme Preference Change of Thermophilic 6-Phosphogluconate Dehydrogenase from NADP(+) to NAD(.).

    Science.gov (United States)

    Huang, Rui; Chen, Hui; Zhong, Chao; Kim, Jae Eung; Zhang, Yi-Heng Percival

    2016-09-02

    Coenzyme engineering that changes NAD(P) selectivity of redox enzymes is an important tool in metabolic engineering, synthetic biology, and biocatalysis. Here we developed a high throughput screening method to identify mutants of 6-phosphogluconate dehydrogenase (6PGDH) from a thermophilic bacterium Moorella thermoacetica with reversed coenzyme selectivity from NADP(+) to NAD(+). Colonies of a 6PGDH mutant library growing on the agar plates were treated by heat to minimize the background noise, that is, the deactivation of intracellular dehydrogenases, degradation of inherent NAD(P)H, and disruption of cell membrane. The melted agarose solution containing a redox dye tetranitroblue tetrazolium (TNBT), phenazine methosulfate (PMS), NAD(+), and 6-phosphogluconate was carefully poured on colonies, forming a second semi-solid layer. More active 6PGDH mutants were examined via an enzyme-linked TNBT-PMS colorimetric assay. Positive mutants were recovered by direct extraction of plasmid from dead cell colonies followed by plasmid transformation into E. coli TOP10. By utilizing this double-layer screening method, six positive mutants were obtained from two-round saturation mutagenesis. The best mutant 6PGDH A30D/R31I/T32I exhibited a 4,278-fold reversal of coenzyme selectivity from NADP(+) to NAD(+). This screening method could be widely used to detect numerous redox enzymes, particularly for thermophilic ones, which can generate NAD(P)H reacted with the redox dye TNBT.

  20. Non-occlusive topical exposure of human skin in vitro as model for cytotoxicity testing of irritant compounds.

    Science.gov (United States)

    Lönnqvist, Susanna; Briheim, Kristina; Kratz, Gunnar

    2016-02-01

    Testing of irritant compounds has traditionally been performed on animals and human volunteers. Animal testing should always be restricted and for skin irritancy mice and rabbits hold poor predictive value for irritant potential in humans. Irritant testing on human volunteers is restricted by the duration subjects can be exposed, and by the subjectivity of interpreting the visual signs of skin irritation. We propose an irritant testing system using viable human full thickness skin with the loss of cell viability in the exposed skin area as end point measurement. Skin was exposed to sodium dodecyl sulfate (SDS) at 20% concentration by non-occluded topical exposure to establish a positive control response and subsequent test compounds were statistically compared with the 20% SDS response. Cell viability and metabolism were measured with 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The model presents correlation between increased concentration of SDS and decreased viability of cells in the exposed skin area (R(2) = 0.76). We propose the model to be used for cytotoxicity testing of irritant compounds. With fully intact barrier function, the model comprises all cells present in the skin with quantifiable end point measurement.

  1. Protective influence of hyaluronic acid on focal adhesion kinase activity in human skin fibroblasts exposed to ethanol.

    Science.gov (United States)

    Donejko, Magdalena; Rysiak, Edyta; Galicka, Elżbieta; Terlikowski, Robert; Głażewska, Edyta Katarzyna; Przylipiak, Andrzej

    2017-01-01

    The aim of this study was to evaluate the effect of ethanol and hyaluronic acid (HA) on cell survival and apoptosis in cultured human skin fibroblasts. Regarding the mechanism of ethanol action on human skin fibroblasts, we investigated cell viability and apoptosis, expression of focal adhesion kinase (FAK), and the influence of HA on those processes. Studies were conducted in confluent human skin fibroblast cultures that were treated with 25 mM, 50 mM, and 100 mM ethanol or with ethanol and 500 µg/mL HA. Cell viability was examined using methyl thiazolyl tetrazolium (MTT) assay and NC-300 Nucleo-Counter. Imaging of the cells using a fluorescence microscope Pathway 855 was performed to measure FAK expression. Depending on the dosage, ethanol decreased cell viability and activated the process of apoptosis in human skin fibroblasts. HA prevented the negative influence of ethanol on cell viability and prevented apoptosis. The analysis of fluorescence imaging using BD Pathway 855 High-Content Bioimager showed the inhibition of FAK migration to the cell nucleus, depending on the increasing concentration of ethanol. This study proves that downregulation of signaling pathway of FAK is involved in ethanol-induced apoptosis in human skin fibroblasts. The work also indicates a protective influence of HA on FAK activity in human skin fibroblasts exposed to ethanol.

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

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    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.

  3. High Efficient Expression, Purification, and Functional Characterization of Native Human Epidermal Growth Factor in Escherichia coli

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    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.

  4. High Efficient Expression, Purification, and Functional Characterization of Native Human Epidermal Growth Factor in Escherichia coli.

    Science.gov (United States)

    Ma, Yi; Yu, Jieying; Lin, Jinglian; Wu, Shaomin; Li, Shan; Wang, Jufang

    2016-01-01

    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-H 10 . 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.

  5. Anticancer Activity of a Hexapeptide from Skate (Raja porosa Cartilage Protein Hydrolysate in HeLa Cells

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    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.

  6. 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.

  7. Interactions Between Fibroblast Cells and Laser Beam Welded AISI 2205 Duplex Stainless Steel

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    Ceyhun KÖSE

    2018-05-01

    Full Text Available Because of their high mechanical strength, excellent corrosion resistance and good weldability, duplex stainless steels are mostly used in industries such as oil, chemistry, petrochemistry, food and occasionally used in medical industry. These properties have enabled us to use duplex stainless steels in biomedical applications recently. Accordingly, duplex stainless steel material can be highly important to examine the toxic effect on the cells. In this study, the effect of the AISI 2205 duplex stainless steels which are joined by CO2 laser beam welding on viability of L929 fibroblast cells has been studied in vitro for the first time. For this aim, the cells were kept in DMEM/F-12 (Thermofisher Scientific 31331-028 medium for 7 days. The viability study was experimentally studied using the MTT (Thiazolyl Blue Tetrazolium Bromide method for 7 days. The cell viability of the laser beam welded sample has been detected to be higher than that of the base metal and the control based on 7th day data. According to the obtained results, it was revealed that laser beam welded and base metal AISI 2205 duplex stainless steel has been found suitable to study for biomedical applications. DOI: http://dx.doi.org/10.5755/j01.ms.24.2.18006

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

    International Nuclear Information System (INIS)

    Cheng Xianmiao; Li Yubao; Zuo Yi; Zhang Li; Li Jidong; Wang Huanan

    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 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

  9. Germination and seedling morphology of four South American Smilax (Smilacaceae

    Directory of Open Access Journals (Sweden)

    Aline Redondo Martins

    2012-03-01

    Full Text Available Species of Smilax, also known as greenbrier, are widely distributed in Brazil and their commercial trades are carried out by the extractivism of native species. We the aim to provide information about the germination and development of seedlings in four Smilax species, different experiments were developed under controlled conditions. We evaluated two germination treatments: temperature (30ºC and 20-30ºC and light (presence/ absence, and for few cases the tetrazolium treatment was applied. A different treatment response was observed among the studied species. Light had a significant influence in S. brasiliensis, with the highest germination rates at 20-30ºC in dark conditions. S. campestris showed significant differences among temperature treatments, but not to light; while S. cissoides showed high germination rates (66-78%, independently of treatment. However, S. polyantha had low germination rates (19-24%. After one year, the expanded leaves showed different characteristics among the studied species. Leaves of S. brasiliensis were ovate, coriaceous, three main veins and prickle-like structures only on the midrib on abaxial face. S. campestris leaves were oblong, coriaceous and prickle-like structures were located at the leaf midrib and margin. S. cissoides had ovate-elliptic, membranaceous leaves, with three main veins with prickle-like structures on the abaxial face. S. polyantha leaves showed ovateelliptic, coriaceous leaves, with three main veins, translucent secondary veins and no prickle-like structures. A seedling identification key was elaborated based on morphological characteristics.

  10. Germination and seedling morphology of four South American Smilax (Smilacaceae).

    Science.gov (United States)

    Martins, Aline Redondo; Soares, Anielca Nascimento; Bombo, Aline Bertolosi; Fidelis, Alessandra; Novembre, Ana Dionisia da Luz Coelho; da Glória, Beatriz Appezzato

    2012-03-01

    Species of Smilax, also known as greenbrier, are widely distributed in Brazil and their commercial trades are carried out by the extractivism of native species. We the aim to provide information about the germination and development of seedlings in four Smilax species, different experiments were developed under controlled conditions. We evaluated two germination treatments: temperature (30 degrees C and 20-30 degrees C) and light (presence/ absence), and for few cases the tetrazolium treatment was applied. A different treatment response was observed among the studied species. Light had a significant influence in S. brasiliensis, with the highest germination rates at 20-30 degrees C in dark conditions. S. campestris showed significant differences among temperature treatments, but not to light; while S. cissoides showed high germination rates (66-78%), independently of treatment. However, S. polyantha had low germination rates (19-24%). After one year, the expanded leaves showed different characteristics among the studied species. Leaves of S. brasiliensis were ovate, coriaceous, three main veins and prickle-like structures only on the midrib on abaxial face. S. campestris leaves were oblong, coriaceous and prickle-like structures were located at the leaf midrib and margin. S. cissoides had ovate-elliptic, membranaceous leaves, with three main veins with prickle-like structures on the abaxial face. S. polyantha leaves showed ovate-elliptic, coriaceous leaves, with three main veins, translucent secondary veins and no prickle-like structures. A seedling identification key was elaborated based on morphological characteristics.

  11. Involvement of TRPV1 and AQP2 in hypertonic stress by xylitol in odontoblast cells.

    Science.gov (United States)

    Tokuda, M; Fujisawa, M; Miyashita, K; Kawakami, Y; Morimoto-Yamashita, Y; Torii, M

    2015-02-01

    To examine the responses of mouse odontoblast-lineage cell line (OLC) cultures to xylitol-induced hypertonic stress. OLCs were treated with xylitol, sucrose, sorbitol, mannitol, arabinose and lyxose. Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium assay. The expression of transient receptor potential vanilloids (TRPV) 1, 3 and 4 was detected using a reverse transcriptase-polymerase chain reaction (RT-PCR) assay. The expression of aquaporin (AQP) 2 was detected using immunofluorescence and Western blotting analysis. The expression of interleukin-6 (IL-6) under xylitol-induced hypertonic stress was assessed using an enzyme-linked immunosorbent assay (ELISA). Small interfering ribonucleic acid (siRNA) for AQP-2 was used to inhibition assay. Xylitol-induced hypertonic stress did not decrease OLC viability, unlike the other sugars tested. OLCs expressed TRPV1, 3 and 4 as well as AQP2. Xylitol inhibited lipopolysaccharide (LPS)-induced IL-6 expression after 3 h of hypertonic stress. TRPV1 mRNA expression was upregulated by xylitol. Costimulation with HgCl2 (AQP inhibitor) and Ruthenium red (TRPV1 inhibitor) decreased cell viability with xylitol stimulation. OLCs treated with siRNA against TRPV1 exhibited decreased cell viability with xylitol stimulation. OLCs have high-cell viability under xylitol-induced hypertonic stress, which may be associated with TRPV1 and AQP2 expressions.

  12. Cytotoxicity and inhibitory properties against topoisomerase II of doxorubicin and its formamidine derivatives.

    Science.gov (United States)

    Kik, Krzysztof; Studzian, Kazimierz; Wasowska-Łukawska, Małgorzata; Oszczapowicz, Irena; Szmigiero, Leszek

    2009-01-01

    This work was undertaken to compare cytotoxicity, DNA damaging properties and effect on DNA cleavage by topoisomerase II of the anthracycline drug doxorubicin (DOX) and its two derivatives with a formamidino group containing a cyclic amine moiety such as morpholine (DOXM) or hexamethyleneimine (DOXH). The tetrazolium dye colorimetric assay was used to determine the cytotoxic activity of anthracyclines toward L1210 leukemia cells. DNA damage was measured by alkaline elution technique. The effect of anthracyclines on DNA cleavage was studied in a cell-free system containing supercoiled pBR322 DNA and purified human topoisomerase II. The cytotoxicity data and the results of studies on the mechanism of DNA break formation by anthracyclines at the cellular level and in the cell-free system showed that the presence of the formamidino group in the doxorubicin molecule reduced its ability to stimulate DNA cleavage by DNA topoisomerase II. DNA topoisomerase II is not a primary cellular target for DOXM or DOXH. An advantageous feature of formamidinoanthracyclines is their mechanism of cytotoxic action which is not related to the inhibition of DNA topoisomerase II. Therefore this class of anthracyclines seems to be a good source for selection of an anticancer drug directed toward cancer cells with the developed multidrug resistance attributed to the presence of altered DNA topoisomerase II.

  13. Estimates of genetics and phenotypics parameters for the yield and quality of soybean seeds.

    Science.gov (United States)

    Zambiazzi, E V; Bruzi, A T; Guilherme, S R; Pereira, D R; Lima, J G; Zuffo, A M; Ribeiro, F O; Mendes, A E S; Godinho, S H M; Carvalho, M L M

    2017-09-27

    Estimating genotype x environment (GxE) parameters for quality and yield in soybean seed grown in different environments in Minas Gerais State was the goal of this study, as well as to evaluate interaction effects of GxE for soybean seeds yield and quality. Seeds were produced in three locations in Minas Gerais State (Lavras, Inconfidentes, and Patos de Minas) in 2013/14 and 2014/15 seasons. Field experiments were conducted in randomized blocks in a factorial 17 x 6 (GxE), and three replications. Seed yield and quality were evaluated for germination in substrates paper and sand, seedling emergence, speed emergency index, mechanical damage by sodium hypochlorite, electrical conductivity, speed aging, vigor and viability of seeds by tetrazolium test in laboratory using completely randomized design. Quadratic component genotypic, GXE variance component, genotype determination coefficient, genetic variation coefficient and environmental variation coefficient were estimated using the Genes software. Percentage analysis of genotypes contribution, environments and genotype x environment interaction were conducted by sites combination two by two and three sites combination, using the R software. Considering genotypes selection of broad adaptation, TMG 1179 RR, CD 2737 RR, and CD 237 RR associated better yield performance at high physical and physiological potential of seed. Environmental effect was more expressive for most of the characters related to soybean seed quality. GxE interaction effects were expressive though genotypes did not present coincidental behavior in different environments.

  14. Bacterial endophytes isolated from plants in natural oil seep soils with chronic hydrocarbon contamination

    Directory of Open Access Journals (Sweden)

    Rhea eLumactud

    2016-05-01

    Full Text Available The bacterial endophytic communities of four plants growing abundantly in soils highly contaminated by hydrocarbons were analyzed through culturable and and culture-independent means. Given their tolerance to the high levels of petroleum contamination at our study site, we sought evidence that Achillea millefolium, Solidago canadensis, Trifolium aureum and Dactylis glomerata support high levels of hydrocarbon degrading endophytes. A total of 190 isolates were isolated from four plant species. The isolates were identified by partial 16S rDNA sequence analysis, with class Actinobacteria as the dominant group in all species except Solidago canadensis, which was dominated by Gammaproteobacteria. Microbacterium foliorum and Plantibacter flavus were present in all the plants, with M. foliorum showing predominance in D. glomerata and both endophytic bacterial species dominated T. aureum. More than 50% of the isolates demonstrated degradative capabilities for octanol, toluene, naphthalene, kerosene or motor oil based on sole carbon source growth screens involving the reduction of tetrazolium dye. P. flavus isolates from all the sampled plants showed growth on all the petroleum hydrocarbons substrates tested. Mineralization of toluene and naphthalene was confirmed using gas-chromatography. 16S based terminal restriction fragment length polymorphism analysis revealed significant differences between the endophytic bacterial communities showing them to be plant host specific at this site. To our knowledge, this is the first account of the degradation potential of bacterial endophytes in these commonly occurring pioneer plants that were not previously known as phytoremediating plants.

  15. Multidetector computed tomography (MDCT) evaluation of myocardial viability: intraindividual comparison of monomeric vs. dimeric contrast media in a rabbit model

    Energy Technology Data Exchange (ETDEWEB)

    Mahnken, Andreas H. [RWTH Aachen University, Department of Diagnostic Radiology, Aachen (Germany)]|[RWTH Aachen University, Applied Medical Engineering, Helmholtz Institute, Aachen (Germany)]|[University Hospital, RWTH Aachen University, Department of Diagnostic Radiology, Aachen (Germany); Jost, Gregor; Sieber, Martin; Seidensticker, Peter R.; Pietsch, Hubertus [Bayer-Schering Pharma AG, Berlin (Germany); Bruners, Philipp [RWTH Aachen University, Department of Diagnostic Radiology, Aachen (Germany)]|[RWTH Aachen University, Applied Medical Engineering, Helmholtz Institute, Aachen (Germany); Guenther, Rolf W. [RWTH Aachen University, Department of Diagnostic Radiology, Aachen (Germany)

    2009-02-15

    To evaluate the influence of different types of iodinated contrast media on the assessment of myocardial viability, acute myocardial infarction (MI) was surgically induced in six rabbits. Over a period of 45 min, contrast-enhanced cardiac MDCT (64 x 0.6 mm, 80 kV, 680mAs{sub eff.}) was repeatedly performed using a contrast medium dose of 600 mg iodine/kg body weight. Animals received randomized iopromide 300 and iodixanol 320, respectively. Attenuation values of healthy and infarcted myocardium were measured. The size of MI was computed and compared with nitroblue tetrazolium (NBT)-stained specimen. The highest attenuation differences between infarcted and healthy myocardium occurred during the arterial phase with 140.0{+-}3.5 HU and 141.0{+-}2.2 HU for iopromide and iodixanol, respectively. For iodixanol the highest attenuation difference on delayed contrast-enhanced images was achieved 3 min post injection (73.5 HU). A slightly higher attenuation difference was observed for iopromide 6 min after contrast medium injection (82.2 HU), although not statistically significant (p=0.6437). Mean infarct volume as measured by NBT staining was 33.5%{+-}13.6%. There was an excellent agreement of infarct sizes among NBT-, iopromide- and iodixanol-enhanced MDCT with concordance-correlation coefficients ranging from {rho}{sub (c)=} 0.9928-0.9982. Iopromide and iodixanol both allow a reliable assessment of MI with delayed contrast-enhanced MDCT. (orig.)

  16. Cytotoxicity of titanium dioxide nanoparticles in mouse fibroblast cells.

    Science.gov (United States)

    Jin, Cheng-Yu; Zhu, Bang-Shang; Wang, Xue-Feng; Lu, Qing-Hua

    2008-09-01

    Nanotitanium dioxide (TiO2) is an important industrial material that is widely used as an additive in cosmetics, pharmaceuticals, and food colorants. Although the small size of the TiO2 nanoparticle is useful in various applications, the biosafety of this material needs to be evaluated. In this study, mouse fibroblast (L929) cells were used to evaluate the cytotoxicity of different concentrations (3-600 microg/mL) of homogeneous and weakly aggregated TiO2 nanoparticles in aqueous solution. The L929 cells became round and even shrank as the concentration of TiO2 nanoparticles increased. Moreover, TiO2 nanoparticle-treated cells had condensed fragmented chromatin or were directly necrosed, as observed by acridine orange (AO) staining. The transmission electron microscopy (TEM) analysis showed that in cells cultured in a medium containing 300 microg/mL TiO2, the number of lysosomes increased, and some cytoplasmic organelles were damaged. In addition, there was a significant increase in oxidative stress at higher TiO2 nanoparticle concentrations (>60 microg/mL). As the concentration of TiO2 nanoparticles increased in the culture medium, the levels of reactive oxygen species (ROS) and lactate dehydrogenase (LDH) increased, while those of methyl tetrazolium cytotoxicity (MTT), glutathione (GSH), and superoxide dismutase (SOD) decreased. A possible mechanism for the cytotoxicity of TiO2 nanoparticles is also discussed.

  17. Magnetic resonance imaging of acute myocardial infarction in pigs using Gd-DTPA

    International Nuclear Information System (INIS)

    Wikstroem, M.; Martinussen, H.J.; Wikstroem, G.; Ericsson, A.; Nyman, R.; Waldenstroem, A.; Hemmingsson, A.; Akademiska Sjukhuset, Uppsala; Akademiska Sjukhuset, Uppsala

    1990-01-01

    Six pigs with coronary artery occlusion were investigated with MR imaging before and subsequently for about 2.5 hours at repeated intervals after the intravenous administration of Gd-DTPA (0.4 mmol/kg). The animals were sacrificed after a total occlusion time of 6 hours and the hearts were excised. The excised hearts were then reexamined in the MR equipment and stained with TTC (triphenyl tetrazolium) in order to define areas of infarction. Four control hearts with 6-hour-old infarctions were only imaged ex vivo without any previous administration of contrast media. In vivo, there was no clear demarcation of infarction with or without Gd-DTPA. Ex vivo, without any contrast media, the infarctions were poorly discriminated with a discretely increased signal intensity relative to normal myocardium in the T2 weighted images. Gd-DTPA was found to accumulate in the infarctions, which caused an elevated signal intensity most pronounced in the T1 weighted images. This considerably improved the delineation of the infarcted area. (orig.)

  18. Galactosylated nanostructured lipid carriers for delivery of 5-FU to hepatocellular carcinoma.

    Science.gov (United States)

    Varshosaz, Jaleh; Hassanzadeh, Farshid; Sadeghi, Hojjat; Khadem, Mostafa

    2012-09-01

    The aim of the present study was to design a targeted delivery system of 5-fluorouracil (5-FU) for hepatocellular carcinoma (HCC). Lactobionic acid (LB) was conjugated to stearyl amine (SA) by a chemical reaction. The nanostructured lipid carriers (NLCs), containing LB conjugate, lecithin, glyceryl monostearate, oil [oleic acid (OA) or Labrafac 5 or 10%], and 5-FU, were dissolved in alcohol/acetone, the oil phase was added to the aqueous phase containing Tween 80 or Solutol(®) HS15 (0.25 or 0.5%), and NLCs were prepared by an emulsification-solvent diffusion method. Physical properties and drug release were studied in NLCs. The thiazolyl blue tetrazolium bromide assay was used to study the cytotoxicity of NLCs on HepG(2) cells, and the cellular uptake of NLCs was determined by flow cytometry. Fourier transform infrared spectroscopy and (1)H-NMR spectra confirmed the successful conjugation of LB and SA. The optimized NLCs consisted of 0.5% Solutol HS15 and 10% OA oil. The particle size of these nanoparticles was 139.2 nm, with a zeta potential of -18 mV, loading efficiency of 34.2%, release efficiency after 2 hours of the release test was 72.6%, and crystallinity was 0.63%. The galactosylated NLCs of 5-FU were cytotoxic on the HepG(2) cell line in a half concentration of 5-FU and seems promising in reducing 5-FU dose in HCC.

  19. 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.

  20. Use of a stainless steel washer platform to study Acinetobacter baumannii adhesion and biofilm formation on abiotic surfaces.

    Science.gov (United States)

    Orsinger-Jacobsen, Samantha J; Patel, Shenan S; Vellozzi, Ernestine M; Gialanella, Phillip; Nimrichter, Leonardo; Miranda, Kildare; Martinez, Luis R

    2013-12-01

    Acinetobacter baumannii is a frequent cause of hospital-acquired pneumonia, and has recently increased in incidence as the causative agent of severe disease in troops wounded in Afghanistan and Iraq. Clinical approaches are limited since A. baumannii strains isolated from patients are extremely resistant to current antimicrobials. A. baumannii can survive desiccation and during outbreaks has been recovered from various sites in the patients' environment. To better understand its prevalence in hospital settings, we used a stainless steel washer (SSW) platform to investigate A. baumannii biofilm formation on abiotic surfaces. Scanning electron microscopy demonstrated that A. baumannii forms strong biofilms on stainless steel surfaces. This platform was combined with a colorimetric 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay to observe the metabolic activity of bacterial cells, and to facilitate the manipulation and comparison of multiple A. baumannii clinical strains. A strong correlation between XTT and c.f.u. assays was demonstrated. To complement the cell viability assays, A. baumannii biofilm mass was measured by crystal violet staining. Furthermore, the effect of commonly used disinfectants and environmental stressors on A. baumannii biofilms and planktonic cells was compared and characterized. Biofilms on SSWs were significantly more resistant than their planktonic counterparts, providing additional evidence that may allow us to understand the high prevalence of this microbe in hospital settings. Our results validate that SSWs are a simple, versatile and innovative method to study A. baumannii biofilms in vitro.

  1. Synthesis, Characterization, and In Vitro Drug Delivery Capabilities of (Zn, Al-Based Layered Double Hydroxide Nanoparticles

    Directory of Open Access Journals (Sweden)

    Vinay J. Nagaraj

    2015-01-01

    Full Text Available There is an urgent need for the development of alternative strategies for effective drug delivery to improve the outcome of patients suffering from deadly diseases such as cancer. Nanoparticles, in particular layered double hydroxide (LDH nanoparticles, have great potential as nanocarriers of chemotherapeutic molecules. In this study, we synthesized (Zn, Al-LDH nanoparticles and report their enhanced pH-dependent stability in comparison to the commonly used (Mg, Al-LDH nanoparticles. Fluorescein isothiocyanate (FITC and valproate (VP were intercalated into (Zn, Al-LDH nanoparticles to study cellular uptake, biocompatibility, and drug delivery capabilities using cultured pancreatic adenocarcinoma BxPC3 cells. Fluorescence measurements indicated that FITC-intercalated LDH nanoparticles showed a greater degree of energy-dependent uptake rather than passive uptake by BxPC3 cells, especially at high concentrations of nanoparticles. Tetrazolium-based colorimetric assays indicated that BxPC3 cells treated with VP-intercalated LDH nanoparticles showed a significant reduction in cell viability along with about 30-fold reduction in IC50 compared to the drug alone. In contrast, the non-drug-intercalated LDH nanoparticles did not affect the cell viability indicating very low innate cytotoxicity. Our research indicates that the superior properties of (Zn, Al-LDH nanoparticles make them ideal candidates for further development as in vivo chemotherapy drug delivery agents.

  2. Factors influencing [F-18]2-fluoro-2-deoxy-D-glucose (F-18 FDG) uptake in melanoma cells. The role of proliferation rate, viability, glucose transporter expression and hexokinase activity

    International Nuclear Information System (INIS)

    Yamada, Kiyoshi; Brink, I.; Bisse, E.; Epting, T.; Engelhardt, R.

    2005-01-01

    Using human (SK-MEL 23, SK-MEL 24 and G361) and murine (B16) melanoma cell lines, the coregulatory potential of the uptake of the positron emission tomography (PET) tracer, [Fluorine-18]2-fluoro-2-deoxy-D-glucose (F-18 FDG) has been investigated in relationship to tumor characteristics. Comparative studies among the four melanoma cell lines demonstrated that the lowest FDG uptake in SK-MEL 24 corresponded strongly to the data for DT (population doubling time) and MTT (tetrazolium salt) cell viability as well as hexokinase (HK) activity, but was not related to the glucose transporter 1 (GLUT 1) expression level. Furthermore, the FDG uptake in each melanoma cell line measured by cell cycle kinetics was significantly positively correlated to both the proliferation index (PI=S/G 2 M phase fractions) and the cell viability, though with one exception relating to the proliferation index (PI) of the lowest FDG uptake cell line, SK-MEL 24. No positive correlation was found between the expression of GLUT 1 and FDG uptake in any individual cell line. However, the HK activities in SK-MEL 23 and 24 showed considerable positive relationships with FDG uptake. Our present study suggests that both the proliferation rate and the cell viability of melanoma cells may be key factors for FDG uptake and that HK activity, rather than GLUT 1 expression, seems to be a major factor. (author)

  3. Estrogenicity of glabridin in Ishikawa cells.

    Directory of Open Access Journals (Sweden)

    Melissa Su Wei Poh

    Full Text Available Glabridin is an isoflavan from licorice root, which is a common component of herbal remedies used for treatment of menopausal symptoms. Past studies have shown that glabridin resulted in favorable outcome similar to 17β-estradiol (17β-E2, suggesting a possible role as an estrogen replacement therapy (ERT. This study aims to evaluate the estrogenic effect of glabridin in an in-vitro endometrial cell line -Ishikawa cells via alkaline phosphatase (ALP assay and ER-α-SRC-1-co-activator assay. Its effect on cell proliferation was also evaluated using Thiazoyl blue tetrazolium bromide (MTT assay. The results showed that glabridin activated the ER-α-SRC-1-co-activator complex and displayed a dose-dependent increase in estrogenic activity supporting its use as an ERT. However, glabridin also induced an increase in cell proliferation. When glabridin was treated together with 17β-E2, synergistic estrogenic effect was observed with a slight decrease in cell proliferation as compared to treatment by 17β-E2 alone. This suggest that the combination might be better suited for providing high estrogenic effects with lower incidences of endometrial cancer that is associated with 17β-E2.

  4. Effects of Ginkgo biloba extract on the apoptosis of oxygen and glucose-deprived SH-SY5Y cells and its mechanism.

    Science.gov (United States)

    Ba, Xiao-Hong; Min, Lian-Qiu

    2015-01-01

    The aim was to observe the effects of the extract of Ginkgo biloba (EGb761) on the apoptosis of oxygen and glucose-deprived (OGD) human neuroblastoma cells (SH-SY5Y) cells and explore its mechanism. SH-SY5Y cells were divided into normal control group, OGD group, OGD for 4 h and EGb761-pretreated groups including very low-concentration (20 μg/ml), low-concentration group (25 μg/ml), moderate-concentration group (50 μg/ml) and high-concentration group (100 μg/ml). Twenty four hours after reoxygenation, cell viability was determined with 3-[4, 5-dimehyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide assay, apoptosis rate was detected with annexin V-fluorescein isothiocyanate/propidium iodide double staining flow cytometry and the protein level of apoptosis-inducing factor (AIF) was observed with immunofluorescence technique in each group. Cell viability was significantly lower in OGD group than in EGb761-pretreated groups, especially in moderate-concentration group (50 μg/ml) (P cells probably through inhibiting AIF nuclear translocation. This study provides a theoretical basis for the application of EGb761 in clinical practice.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. UV irradiation of the blood in the therapy of torpid forms of gonorrheal uretritis

    International Nuclear Information System (INIS)

    Vasil'ev, M.M.; Zorin, S.P.

    1987-01-01

    Clinical efficiency of the IR-irradiation proper blood reinfusion (IRIPBR) method in therapy of torpid forms of gonorrheal uretritis, dynamics of such factors of the oxygen-dependent bactericidal system of polymorphonuclear neutrophils leukocytes (PMNL) as myeloperoxidases (MPO) and test of nitroblue tetrazolium (NBT-test) reduction as well as general quantity of PMNL in the course of immunotherapy by gonovaccine and pyrogenal and by the IRIPBR method have been studied in two groups of patients with gonorrheal uretritis (54 men). The patients of the first group (32) received peniciline, pyrogenal and gonovaccine injections, as well as local treatment according to the instruction, the patients of the second group (22) were prescribed instead of pyrogenal injections 3 procedures of IRIPBR on 2 ml autoblood basis per 1 kg of body mass (in average 130-150 ml) with further treatment by antibiotics. LK-5E has been used to carry out IRIPBR for blood UV irradiation. Blood has been irradiated by MD-73 M ''Izol'lda'' device. It is shown that IRIPBR causes statistically reliable increase of the number of circulating blood leucocytes including PMNL with simultaneous increase of MPO PMNL activity and the capability to generate pro-stimulations of O 2 - determined by the NBT-test

  10. Cytotoxic and genotoxic studies of essential oil from Rosa damascene Mill., Kashan, Iran.

    Science.gov (United States)

    Shokrzadeh, Mohammad; Habibi, Emran; Modanloo, Mona

    2017-08-01

    Aim Rosa damascene Mill. belongs to the family of Roseaceae and its essential oil is produced in large amounts in Iran. The wide application of rose oil has raised questions about potential adverse health effects. We have investigated cytotoxic activity and genotoxic effects of Rosa oil from Kashan, Iran. Methods The cytotoxic effect and IC50 of the essential oil on the cell lines was studied followed by MTT assay. In this assay mitochondrial oxidoreductase enzymes with reducing the tetrazolium dye MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) reflect the number of viable cells. Genotoxic effect of the oil was evaluated by micronucleus assay by evaluating produced micronuclei due to cytogenetic damage in binucleated lymphocytes. Results The results showed that essential oil significantly had cytotoxic and genotoxic effects at doses over 10µg/mL (pessential oil of Rose showed lower IC50 in cancer cell line (A549) in comparison with the normal cell line (NIH3T3). Conclusion Cytotoxic and genotoxic properties of essential oil of Rose in Kashan, Iran, are safe at a dose of 10µg/mL. Also, a good cytotoxic effect was shown and could be introduced as an anticancer compound. Further studies are needed with regard to anti-cancer effects of Rose essential oil. Copyright© by the Medical Assotiation of Zenica-Doboj Canton.

  11. In vitro effects of three blood derivatives on human corneal epithelial cells.

    Science.gov (United States)

    Freire, Vanesa; Andollo, Noelia; Etxebarria, Jaime; Durán, Juan A; Morales, María-Celia

    2012-08-15

    We compared the effects of three blood derivatives, autologous serum (AS), platelet-rich plasma (PRP), and serum derived from plasma rich in growth factors (PRGF), on a human corneal epithelial (HCE) cell line to evaluate their potential as an effective treatment for corneal epithelial disorders. The concentrations of epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), and fibronectin were quantified by ELISA. The proliferation and viability of HCE cells were measured by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay. Cell morphology was assessed by phase-contrast microscopy. The patterns of expression of several connexin, involucrin, and integrin α6 genes were analyzed by real-time RT-PCR. We found significantly higher levels of EGF in PRGF compared to AS and PRP. However, AS and PRGF induced robust proliferation of HCE cells. In addition, PRGF cultured cells grew as heterogeneous colonies, exhibiting differentiated and non-differentiated cell phenotypes, whereas AS- and PRP-treated cultures exhibited quite homogeneous colonies. Finally, PRGF upregulated the expression of several genes associated with communication and cell differentiation, in comparison to AS or PRP. PRGF promotes biological processes required for corneal epithelialization, such as proliferation and differentiation. Since PRGF effects are similar to those associated with routinely used blood derivatives, the present findings warrant further research on PRGF as a novel alternative treatment for ocular surface diseases.

  12. Detection of alkaline phosphatase in canine cells previously stained with Wright-Giemsa and its utility in differentiating osteosarcoma from other mesenchymal tumors.

    Science.gov (United States)

    Ryseff, Julia K; Bohn, Andrea A

    2012-09-01

    Osteosarcoma (OSA) is a common primary bone tumor in dogs. Demonstration of alkaline phosphatase (ALP) reactivity by tumor cells on unstained slides is useful in differentiating osteosarcoma from other types of sarcoma. However, unstained slides are not always available. The objectives of this study were to evaluate the diagnostic utility of detecting ALP expression in differentiating osteosarcoma from other sarcomas in dogs using cytologic material previously stained with Wright-Giemsa stain and to assess the sensitivity and specificity of ALP expression for diagnosing osteosarcoma using a specific protocol. Archived aspirates of histologically confirmed sarcomas in dogs that had been previously stained with Wright-Giemsa stain were treated with 5-bromo, 4-chloro, 3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) as a substrate for ALP. Cells were evaluated for expression of ALP after incubation with BCIP/NBT for 1 hour. Sensitivity and specificity of ALP expression for diagnosis of OSA were calculated. In samples from 83 dogs, cells from 15/17 OSAs and from 4/66 tumors other than OSA (amelanotic melanoma, gastrointestinal stromal tumor, collision tumor, and anaplastic sarcoma) expressed ALP. Sensitivity and specificity of ALP expression detected using BCIP/NBT substrate applied to cells previously stained with Wright-Giemsa stain for OSA were 88 and 94%, respectively. ALP expression detected using BCIP/NBT substrate applied to previously stained cells is useful in differentiating canine OSA from other mesenchymal neoplasms. © 2012 American Society for Veterinary Clinical Pathology.

  13. In Vitro/In Vivo Evaluation of Dexamethasone--PAMAM Dendrimer Complexes for Retinal Drug Delivery.

    Science.gov (United States)

    Yavuz, Burçin; Pehlivan, Sibel Bozdağ; Vural, İmran; Ünlü, Nurşen

    2015-11-01

    Current treatment options for diabetic retinopathy (DR) have side effects because of invasive application and topical application does not generally result in therapeutic levels in the target tissue. Therefore, improving the drug delivery to retina, following topical administration, might be a solution to DR treatment problems. The purpose of this study was to investigate the complexation effects of poly(amidoamine) (PAMAM) dendrimers on ocular absorption of dexamethasone (DEX). Using different PAMAM generations, complex formulations were prepared and characterized. Formulations were evaluated in terms of cytotoxicity and cell permeability, as well as ex vivo transport across ocular tissues. The ocular pharmacokinetic properties of DEX formulations were studied in Sprague-Dawley rats following topical and subconjunctival applications, to evaluate the effect of PAMAM on retinal delivery of DEX. Methyl-thiazol-tetrazolium (MTT) assay indicated that all groups resulted in cell viability comparable to DEX solution (87.5%), with the cell viability being the lowest for G3 complex at 73.5%. Transport study results showed that dendrimer complexation increases DEX transport across both cornea and sclera tissues. The results of in vivo studies were also indicated that especially anionic DEX-PAMAM complex formulations have reached higher DEX concentrations in ocular tissues compared with plain DEX suspension. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

  14. Mechanism of gene transfection by polyamidoamine (PAMAM) dendrimers modified with ornithine residues.

    Science.gov (United States)

    Kumar, Ajay; Yellepeddi, Venkata K; Vangara, Kiran K; Strychar, Kevin B; Palakurthi, Srinath

    2011-11-01

    The aim of this study was to prepare and investigate the mechanism of uptake of the dendriplexes prepared with ornithine-conjugated polyamidoamine (PAMAM) G4 dendrimers. Ornithine-conjugated PAMAMG4 dendrimers were prepared by Fmoc synthesis. A comparative transfection study in NCI H157G cells and polyamine transport-deficient cell line NCI H157R was performed to confirm the role of the polyamine transporter system (PAT) in the dendriplex uptake. Transfection efficiency significantly increased with increase in generation number and extent of ornithine conjugation. Transfection efficiency of the PAMAMG4-ORN60 dendrimers significantly decreased in presence of excess of ornithine (P dendrimers. Transfection efficiency of PAMAMG4-ORN60 was significantly low in NCI H157R (31.66 ± 3.95%, RFU: 17.87 ± 1.34) as compared to NCI H157G cell line (63.07 ± 6.8%, relative fluorescence units (RFU): 23.28 ± 0.66). Results indicate the role of PAT in addition to charge-mediated endocytosis in the internalization of ornithine-conjugated PAMAMG4 dendrimers. Cytotoxicity analysis (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay) in human embryonic kidney cell line (HEK) 293T cells showed that the dendriplexes were non-toxic at N/P 10.

  15. Antiproliferative Activities of Bouea Macrophylla Seed Extracts

    International Nuclear Information System (INIS)

    Arapoc, D.J.; Mohamed Zaffar Ali Mohamed Amiroudine; Zainah Adam; Rosniza Razali; Shafii Khamis

    2016-01-01

    Bouea macrophylla or commonly known as kundang fruit in Malaysia is a tropical fruit tree native to Southeast Asia. This plant belongs to the family Anacardiaceae which are cultivated for their edible fruits, seeds and medicinal compounds. The present study was conducted to evaluate the anti proliferation activities of aqueous, methanolic, chloroform and hexane extracts from the seed of B. macrophylla. The extracts were screened on human squamous cell carcinoma (HTB-43), breast cancer (MCF7) and (MDA-MB-231) cell lines by using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Most effective concentration were screened for apoptosis induction in cells using Hoechst stain. Our present study has shown that aqueous, methanolic, chloroform dan hexane extracts exhibited promising inhibition activity against HTB43 cell lines with the IC50 values were 29.32±5.80, 18.65±2.94, 21.14±6.97 and 34.36±16.50 μg/ mL, respectively. Meanwhile, only hexane extract showed inhibition against MCF7 (59.07±5.76) and MDA-MB-231(123.35±28.65). Besides that, the results also indicate that promising anticancer activity and causes loss in cancer cell viability by activating the apoptotic process. These findings suggest that B. macrophylla may have novel therapeutic applications for the treatment of different cancer types. (author)

  16. The identification and characterization of osmotolerant yeast isolates from chemical wastewater evaporation ponds.

    Science.gov (United States)

    Lahav, R; Fareleira, P; Nejidat, A; Abeliovich, A

    2002-04-01

    Ramat Hovav is a major chemical industrial park manufacturing pharmaceuticals, pesticides, and various aliphatic and aromatic halogens. All wastewater streams are collected in large evaporation ponds. Salinity in the evaporation ponds fluctuates between 3% (w/v) and saturation and pH values range between 2.0 and 10.0. We looked for microorganisms surviving in these extreme environmental conditions and found that 2 yeast strains dominate this biotope. 18S rDNA sequence analysis identified the isolates as Pichia guilliermondii and Rhodotorula mucilaginosa. Both isolates grew in NaCl concentrations ranging up to 3.5 M and 2.5 M, respectively, and at a pH range of 2-10. There was a distinct difference between the Rhodotorula and Pichia strains and S. cerevisiae RS16 that served as a control strain with respect to accumulation of osmoregulators and internal ion concentrations when exposed to osmotic stress. The Pichia and Rhodotorula strains maintained high glycerol concentration also in media low in NaCl. Utilization of various carbon sources was examined. Using a tetrazolium-based assay we show that the Rhodotorula and Pichia strains are capable of utilizing a wide range of different carbon sources including anthracene, phenanthrene, and other cyclic aromatic hydrocarbons.

  17. Removal of Zn(II) from electroplating effluent using yeast biofilm formed on gravels: batch and column studies

    Science.gov (United States)

    2014-01-01

    Background Present study deals with the removal of Zn(II) ions from effluent using yeast biofilm formed on gravels. Methods The biofilm forming ability of Candida rugosa and Cryptococcus laurentii was evaluated using XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) reduction assay and monitored by scanning electron microscopy (SEM), and Confocal laser scanning microscopy (CLSM). Copious amount of extracellular polymeric substances (EPS) produced by yeast species was quantified and characterized by Fourier transform infrared spectroscopy (FT-IR). Results Yeast biofilm formed on gravels by C. rugosa and C. laurentii showed 88% and 74.2% removal of Zn(II) ions respectively in batch mode. In column mode, removal of Zn(II) ions from real effluent was found to be 95.29% by C. rugosa biofilm formed on gravels. Conclusion The results of the present study showed that there is a scope to develop a cost effective method for the efficient removal of Zn(II) from effluent using gravels coated with yeast biofilm. PMID:24397917

  18. Induction of Mitochondrial Dependent Apoptosis in Human Leukemia K562 Cells by Meconopsis integrifolia: A Species from Traditional Tibetan Medicine

    Directory of Open Access Journals (Sweden)

    Jianping Fan

    2015-06-01

    Full Text Available Objectives: Meconopsis integrifolia (M. integrifolia is one of the most popular members in Traditional Tibetan Medicine. This study aimed to investigate the anticancer effect of M. integrifolia and to detect the underlying mechanisms of these effects. Methods: 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT assay and trypan blue assay were used to evaluate the cytotoxicity of M. integrifolia. Changes in cell nuclear morphology and reactive oxygen species (ROS level were observed by fluorescent microscopy. Apoptosis ratio, DNA damage and mitochondrial membrane potential (MMP loss were analyzed by flow cytometry. Western blotting assay was adopted to detect the proteins related to apoptosis. Immunofluorescence was used to observe the release of cytochrome C. Results: The obtained data revealed that M. integrifolia could significantly inhibit K562 cell viability, mainly by targeting apoptosis induction and cell cycle arrest in G2/M phase. Collapse in cell morphology, chromatin condensation, DNA damage and ROS accumulation were observed. Further mechanism detection revealed that mitochondrion might be a key factor in M. integrifolia-induced apoptosis. Conclusions: M. integrifolia could induce mitochondria mediated apoptosis and cell cycle arrest in G2/M phase with little damage to normal cells, suggesting that M. integrifolia might be a potential and efficient anticancer agent that deserves further investigation.

  19. A novel staining protocol for multiparameter assessment of cell heterogeneity in Phormidium populations (cyanobacteria employing fluorescent dyes.

    Directory of Open Access Journals (Sweden)

    Daria Tashyreva

    Full Text Available Bacterial populations display high heterogeneity in viability and physiological activity at the single-cell level, especially under stressful conditions. We demonstrate a novel staining protocol for multiparameter assessment of individual cells in physiologically heterogeneous populations of cyanobacteria. The protocol employs fluorescent probes, i.e., redox dye 5-cyano-2,3-ditolyl tetrazolium chloride, 'dead cell' nucleic acid stain SYTOX Green, and DNA-specific fluorochrome 4',6-diamidino-2-phenylindole, combined with microscopy image analysis. Our method allows simultaneous estimates of cellular respiration activity, membrane and nucleoid integrity, and allows the detection of photosynthetic pigments fluorescence along with morphological observations. The staining protocol has been adjusted for, both, laboratory and natural populations of the genus Phormidium (Oscillatoriales, and tested on 4 field-collected samples and 12 laboratory strains of cyanobacteria. Based on the mentioned cellular functions we suggest classification of cells in cyanobacterial populations into four categories: (i active and intact; (ii injured but active; (iii metabolically inactive but intact; (iv inactive and injured, or dead.

  20. Electrical stimulation with periodic alternating intervals stimulates neuronal cells to produce neurotrophins and cytokines through activation of mitogen-activated protein kinase pathways.

    Science.gov (United States)

    Yamamoto, Kenta; Yamamoto, Toshiro; Honjo, Kenichi; Ichioka, Hiroaki; Oseko, Fumishige; Kishida, Tsunao; Mazda, Osam; Kanamura, Narisato

    2015-12-01

    Peripheral neuropathy is a representative complication of dental surgery. Electrical therapy, based on electrical stimulation with periodic alternating intervals (ES-PAI), may promote nerve regeneration after peripheral nerve injury in a non-invasive manner, potentially providing an effective therapy for neuropathy. This study aimed to analyze the molecular mechanisms underlying the nerve recovery stimulated by ES-PAI. In brief, ES-PAI was applied to a neuronal cell line, Neuro2A, at various intensities using the pulse generator apparatus, FREUDE. Cell viability, neurotrophin mRNA expression, and cytokine production were examined using a tetrazolium-based assay, real-time RT-PCR, and ELISA, respectively. Mitogen-activated protein kinase (MAPK) signaling was assessed using flow cytometry. It was found that ES-PAI increased the viability of cells and elevated expression of nerve growth factor (NGF) and neurotrophin-3 (NT-3); ESPAI also augmented vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) expression, which was restored by addition of p38 inhibitors. Phosphorylation of p38 and extracellular signal-regulated kinase 1/2 (ERK-1/2) was augmented by ES-PAI. Hence, ES-PAI may ameliorate peripheral neuropathy by promoting neuronal cell proliferation and production of neurogenic factors by activating p38 and ERK-1/2 pathways. © 2015 Eur J Oral Sci.

  1. Antiproliferative terpenoids from almond hulls (Prunus dulcis): identification and structure-activity relationships.

    Science.gov (United States)

    Amico, Vincenzo; Barresi, Vincenza; Condorelli, Daniele; Spatafora, Carmela; Tringali, Corrado

    2006-02-08

    Bioassay-guided fractionation of the EtOAc crude extract from Sicilian almond hulls, a waste material from Prunus dulcis crop, allowed identification of 10 constituents, isolated as pure compounds (1-5, 7, and 10) or unseparable mixtures (5 + 6 and 8 + 9). All compounds were subjected to spectroscopic analysis and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide bioassay on MCF-7 human breast cancer cells. In addition to the main components oleanolic (1), ursolic (2), and betulinic (3) acids, the 2-hydroxy analogues alphitolic (4), corosolic (5), and maslinic (6) acids, as well as the related aldehydes, namely, betulinic (7), oleanolic (8), and ursolic (9), were identified. From a more polar fraction, the beta-sitosterol 3-O-glucoside (10) was also identified. A sample of commercially available betulin (11) was also included in bioassays as further support to a structure-activity relationship study. Betulinic acid showed antiproliferative activity toward MCF-7 cells (GI50 = 0.27 microM), higher than the anticancer drug 5-fluorouracil.

  2. In vitro evaluation of the genotoxicity of a family of novel MeO-PEG-poly(D,L-lactic-co-glycolic acid)-PEG-OMe triblock copolymer and PLGA nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    He Lili; Yang Likai; Zhang Zhirong; Gong Tao; Deng Li; Sun Xun [Key Laboratory of Drug Targeting and Novel Drug Delivery Systems, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu, Sichuan 610041 (China); Gu Zhongwei, E-mail: xunsun22@gmail.co [National Engineering Research Center for Biomaterials, Engineering Research Center of Biomaterials, Sichuan University, Chengdu 610064 (China)

    2009-11-11

    Despite the booming development of nanoparticle materials for pharmaceutical applications, studies on their genotoxicity are few. In our previous efforts to develop an intravenous nanoparticle material, a family of novel monomethoxy(polyethylene glycol)-poly(D,L-lactic-co-glycolic acid)-monomethoxy (PELGE) polymers was synthesized. The cytotoxicity and genotoxicity of nine kinds of selected blank PELGE and PLGA (poly(D,L-lactic and glycolic acid)) nanoparticles were evaluated using methyl thiazolyl tetrazolium (MTT), micronucleus (MN) and sister chromatid exchange (SCE) assays with or without the addition of a metabolic activation system (S9 mix), using Chinese hamster ovary (CHO) cells. The cytotoxicity of nanoparticles exhibited a dose-dependent response, with a concentration of 5 mg ml{sup -1} being the turning point. The frequencies of MN observed in samples treated with various nanoparticles were not statistically different from those seen in the negative controls in the presence or absence of the S9 mix. Also, no cell cycle delay was observed. The numbers of SCE per cell observed in samples treated with five kinds of PELGE nanoparticles were significantly greater than those found in the negative controls with or without the S9 mix. The discrepancies found in the two assays suggest that the five kinds of nanoparticles may produce only a weakly clastogenic response.

  3. Modification of the cellular heat sensitivity of cucumber by growth under supplemental ultraviolet-B radiation

    International Nuclear Information System (INIS)

    Caldwell, C.R.

    1994-01-01

    The effect of ultraviolet B (UV-B) radiation on the thermal sensitivity of cucumber (Cucumis sativus L.) was studied using UV-B-sensitive cv Poinsett 76 and UV-B-resistant cv Ashley grown under control and elevated (300 mW m -2 ) UV-B radiation levels. Using both cotyledon and leaf discs, the ability of the tissue to reduce triphenyl tetrazolium chloride (TTC) was determined after treatment at 50 degrees C for various times. Semilogarithmic plots of TTC reduction as a function of time at 50 degrees were curvilinear. They were monophasic for the control cucumber and biphasic for cucumber grown in the presence of elevated UV-B. Treatment of cucumber plants at 37 degrees C for 24 h or of tissue discs at acute UV-B levels for 1 h further modified their response to elevated temperature. These results suggest that growth of cucumber under enhanced UV-B radiation levels increased its ability to withstand elevated temperatures. 19 refs., 2 figs., 2 tabs

  4. The anti-biofilm activity of lemongrass (Cymbopogon flexuosus) and grapefruit (Citrus paradisi) essential oils against five strains of Staphylococcus aureus.

    Science.gov (United States)

    Adukwu, E C; Allen, S C H; Phillips, C A

    2012-11-01

    To determine the sensitivity of five strains of Staphylococcus aureus to five essential oils (EOs) and to investigate the anti-biofilm activity of lemongrass and grapefruit EOs. Antimicrobial susceptibility screening was carried out using the disk diffusion method. All of the strains tested were susceptible to lemongrass, grapefruit, bergamot and lime EOs with zones of inhibition varying from 2·85 to 8·60 cm although they were resistant to lemon EO. Lemongrass EO inhibited biofilm formation at 0·125% (v/v) as measured by colorimetric assay and at 0·25% (v/v) no metabolic activity was observed as determined by 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction. Grapefruit EO did not show any anti-biofilm activity. Following exposure to lemongrass EO extensive disruption to Staph. aureus biofilms was shown under scanning electron microscopy. In comparison to the other EOs tested, lemongrass exhibited the most effective antimicrobial and anti-biofilm activity. The effect of lemongrass EO highlights its potential against antibiotic resistant Staph. aureus in the healthcare environment. © 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

  5. Antimicrobial effects of Piper hispidum extract, fractions and chalcones against Candida albicans and Staphylococcus aureus.

    Science.gov (United States)

    Costa, G M; Endo, E H; Cortez, D A G; Nakamura, T U; Nakamura, C V; Dias Filho, B P

    2016-09-01

    Three chalcones, 2'-hydroxy-4,4',6'-trimethoxychalcone, 2'-hydroxy-4,4',6'-tetramethoxychalcone, and 3,2'-dihydroxy-4,4',6'-trimethoxychalcone, were isolated from the leaves of Piper hispidum in a bioguided fractionation of crude extract. The antimicrobial activity of crude extract of P. hispidum leaves was determined against bacteria Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus and yeasts Candida albicans, C. parapsilosis and C. tropicalis. Fractions and chalcones were tested against C. albicans and S. aureus. The checkerboard assay was performed to assess synergic interactions between extract and antifungal drugs, and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay was used to evaluate anti-biofilm effects of extract. The extract was active against yeasts, S. aureus and B. subtilis with MIC values between 15.6 and 62.5μg/mL. Synergistic effects of extract associated with fluconazole and nystatin were observed against C. albicans, with fractional inhibitory concentration indices of 0.37 and 0.24, respectively. The extract was also effective against C. albicans and S. aureus biofilm cells at concentrations of 62.5 and 200μg/mL, respectively. Thus, P. hispidum may be a possible source of bioactive substances with antimicrobial properties. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  6. [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.

  7. The role of adenosine in preconditioning by brief pressure overload in rats.

    Science.gov (United States)

    Huang, Cheng-Hsiung; Tsai, Shen-Kou; Chiang, Shu-Chiung; Lai, Chang-Chi; Weng, Zen-Chung

    2015-08-01

    Brief pressure overload of the left ventricle reduced myocardial infarct (MI) size in rabbits has been previously reported. Its effects in other species are not known. This study investigates effects of pressure overload and the role of adenosine in rats in this study. MI was induced by 40-minute occlusion of the left anterior descending coronary artery followed by 3-hour reperfusion. MI size was determined by triphenyl tetrazolium chloride staining. Brief pressure overload was induced by two 10-minute episodes of partial snaring of the ascending aorta. Systolic left ventricular pressure was raised 50% above the baseline value. Ischemic preconditioning was elicited by two 10-minute coronary artery occlusions. The MI size (mean ± standard deviation), expressed as percentage of area at risk, was significantly reduced in the pressure overload group as well as in the ischemic preconditioning group (17.4 ± 3.0% and 18.2 ± 1.5% vs. 26.6 ± 2.4% in the control group, p overload and ischemic preconditioning (18.3 ± 1.5% and 18.2 ± 2.0%, respectively, p overload of the left ventricle preconditioned rat myocardium against infarction. Because SPT did not significantly alter MI size reduction, our results did not support a role of adenosine in preconditioning by pressure overload in rats. Copyright © 2013. Published by Elsevier B.V.

  8. Effect of citrus flavonoids on HL-60 cell differentiation.

    Science.gov (United States)

    Kawaii, S; Tomono, Y; Katase, E; Ogawa, K; Yano, M

    1999-01-01

    Twenty-seven Citrus flavonoids were examined for their activity of induction of terminal differentiation of human promyelocytic leukemia cells (HL-60) by nitro blue tetrazolium (NBT) reducing, nonspecific esterase, specific esterase, and phagocytic activities. 10 flavonoids were judged to be active (percentage of NBT reducing cells was more than 40% at a concentration of 40 microM), and the rank order of potency was natsudaidain, luteolin, tangeretin, quercetin, apigenin, 3, 3, '4, '5, 6, 7, 8-heptamethoxyflavone, nobiletin, acacetin, eriodictyol, and taxifolin. These flavonoids exerted their activity in a dose-dependent manner. HL-60 cells treated with these flavonoids differentiated into mature monocyte/macrophage. The structure-activity relationship established from comparison between flavones and flavanones revealed that ortho-catechol moiety in ring B and C2-C3 double bond had an important role for induction of differentiation of HL-60. In polymethoxylated flavones, hydroxyl group at C3 and methoxyl group at C8 enhanced the differentiation-inducing activity.

  9. Differentiation-promoting activity of pomegranate (Punica granatum) fruit extracts in HL-60 human promyelocytic leukemia cells.

    Science.gov (United States)

    Kawaii, Satoru; Lansky, Ephraim P

    2004-01-01

    Differentiation refers to the ability of cancer cells to revert to their normal counterparts, and its induction represents an important noncytotoxic therapy for leukemia, and also breast, prostate, and other solid malignancies. Flavonoids are a group of differentiation-inducing chemicals with a potentially lower toxicology profile than retinoids. Flavonoid-rich polyphenol fractions from the pomegranate (Punica granatum) fruit exert anti-proliferative, anti-invasive, anti-eicosanoid, and pro-apoptotic actions in breast and prostate cancer cells and anti-angiogenic activities in vitro and in vivo. Here we tested flavonoid-rich fractions from fresh (J) and fermented (W) pomegranate juice and from an aqueous extraction of pomegranate pericarps (P) as potential differentiation-promoting agents of human HL-60 promyelocytic leukemia cells. Four assays were used to assess differentiation: nitro blue tetrazolium reducing activity, nonspecific esterase activity, specific esterase activity, and phagocytic activity. In addition, the effect of these extracts on HL-60 proliferation was evaluated. Extracts W and P were strong promoters of differentiation in all settings, with extract J showing only a relatively mild differentiation-promoting effect. The extracts had proportional inhibitory effects on HL-60 cell proliferation. The results highlight an important, previously unknown, mechanism of the cancer preventive and suppressive potential of pomegranate fermented juice and pericarp extracts.

  10. Radiosensitization of non-small cell lung cancer by kaempferol.

    Science.gov (United States)

    Kuo, Wei-Ting; Tsai, Yuan-Chung; Wu, His-Chin; Ho, Yung-Jen; Chen, Yueh-Sheng; Yao, Chen-Han; Yao, Chun-Hsu

    2015-11-01

    The aim of the present study was to determine whether kaempferol has a radiosensitization potential for lung cancer in vitro and in vivo. The in vitro radio-sensitization activity of kaempferol was elucidated in A-549 lung cancer cells by using an MTT (3-(4 5-dimethylthiazol-2-yl)-25-diphenyl-tetrazolium bromide) assay, cell cycle analysis and clonogenic assay. The in vivo activity was evaluated in the BALB/c nude mouse xenograft model of A-549 cells by hematoxylin and eosin staining and immunohistochemistry, and the tumor volume was recorded. Protein levels of the apoptotic pathway were detected by western blot analysis. Treatment with kaempferol inhibited the growth of A-549 cells through activation of apoptotic pathway. However, the same doses did not affect HFL1 normal lung cell growth. Kaempferol induced G2/M cell cycle arrest and the enhancement of radiation-induced death and clonogenic survival inhibition. The in vivo data showed that kaempferol increased tumor cell apoptosis and killing of radiation. In conclusion, the findings demonstrated that kaempferol increased tumor cell killing by radiation in vitro and in vivo through inhibition of the AKT/PI3K and ERK pathways and activation of the mitochondria apoptosis pathway. The results of the present study provided solid evidence that kaempferol is a safe and potential radiosensitizer.

  11. Kaempferol Inhibits Angiogenesis by Suppressing HIF-1α and VEGFR2 Activation via ERK/p38 MAPK and PI3K/Akt/mTOR Signaling Pathways in Endothelial Cells.

    Science.gov (United States)

    Kim, Gi Dae

    2017-12-01

    Kaempferol has been shown to inhibit vascular formation in endothelial cells. However, the underlying mechanisms are not fully understood. In the present study, we evaluated whether kaempferol exerts antiangiogenic effects by targeting extracellular signal-regulated kinase (ERK)/p38 mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) signaling pathways in endothelial cells. Endothelial cells were treated with various concentrations of kaempferol for 24 h. Cell viability was determined by the 3- (4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay; vascular formation was analyzed by tube formation, wound healing, and mouse aortic ring assays. Activation of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor receptor 2 (VEGFR2), ERK/p38 MAPK, and PI3K/Akt/mTOR was analyzed by Western blotting. Kaempferol significantly inhibited cell migration and tube formation in endothelial cells, and suppressed microvessel sprouting in the mouse aortic ring assay. Moreover, kaempferol suppressed the activation of HIF-1α, VEGFR2, and other markers of ERK/p38 MAPK and PI3K/Akt/mTOR signaling pathways in endothelial cells. These results suggest that kaempferol inhibits angiogenesis by suppressing HIF-1α and VEGFR2 activation via ERK/p38 MAPK and PI3K/Akt/mTOR signaling in endothelial cells.

  12. 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.

  13. Effects of drinking desalinated seawater on cell viability and proliferation.

    Science.gov (United States)

    Macarrão, Camila Longhi; Bachi, André Luis Lacerda; Mariano, Mario; Abel, Lucia Jamli

    2017-06-01

    Desalination of seawater is becoming an important means to address the increasing scarcity of freshwater resources in the world. Seawater has been used as drinking water in the health, food, and medical fields and various beneficial effects have been suggested, although not confirmed. Given the presence of 63 minerals and trace elements in drinking desalinated seawater (63 DSW), we evaluated their effects on the behavior of tumorigenic and nontumorigenic cells through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and annexin-V-fluorescein isothiocyanate/propidium iodide staining. Our results showed that cell viability and proliferation in the presence of 63 DSW were significantly greater than in mineral water and in the presence of fetal bovine serum in a dose-dependent manner. Furthermore, 63 DSW showed no toxic effect on murine embryonic fibroblast (NIH-3T3) and murine melanoma (B16-F10) cells. In another assay, we also showed that pre-treatment of non-adherent THP-1 cells with 63 DSW reduces apoptosis incidence, suggesting a protective effect against cell death. We conclude that cell viability and proliferation were improved by the mineral components of 63 DSW and this effect can guide further studies on health effects associated with DSW consumption.

  14. 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.

  15. 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.

  16. 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.

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

    International Nuclear Information System (INIS)

    Lee, Sam Sun; You Dong Soo

    1997-01-01

    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.

  18. Oxidative stress and maternal obesity: feto-placental unit interaction.

    Science.gov (United States)

    Malti, N; Merzouk, H; Merzouk, S A; Loukidi, B; Karaouzene, N; Malti, A; Narce, M

    2014-06-01

    To determine oxidative stress markers in maternal obesity during pregnancy and to evaluate feto-placental unit interaction, especially predictors of fetal metabolic alterations. 40 obese pregnant women (prepregnancy BMI > 30 kg/m²) were compared to 50 control pregnant women. Maternal, cord blood and placenta samples were collected at delivery. Biochemical parameters (total cholesterol and triglycerides) and oxidative stress markers (malondialdehyde, carbonyl proteins, superoxide anion expressed as reduced Nitroblue Tetrazolium, nitric oxide expressed as nitrite, reduced glutathione, catalase, superoxide dismutase) were assayed by biochemical methods. Maternal, fetal and placental triglyceride levels were increased in obese group compared to control. Maternal malondialdehyde, carbonyl proteins, nitric oxide and superoxide anion levels were high while reduced glutathione concentrations and superoxide dismutase activity were low in obesity. In the placenta and in newborns of these obese mothers, variations of redox balance were also observed indicating high oxidative stress. Maternal and placental interaction constituted a strong predictor of fetal redox variations in obese pregnancies. Maternal obesity compromised placental metabolism and antioxidant status which strongly impacted fetal redox balance. Oxidative stress may be one of the key downstream mediators that initiate programming of the offspring. Maternal obesity is associated with metabolic alterations and dysregulation of redox balance in the mother-placenta - fetus unit. These perturbations could lead to maternal and fetal complications and should be carefully considered. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. 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

    2017-01-01

    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. PMID:28772455

  20. Zirconia/Hydroxyapatite Composites Synthesized Via Sol-Gel: Influence of Hydroxyapatite Content and Heating on Their Biological Properties

    Science.gov (United States)

    Bollino, Flavia; Armenia, Emilia; Tranquillo, Elisabetta

    2017-01-01

    Zirconia (ZrO2) and zirconia-based glasses and ceramics are materials proposed for use in the dental and orthopedic fields. In this work, ZrO2 glass was modified by adding different amounts of bioactive and biocompatible hydroxyapatite (HAp). ZrO2/HAp composites were synthesized via the sol-gel method and heated to different temperatures to induce modifications of their chemical structure, as ascertained by Fourier transform infrared spectroscopy (FTIR) analysis. The aim was to investigate the effect of both HAp content and heating on the biological performances of ZrO2. The materials’ bioactivity was studied by soaking samples in a simulated body fluid (SBF). FTIR and scanning electron microscopy (SEM)) analyses carried out after exposure to SBF showed that all materials are bioactive, i.e., they are able to form a hydroxyapatite layer on their surface. Moreover, the samples were soaked in a solution containing bovine serum albumin (BSA). FTIR analysis proved that the synthesized materials are able to adsorb the blood protein, the first step of cell adhesion. WST-8 ([2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt]) assay showed that no cytotoxicity effects were induced by the materials’ extract. However, the results proved that bioactivity increases with both the HAp content and the temperature used for the thermal treatment, whereas biocompatibility increases with heating but is not affected by the HAp content. PMID:28773116

  1. Pharmacological blocking of the osteoclastic biocorrosion of surgical stainless steel in vitro.

    Science.gov (United States)

    Lionetto, S; Little, A; Moriceau, G; Heymann, D; Decurtins, M; Plecko, M; Filgueira, L; Cadosch, D

    2013-04-01

    In vitro studies suggest that human osteoclasts (OC) are able to corrode surgical stainless steel 316L (SS). The aim of this study was to investigate whether osteoclastic biocorrosion can be blocked pharmacologically. Human OCs were generated in vitro from peripheral blood monocytic cells (PBMCs) in the presence of OC differentiation cytokines. The osteoclastic viability, differentiation, and resorptive function (on both bone and SS) were assessed using standard colorimetric cell viability assay 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenil)-2H-tetrazolium, inner salt (MTS), fluorescence microscopy, tartrate-resistant acid phosphatase expression (flow cytometry), and scanning electron microscopy. OCs cultured on SS were exposed to nontoxic concentrations of bafilomycin A1, amiloride hydrochloride, or zoledronic acid. The extent of biocorrosion was quantified using atomic emission spectrometry (to measure the concentration of metal ions released into the supernatant) and scanning electron microscopy. PBMCs differentiated into mature and functional OC in the presence of all the drugs used. Osteoclastic resorption of SS was noted with differences in the resorption pattern for all drug treatments. Under the drug treatments, single areas of osteoclastic resorption were larger in size but less abundant when compared with positive controls. None of the drugs used were able to inhibit osteoclastic biocorrosion of SS. Copyright © 2012 Wiley Periodicals, Inc.

  2. 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.

  3. Multinucleated Giant Cancer Cells Produced in Response to Ionizing Radiation Retain Viability and Replicate Their Genome

    Directory of Open Access Journals (Sweden)

    Razmik Mirzayans

    2017-02-01

    Full Text Available Loss of wild-type p53 function is widely accepted to be permissive for the development of multinucleated giant cells. However, whether therapy-induced multinucleation is associated with cancer cell death or survival remains controversial. Herein, we demonstrate that exposure of p53-deficient or p21WAF1 (p21-deficient solid tumor-derived cell lines to ionizing radiation (between 2 and 8 Gy results in the development of multinucleated giant cells that remain adherent to the culture dish for long times post-irradiation. Somewhat surprisingly, single-cell observations revealed that virtually all multinucleated giant cells that remain adherent for the duration of the experiments (up to three weeks post-irradiation retain viability and metabolize 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl-tetrazolium bromide (MTT, and the majority (>60% exhibit DNA synthesis. We further report that treatment of multinucleated giant cells with pharmacological activators of apoptosis (e.g., sodium salicylate triggers their demise. Our observations reinforce the notion that radiation-induced multinucleation may reflect a survival mechanism for p53/p21-deficient cancer cells. With respect to evaluating radiosensitivity, our observations underscore the importance of single-cell experimental approaches (e.g., single-cell MTT as the creation of viable multinucleated giant cells complicates the interpretation of the experimental data obtained by commonly-used multi-well plate colorimetric assays.

  4. 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. IC 50 obtained was 36.6  μ g/mL for CEO and 129.9  μ g/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  μ g/mL of CEO and 32.12  μ g/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.

  5. 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.

  6. The Effects of Royal Jelly on In-Vitro Cytotoxicity of K562 Cells and Peripheral Blood Mononuclear Cells

    Directory of Open Access Journals (Sweden)

    SE Hosseini

    2014-02-01

    Full Text Available Abstract Background & aim: Royal jelly, secreted by worker bees, has different biological activities on cells and tissues. The aim of this study was to evaluate the effects of royal jelly on peripheral blood mononuclear cells and on the tumor category of K562 cell line. Methods: In the present experimental study, three subjects were selected separately with three repetitions. K562 (104 cells and PBMC (105 cells with different concentrations of royal jelly (5, 10, 25, 50 and 100 mg/ml were cultured under standard conditions for 48 and 72 h separately. The fatality rate on PBMC cells and K562 cancer cells was evaluated by using MTT (Tetrazolium Dye-Reduction Assay. The number of viable cells in PBMC that were exposed for 48 hours with Royal Jelly was evaluated by trypan blue staining. Data were analyzed by ANOVA. Results: The royal jelly had no cytotoxicity effect on PBMC cells but at concentration of 50 and 100 mg/mL the cytotoxicity effect were observed on k562 cells whereas, at 10 and 25 mg/ml the number of PBMC viable cells increased. Conclusion: Due to the lack of lethality of royal jelly on PBMC cells and PBMC cell viability and an increase in the fatality rate of cancer cells in the future, royal jelly can be used as a potential candidate for treatment of leukemia. Keywords: Royal jelly, K562, peripheral blood mononuclear cell

  7. Assessment of antibacterial and cytotoxic effects of orthodontic stainless steel brackets coated with different phases of titanium oxide: An in-vitro study.

    Science.gov (United States)

    Baby, Roshen Daniel; Subramaniam, Siva; Arumugam, Ilakkiya; Padmanabhan, Sridevi

    2017-04-01

    Our objective was to assess the antibacterial and cytotoxic effects of orthodontic stainless steel brackets coated with different phases of photocatalytic titanium oxide. From a total sample of 115 brackets, 68 orthodontic stainless steel brackets were coated with titanium oxide using a radiofrequency magnetron sputtering machine. The coated brackets were then converted into 34 each of the anatase and rutile phases of titanium oxide. These brackets were subdivided into 4 groups for antibacterial study and 3 groups for cytotoxicity study. Brackets for the antibacterial study were assessed against the Streptococcus mutans species using microbiologic tests. Three groups for the cytotoxicity study were assessed using the thiazolyl tetrazolium bromide assay. The antibacterial study showed that both phases were effective, but the rutile phase of photocatalytic titanium oxide had a greater bactericidal effect than did the anatase phase. The cytotoxicity study showed that the rutile phase had a greater decrease in viability of cells compared with the anatase phase. It is recommended that orthodontic brackets be coated with the anatase phase of titanium oxide since they exhibited a significant antibacterial property and were only slightly cytotoxic. Copyright © 2016 American Association of Orthodontists. Published by Elsevier Inc. All rights reserved.

  8. Antimicrobial Efficacy of Salvadora persica Extracts on a Monospecies Biofilm on Orthodontic Brackets In Vitro.

    Science.gov (United States)

    Halawany, Hassan S; Abraham, Nimmi B; Siddiqui, Yunus M; Balto, Hanan A; Jacob, Vimal

    2016-01-01

    The oral cavity is a rich ecosystem with a plethora of microorganisms, and different components of fixed orthodontic appliances may contribute to a shift in the balance of oral ecology. The purpose of this study was to investigate the antimicrobial potential of hexane and ethanol extracts of Salvadora persica on a monospecies biofilm model established on orthodontic brackets in vitro. Streptococcus mutans biofilm was formed on mini diamond orthodontic brackets following three days of anaerobic incubation at 37˚C. The bacterial cell viability of this biofilm was measured after their exposure to saline, hexane extract of S. persica, ethanol extract of S. persica and 0.2% chlorhexidine using 3-(4, 5-dimethylthiazol- 2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS) assay. On half of the brackets, the colony forming units (CFU) were counted. Both experiments were performed in triplicate. The absorbance values obtained from the MTS reduction assay after exposure to the different test agents showed a decline in the bacterial cell viability of the S. mutans biofilm as follows: chlorhexidine (+)0.05). The CFU counts of S. mutans obtained from chlorhexidine exposure were lower than from hexane and ethanol extracts. S. persica extracts were found to have antimicrobial effects on S. mutans biofilm established in vitro on orthodontic brackets suggestive of its potential use as an oral antimicrobial agent for orthodontic patients.

  9. EGCG protects against homocysteine-induced human umbilical vein endothelial cells apoptosis by modulating mitochondrial-dependent apoptotic signaling and PI3K/Akt/eNOS signaling pathways.

    Science.gov (United States)

    Liu, Shumin; Sun, Zhengwu; Chu, Peng; Li, Hailong; Ahsan, Anil; Zhou, Ziru; Zhang, Zonghui; Sun, Bin; Wu, Jingjun; Xi, Yalin; Han, Guozhu; Lin, Yuan; Peng, Jinyong; Tang, Zeyao

    2017-05-01

    Homocysteine (Hcy) induced vascular endothelial injury leads to the progression of endothelial dysfunction in atherosclerosis. Epigallocatechin gallate (EGCG), a natural dietary antioxidant, has been applied to protect against atherosclerosis. However, the underlying protective mechanism of EGCG has not been clarified. The present study investigated the mechanism of EGCG protected against Hcy-induced human umbilical vein endothelial cells (HUVECs) apoptosis. Methyl thiazolyl tetrazolium assay (MTT), transmission electron microscope, fluorescent staining, flow cytometry, western blot were used in this study. The study has demonstrated that EGCG suppressed Hcy-induced endothelial cell morphological changes and reactive oxygen species (ROS) generation. Moreover, EGCG dose-dependently prevented Hcy-induced HUVECs cytotoxicity and apoptotic biochemical changes such as reducing mitochondrial membrane potential (MMP), decreasing Bcl-2/Bax protein ratio and activating caspase-9 and 3. In addition, EGCG enhanced the protein ratio of p-Akt/Akt, endothelial nitric oxide synthase (eNOS) activation and nitric oxide (NO) formation in injured cells. In conclusion, the present study shows that EGCG prevents Hcy-induced HUVECs apoptosis via modulating mitochondrial apoptotic and PI3K/Akt/eNOS signaling pathways. Furthermore, the results indicate that EGCG is likely to represent a potential therapeutic strategy for atherosclerosis associated with Hyperhomocysteinemia (HHcy).

  10. Antibacterial activity of food-grade chitosan against Vibrio parahaemolyticus biofilms.

    Science.gov (United States)

    Xie, Ting; Liao, Zhenlin; Lei, Huan; Fang, Xiang; Wang, Jie; Zhong, Qingping

    2017-09-01

    Biofilm is a community composed of microbes and the extracellular polymeric substances. This special architecture poses a significant public health risk as it increases the fitness of bacteria in harsh conditions and renders bacterial resistance to antimicrobial agents and cleaning. In this study, we investigated the inhibition and eradication effects of chitosan on the biofilm of Vibrio parahaemolyticus, an important food-borne pathogen. The crystal violet staining, [2, 3-bis (2-methoxy-4-nitro-5- sulfophenyl)-2H-tetrazolium-5-carboxanilide] (XTT) reduction method, phenol-sulfuric acid method, fluorescence microscope and confocal laser scanning microscope (CLSM) observation were conducted. The results indicated that the minimum inhibitory concentration (MIC) of chitosan was 1.25 mg/mL. Sub-MIC of chitosan could significantly inhibit biofilm formation, reduce the metabolic activities and the secretion of extracellular polysaccharide (EPS). Moreover, chitosan at 4MIC could eradicate 85.06% mature biofilm of V. parahaemolyticus, and decrease 81.43% EPS in mature biofilm. These results were also confirmed by the visual images obtained from fluorescence microscopy and CLSM. This study elucidated that chitosan was not only effective to prevent biofilm formation, but also eradicate mature biofilms of V. parahaemolyticus. Copyright © 2017. Published by Elsevier Ltd.

  11. 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.

  12. Anti-HIV-1 activities of the extracts from the medicinal plant Linum grandiflorum Desf

    DEFF Research Database (Denmark)

    Mohammed, Magdy M. D.; Christensen, Lars Porskjær; Ibrahim, Nabaweya A.

    2009-01-01

    As part of our screening of anti-AIDS agents from natural sources e.g. Ixora undulata, Paulownia tomentosa, Fortunella margarita, Aegle marmelos and Erythrina abyssinica, the different organic and aqueous extracts of Linum grandiflorum leaves and seeds were evaluated in vitro by the microculture...... tetrazolium (MTT) assay. The activity of the tested extracts against multiplication of HIV-1 wild type IIIB, N119, A17, and EFVR in acutely infected cells was based on inhibition of virus-induced cytopathicity in MT-4 cells. Results revealed that both the MeOH and the CHCl3 extracts of L. grandiflorum have...... significant inhibitory effects against HIV-1 induced infection with MT-4 cells. The MeOH extract of the leaves is more potent than other extracts against MT-4 cell cultures infected with the wild type HIV-1, strain IIIB with an ED50 of 46 ± 6 µM, while the CHCl3 extract of the seeds is more potent than other...

  13. Development of test models to quantify encapsulated bioburden in spacecraft polymer materials by cultivation-dependent and molecular methods

    Science.gov (United States)

    Bauermeister, Anja; Moissl-Eichinger, Christine; Mahnert, Alexander; Probst, Alexander; Flier, Niwin; Auerbach, Anna; Weber, Christina; Haberer, Klaus; Boeker, Alexander

    Bioburden encapsulated in spacecraft polymers (such as adhesives and coatings) poses a potential risk to scientific exploration of other celestial bodies, but it is not easily detectable. In this study, we developed novel testing strategies to estimate the quantity of intrinsic encapsulated bioburden in polymers used frequently on spaceflight hardware. In particular Scotch-Weld (TM) 2216 B/A (Epoxy adhesive); MAP SG121FD (Silicone coating), Solithane (®) 113 (Urethane resin); ESP 495 (Silicone adhesive); and Dow Corning (®) 93-500 (Silicone encapsulant) were investigated. As extraction of bioburden from polymerized (solid) materials did not prove feasible, a method was devised to extract contaminants from uncured polymer precursors by dilution in organic solvents. Cultivation-dependent analyses showed less than 0.1-2.5 colony forming units (cfu) per cm³ polymer, whereas quantitative PCR with extracted DNA indicated considerably higher values, despite low DNA extraction efficiency. Results obtained by this method reflected the most conservative proxy for encapsulated bioburden. To observe the effect of physical and chemical stress occurring during polymerization on the viability of encapsulated contaminants, Bacillus safensis spores were embedded close to the surface in cured polymer, which facilitated access for different analytical techniques. Staining by AlexaFluor succinimidyl ester 488 (AF488), propidium monoazide (PMA), CTC (5-cyano-2,3-diotolyl tetrazolium chloride) and subsequent confocal laser scanning microscopy (CLSM) demonstrated that embedded spores retained integrity, germination and cultivation ability even after polymerization of the adhesive Scotch-Weld™ 2216 B/A.

  14. 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.

  15. Some South African Rubiaceae Tree Leaf Extracts Have Antimycobacterial Activity Against Pathogenic and Non-pathogenic Mycobacterium Species.

    Science.gov (United States)

    Aro, Abimbola O; Dzoyem, Jean P; Hlokwe, Tiny M; Madoroba, Evelyn; Eloff, Jacobus N; McGaw, Lyndy J

    2015-07-01

    Tuberculosis (TB) caused by Mycobacterium tuberculosis remains an ongoing threat to human health. Many plant species contain antimycobacterial compounds, which may serve as template molecules for new anti-TB drugs. The Rubiaceae family is the largest family of trees in southern Africa, and preliminary evidence revealed antimycobacterial activity in several species of the genus, motivating further studies. Leaf extracts of 15 tree species from the Rubiaceae family were screened for antimycobacterial activity against pathogenic M. tuberculosis and non-pathogenic Mycobacterium smegmatis, Mycobacterium aurum and Mycobacterium bovis BCG (Bacillus Calmette-Guérin) using a twofold serial microdilution assay. Cytotoxicity was determined using a tetrazolium-based colorimetric assay against C3A liver cells and Vero kidney cells. Minimum inhibitory concentration values as low as 0.04 mg/mL against M. smegmatis and M. tuberculosis were recorded. Activity against M. aurum was the best predictor of activity against pathogenic M. tuberculosis (correlation coefficient = 0.9). Bioautography indicated at least 40 different antimycobacterial compounds in the extracts. Cytotoxicity of the extracts varied, and Oxyanthus speciosus had the most promising selectivity index values. Copyright © 2015 John Wiley & Sons, Ltd.

  16. Short Term INT-Formazan Production as a Proxy for Marine Prokaryote Respiration

    Science.gov (United States)

    Cajal-Medrano, R.; Villegas-Mendoza, J.; Maske, H.

    2016-02-01

    Prokaryotes are poisoned by the tetrazolium electron transport probe INT on time scales of less than one hour, invalidating the interpretation of the rate of in vivo INT reduction to formazan as a proxy for oxygen consumption rates (Villegas-Mendoza et al. 2015). We measured oxygen consumption rate (R; µM O2 hour-1) and electron transport activity with in vivo INT formazan production (IFP, mM formazan) at 0.5 mM INT during 1 hour exposure time of natural communities and cultures of the marine bacteria Vibrio harveyi growing in batch and continuous cultures. A strong exponential relationship R = 0.20 IFP2.15 (pgrowth rates under aerobic condition. We find that IFP and oxygen consumption increase with bacterial specific growth rates and temperature as expected from basic principles of physiology and biochemistry. Oxygen and nitrogen saturated batch cultures of V. harveyi showed that both, IFP and oxygen consumption increased for 0.8 hours but then stopped similar to natural bacterial communities supporting the above relationship of IFP to prokaryote respiration. Our method implies adding 0.5 mM INT to a plankton sample and incubating for less than 1 hour. After prokaryote separation by size filtration (0.8 mm), the formazan crystals are collected by filtration (0.2 mm) and dissolved in propanol. The absorbance at 485 nm per sample volume yields the formazan potential that is related to prokaryote respiration in the sample.

  17. The citotoxicity of calcium hydroxide intracanal dressing by MTT assay

    Directory of Open Access Journals (Sweden)

    Nanik Zubaidah

    2007-12-01

    Full Text Available Calcium hydroxide had been used as the intracanal dressing in endodontic treatment due to its high alkaline and high antimicrobial capacity. It also be able to dissolve the necrotic tissue, prevent the root resorbtion and regenerate a new hard tissue. The aim of this study is to identify the concentration of calcium hydroxide that has the lowest citotoxicity. There are 5 groups, each group had 8 samples with different concentration of calcium hydroxide. Group I: 50%, Group II: 55%, Group III: 60%, Group IV: 65% and Group V: 70%. The citotoxicity test by using enzymatic assay of MTT [3-(4.5- dimethylthiazol-2yl ]-2.5 diphenyl tetrazolium bromide, against fibroblast cell (BHK-21. The result of susceptibility test was showed by the citotoxicity detection of the survive cell of fibroblast that was measured spectrophotometrically using 595 nm beam. The data was analyzed using One-Way ANOVA test with significant difference α = 0.05 and subsequently LSD test. The result showed that in concentration 50%, 55%, 60%, 65%, and 70% calcium hydroxide had low toxicity, but calcium hydroxide 60%, had the lowest toxicity.

  18. Glycation-assisted synthesized gold nanoparticles inhibit growth of bone cancer cells.

    Science.gov (United States)

    Rahim, Moniba; Iram, Sana; Khan, Mohd Sajid; Khan, M Salman; Shukla, Ankur R; Srivastava, A K; Ahmad, Saheem

    2014-05-01

    This study presents a novel approach to synthesize glycogenic gold nanoparticles (glycogenic GNps) capped with glycated products (Schiff's base, Heyns products, fructosylamine etc.). These glycogenic GNps have been found to be active against human osteosarcoma cell line (Saos-2) with an IC50 of 0.187 mM, while the normal human embryonic lung cell line (L-132) remained unaffected up to 1mM concentration. The size of glycogenic GNps can also be controlled by varying the time of incubation of gold solution. Glycation reactions involving a combination of fructose and HSA (Human Serum Albumin) were found to be effective in the reduction of gold to glycogenic GNps whereas glucose in combination with HSA did not result in the reduction of gold. The progress of the reaction was followed using UV-visible spectroscopy and NBT (Nitroblue tetrazolium) assay. The glycogenic GNps were found to be spherical in shape with an average size of 24.3 nm, in a stable emulsion. These GNps were characterized using UV-visible spectroscopy, zeta potential analysis, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Copyright © 2013 Elsevier B.V. All rights reserved.

  19. Development and validation of a quick easily used biochemical assay for evaluating the viability of small immobile arthropods.

    Science.gov (United States)

    Phillips, Craig B; Iline, Ilia I; Richards, Nicola K; Novoselov, Max; McNeill, Mark R

    2013-10-01

    Quickly, accurately, and easily assessing the efficacy of treatments to control sessile arthropods (e.g., scale insects) and stationary immature life stages (e.g., eggs and pupae) is problematic because it is difficult to tell whether treated organisms are alive or dead. Current approaches usually involve either maintaining organisms in the laboratory to observe them for development, gauging their response to physical stimulation, or assessing morphological characters such as turgidity and color. These can be slow, technically difficult, or subjective, and the validity of methods other than laboratory rearing has seldom been tested. Here, we describe development and validation of a quick easily used biochemical colorimetric assay for measuring the viability of arthropods that is sufficiently sensitive to test even very small organisms such as white fly eggs. The assay was adapted from a technique for staining the enzyme hexokinase to signal the presence of adenosine triphosphate in viable specimens by reducing a tetrazolium salt to formazan. Basic laboratory facilities and skills are required for production of the stain, but no specialist equipment, expertise, or facilities are needed for its use.

  20. Surface characterization and cytotoxicity response of biodegradable magnesium alloys

    International Nuclear Information System (INIS)

    Pompa, Luis; Rahman, Zia Ur; Munoz, Edgar; Haider, Waseem

    2015-01-01

    Magnesium alloys have raised an immense amount of interest to many researchers because of their evolution as a new kind of third generation materials. Due to their biocompatibility, density, and mechanical properties, magnesium alloys are frequently reported as prospective biodegradable implant materials. Moreover, magnesium alloys experience a natural phenomenon to biodegrade in aqueous solutions due to its corrosion activity, which is excellent for orthopedic and cardiovascular applications. However, a major concern with such alloys is fast and non-uniform corrosion degradation. Controlling the degradation rate in the physiological environment determines the success of biodegradable implants. In this investigation, three different grades of magnesium alloys: AZ31B, AZ91E and ZK60A were studied for their corrosion resistance and biocompatibility. Scanning electron microscopy, energy dispersive spectroscopy, atomic force microscopy and contact angle meter are used to study surface morphology, chemistry, roughness and wettability, respectively. Additionally, the cytotoxicity of the leached metal ions was evaluated by using a tetrazolium based bio-assay, MTS. - Highlights: • Micro-textured features formed after the anodization of magnesium alloys. • Contact angle increased and surface free energy decreased by anodization. • Corrosion rate increased for anodized surfaces compared to untreated samples. • Cell viability was greater than 75% implying the cytocompatibility of Mg alloys

  1. Titania-polymeric powder coatings with nano-topography support enhanced human mesenchymal cell responses.

    Science.gov (United States)

    Mozumder, Mohammad Sayem; Zhu, Jesse; Perinpanayagam, Hiran

    2012-10-01

    Titanium implant osseointegration is dependent on the cellular response to surface modifications and coatings. Titania-enriched nanocomposite polymeric resin coatings were prepared through the application of advanced ultrafine powder coating technology. Their surfaces were readily modified to create nano-rough (topographies that supported human embryonic palatal mesenchymal cell responses. Energy dispersive x-ray spectroscopy confirmed continuous and homogenous coatings with a similar composition and even distribution of titanium. Scanning electron microscopy (SEM) showed complex micro-topographies, and atomic force microscopy revealed intricate nanofeatures and surface roughness. Cell counts, mitochondrial enzyme activity reduction of yellow 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to dark purple, SEM, and inverted fluorescence microscopy showed a marked increase in cell attachment, spreading, proliferation, and metabolic activity on the nanostructured surfaces. Reverse Transcription- Polymerase Chain Reaction (RT-PCR) analysis showed that type I collagen and Runx2 expression were induced, and Alizarin red staining showed that mineral deposits were abundant in the cell cultures grown on nanosurfaces. This enhancement in human mesenchymal cell attachment, growth, and osteogenesis were attributed to the nanosized surface topographies, roughness, and moderate wetting characteristics of the coatings. Their dimensional similarity to naturally occurring matrix proteins and crystals, coupled with their increased surface area for protein adsorption, may have facilitated the response. Therefore, this application of ultrafine powder coating technology affords highly biocompatible surfaces that can be readily modified to accentuate the cellular response. Copyright © 2012 Wiley Periodicals, Inc.

  2. A smart multifunctional nanocomposite for intracellular targeted drug delivery and self-release

    Science.gov (United States)

    Wang, Chan; Lv, Piping; Wei, Wei; Tao, Shengyang; Hu, Tao; Yang, Jingbang; Meng, Changgong

    2011-10-01

    A multifunctional 'all-in-one' nanocomposite is fabricated using a colloid, template and surface-modification method. This material encompasses magnetic induced target delivery, cell uptake promotion and controlled drug release in one system. The nanocomposite is characterized by scanning electron microscopy, transmission electron microscopy, x-ray diffraction, N2 adsorption and vibrating sample magnetometry. The prepared material has a diameter of 350-400 nm, a high surface area of 420.29 m2 g - 1, a pore size of 1.91 nm and a saturation magnetization of 32 emu g - 1. Doxorubicin (DOX) is loaded in mesopores and acid-sensitive blockers are introduced onto the orifices of the mesopores by a Schiff base linker to implement pH-dependent self-release. Folate was also introduced to improve DOX targeted delivery and endocytosis. The linkers remained intact to block pores with ferrocene valves and inhibit the diffusion of DOX at neutral pH. However, in lysosomes of cancer cells, which have a weak acidic pH, hydrolysis of the Schiff base group removes the nanovalves and allows the trapped DOX to be released. These processes are demonstrated by UV-visible absorption spectra, confocal fluorescence microscopy images and methyl thiazolyl tetrazolium assays in vitro, which suggest that the smart nanocomposite successfully integrates targeted drug delivery with internal stimulus induced self-release and is a potentially useful material for nanobiomedicine.

  3. Folic Acid supplementation stimulates notch signaling and cell proliferation in embryonic neural stem cells.

    Science.gov (United States)

    Liu, Huan; Huang, Guo-Wei; Zhang, Xu-Mei; Ren, Da-Lin; X Wilson, John

    2010-09-01

    The present study investigated the effect of folic acid supplementation on the Notch signaling pathway and cell proliferation in rat embryonic neural stem cells (NSCs). The NSCs were isolated from E14-16 rat brain and grown as neurospheres in serum-free suspension culture. Individual cultures were assigned to one of 3 treatment groups that differed according to the concentration of folic acid in the medium: Control (baseline folic acid concentration of 4 mg/l), low folic acid supplementation (4 mg/l above baseline, Folate-L) and high folic acid supplementation (40 mg/l above baseline, Folate-H). NSCs were identified by their expression of immunoreactive nestin and proliferating cells by incorporation of 5'bromo-2'deoxyuridine. Cell proliferation was also assessed by methyl thiazolyl tetrazolium assay. Notch signaling was analyzed by real-time PCR and western blot analyses of the expression of Notch1 and hairy and enhancer of split 5 (Hes5). Supplementation of NSCs with folic acid increased the mRNA and protein expression levels of Notch1 and Hes5. Folic acid supplementation also stimulated NSC proliferation dose-dependently. Embryonic NSCs respond to folic acid supplementation with increased Notch signaling and cell proliferation. This mechanism may mediate the effects of folic acid supplementation on neurogenesis in the embryonic nervous system.

  4. A smart multifunctional nanocomposite for intracellular targeted drug delivery and self-release

    Energy Technology Data Exchange (ETDEWEB)

    Wang Chan; Tao Shengyang; Hu Tao; Yang Jingbang; Meng Changgong [School of Chemical Engineering, Dalian University of Technology, Dalian, Liaoning (China); Lv Piping; Wei Wei, E-mail: taosy@dlut.edu.cn [National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing (China)

    2011-10-14

    A multifunctional 'all-in-one' nanocomposite is fabricated using a colloid, template and surface-modification method. This material encompasses magnetic induced target delivery, cell uptake promotion and controlled drug release in one system. The nanocomposite is characterized by scanning electron microscopy, transmission electron microscopy, x-ray diffraction, N{sub 2} adsorption and vibrating sample magnetometry. The prepared material has a diameter of 350-400 nm, a high surface area of 420.29 m{sup 2} g{sup -1}, a pore size of 1.91 nm and a saturation magnetization of 32 emu g{sup -1}. Doxorubicin (DOX) is loaded in mesopores and acid-sensitive blockers are introduced onto the orifices of the mesopores by a Schiff base linker to implement pH-dependent self-release. Folate was also introduced to improve DOX targeted delivery and endocytosis. The linkers remained intact to block pores with ferrocene valves and inhibit the diffusion of DOX at neutral pH. However, in lysosomes of cancer cells, which have a weak acidic pH, hydrolysis of the Schiff base group removes the nanovalves and allows the trapped DOX to be released. These processes are demonstrated by UV-visible absorption spectra, confocal fluorescence microscopy images and methyl thiazolyl tetrazolium assays in vitro, which suggest that the smart nanocomposite successfully integrates targeted drug delivery with internal stimulus induced self-release and is a potentially useful material for nanobiomedicine.

  5. DNA methyltransferase mediates dose-dependent stimulation of neural stem cell proliferation by folate.

    Science.gov (United States)

    Li, Wen; Yu, Min; Luo, Suhui; Liu, Huan; Gao, Yuxia; Wilson, John X; Huang, Guowei

    2013-07-01

    The proliferative response of neural stem cells (NSCs) to folate may play a critical role in the development, function and repair of the central nervous system. It is important to determine the dose-dependent effects of folate in NSC cultures that are potential sources of transplantable cells for therapies for neurodegenerative diseases. To determine the optimal concentration and mechanism of action of folate for stimulation of NSC proliferation in vitro, NSCs were exposed to folic acid or 5-methyltetrahydrofolate (5-MTHF) (0-200 μmol/L) for 24, 48 or 72 h. Immunocytochemistry and methyl thiazolyl tetrazolium assay showed that the optimal concentration of folic acid for NSC proliferation was 20-40 μmol/L. Stimulation of NSC proliferation by folic acid was associated with DNA methyltransferase (DNMT) activation and was attenuated by the DNMT inhibitor zebularine, which implies that folate dose-dependently stimulates NSC proliferation through a DNMT-dependent mechanism. Based on these new findings and previously published evidence, we have identified a mechanism by which folate stimulates NSC growth. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. 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

  7. New markers of urinary tract infection.

    Science.gov (United States)

    Masajtis-Zagajewska, Anna; Nowicki, Michal

    2017-08-01

    Urinary tract infection (UTI) is the most common bacterial infection independent of age. It is also one of the most common causes of hospitalizations for infections among elderly people and the most common indication for antibiotic prescriptions in primary care. Both diagnostics and management of lower and upper urinary tract infections provide challenges in clinical practice due to their high prevalence and recurrence, and worldwide increase of antibiotic resistance. The clinical symptoms of UTI are often uncharacteristic or asymptomatic. The accurate diagnosis and early treatment are crucial due to risk of septicaemia and long-term consequences. Currently the diagnosis of urinary tract infection is based on the presence of clinical symptoms in combination with the results of nitrite strip test indicating the presence of bacteria in urine and semi-quantitative measurement of white blood cells count in urine. Although urine culture is the gold standard in UTI diagnostics it is both time-consuming and costly. Searching for novel biomarkers of UTI has attracted much attention in recent years. The article reviews several promising serum and urine biomarkers of UTI such as leukocyte esterase, C-reactive protein, procalcitonin, interleukins, elastase alpha (1)-proteinase inhibitor, lactofferin, secretory immunoglobulin A, heparin-binding protein, xanthine oxidase, myeloperoxidase, soluble triggering receptor expressed on myeloid cells-1, α-1 microglobulin (α1Mg) and tetrazolium nitroblue test (TNB). Copyright © 2017 Elsevier B.V. All rights reserved.

  8. 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.

  9. Both Creatine and Its Product Phosphocreatine Reduce Oxidative Stress and Afford Neuroprotection in an In Vitro Parkinson’s Model

    Science.gov (United States)

    Martín-de-Saavedra, Maria D.; Romero, Alejandro; Egea, Javier; Ludka, Fabiana K.; Tasca, Carla I.; Farina, Marcelo; Rodrigues, Ana Lúcia S.; López, Manuela G.

    2014-01-01

    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. PMID:25424428

  10. PRODUCTION AND PHYSIOLOGICAL QUALITY OF SOYBEAN SEEDS IN ORTHIC QUARTZARENIC NEOSOIL OF THE CERRADO REGION

    Directory of Open Access Journals (Sweden)

    CHARLINE ZARATIN ALVES

    2015-01-01

    Full Text Available The aim of this study was to evaluate the effect of the sowing periods in production , productivity component and physiological quality of seeds of three soybean cultivars (TMG133RR, P98Y70RR and NS7670RR in Orthic Quartzarenic Neosoil in the cerrado region. The design used was a randomized block a factorial scheme design with four repetitions, and each plot with useful space consisted of three rows of four meters length, spaced at 0.45 meters. Field evaluations were the final stand, the height of the plants, height of the first pod insertion, the mass of 100 seeds and productivity. Already in the lab seeds were evaluated for germination and vigor (first germination count, emergency, emergence speed index, length and dry mass of the aerial part of the plant and roots, electrical conductivity, accelerated aging and tetrazolium test. It concludes that it is possible to use Orthic Quartzarenic Neosoils , located in the Cerrado region at altitudes higher than 600 m, in years and places with good water distribution in the spring - summer seasons, for grain and soybeans production, but as a first goal, the crop should be sown in the first 20 days of November otherwise it should take place in early December.

  11. Silicalite nanoparticles that promote transgene expression

    International Nuclear Information System (INIS)

    Pearce, Megan E; Mai, Hoang Q; Salem, Aliasger K; Lee, Namhoon; Larsen, Sarah C

    2008-01-01

    Here, we report on a new zeolite-based silicalite nanoparticle that can enhance the transfection efficiencies generated by poly ethylene imine-plasmid DNA (PEI-pDNA) complexes via a sedimentation mechanism and can enhance the transfection efficiencies of pDNA alone when surface functionalized with amine groups. The silicalite nanoparticles have a mean size of 55 nm. Functionalizing the silicalite nanoparticles with amine groups results in a clear transition in zeta potential from -25.9 ± 2.3 mV (pH 7.4) for unfunctionalized silicalite nanoparticles to 4.9 ± 0.7 mV (pH 7.4) for amine functionalized silicalite nanoparticles. We identify that silicalite nanoparticles used to promote non-viral vector acceleration to the cell surface are found in acidic vesicles or the cytoplasm but not the nucleus. An MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay showed that the silicalite nanoparticles were non-toxic at the concentrations tested for transfection. We show that surface functionalization of silicalite nanoparticles with amine groups results in a significant (230%) increase in transfection efficiency of pDNA when compared to unfunctionalized silicalite nanoparticles. Silicalite nanoparticles enhanced pDNA-PEI induced transfection of human embryonic kidney (HEK-293) cells by over 150%

  12. In Vitro and In Vivo Antimalarial Evaluations of Myrtle Extract, a Plant Traditionally Used for Treatment of Parasitic Disorders

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    Farzaneh Naghibi

    2013-01-01

    Full Text Available Based on the collected ethnobotanical data from the Traditional Medicine and Materia Medica Research Center (TMRC, Iran, Myrtus communis L. (myrtle was selected for the assessment of in vitro and in vivo antimalarial and cytotoxic activities. Methanolic extract of myrtle was prepared from the aerial parts and assessed for antiplasmodial activity, using the parasite lactate dehydrogenase (pLDH assay against chloroquine-resistant (K1 and chloroquine-sensitive (3D7 strains of Plasmodium falciparum. The 4-day suppressive test was employed to determine the parasitemia suppression of the myrtle extract against P. berghei  in vivo. The IC50 values of myrtle extract were 35.44 µg/ml against K1 and 0.87 µg/ml against 3D7. Myrtle extract showed a significant suppression of parasitaemia (84.8 ± 1.1% at 10 mg/kg/day in mice infected with P. berghei after 4 days of treatment. Cytotoxic activity was carried out against mammalian cell lines using methyl thiazol tetrazolium (MTT assay. No cytotoxic effect on mammalian cell lines up to 100 µg/mL was shown. The results support the traditional use of myrtle in malaria. Phytochemical investigation and understanding the mechanism of action would be in our upcoming project.

  13. Metabolic activity of tree saps of different origin towards cultured human cells in the light of grade correspondence analysis and multiple regression modeling

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    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.

  14. Biocompatibility Assessment of Poly(lactic acid Films after Sterilization with Ethylene Oxide in Histological Study In Vivo with Wistar Rats and Cellular Adhesion of Fibroblasts In Vitro

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    Michele Savaris

    2017-01-01

    Full Text Available Biomaterials must meet certain fundamental requirements for their usage in living beings, such as biocompatibility, bifunctionality, and sterilizability, without having chemical and structural changes. The biocompatibility of poly(lactic acid (PLA films, shaped by compression, was evaluated after sterilization by ethylene oxide by a histological in vivo test with Wistar rats and cytotoxicity in cell adhesion in vitro. The cytotoxicity test was performed by the reduction of tetrazolium salt (MTT. Thermal and chemical changes in PLA films concerning the proposed sterilization process and characteristics were not observed to evidence polymer degradation due to sterilization. The analysis of the cytotoxicity by the MTT method has shown that the sterilized PLA films are not cytotoxic. The adhesion and proliferation of fibroblasts on PLA films were homogeneously distributed over the evaluation period, showing an elongated appearance with unnumbered cytoplasmic extensions and cell-cell interactions. By examining the biocompatibility in a histological study, a mild tissue inflammation was observed with the presence of fibrosis in the samples that had been exposed for 21 days in the rats’ bodies. PLA films sterilized with ethylene oxide did not exhibit cell adhesion in vitro and toxicity to the surrounding tissue in vivo and they may be used in future in vivo testing, according to histological findings in Wistar rats in the present study.

  15. Alterations in Sensitivity to Estrogen, Dihydrotestosterone, and Xenogens in B-Lymphocytes from Children with Autism Spectrum Disorder and Their Unaffected Twins/Siblings

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    Martyn A. Sharpe

    2013-01-01

    Full Text Available It has been postulated that androgen overexposure in a susceptible person leads to excessive brain masculinization and the autism spectrum disorder (ASD phenotype. In this study, the responses to estradiol (E2, dihydrotestosterone (DHT, and dichlorodiphenyldichloroethylene (DDE on B-lymphocytes from ASD subjects and controls are compared. B cells were obtained from 11 ASD subjects, their unaffected fraternal twins, and nontwin siblings. Controls were obtained from a different cell bank. Lactate dehydrogenase (LDH and sodium 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl-2H-tetrazolium-5-carboxanilide (XTT reduction levels were measured after incubation with different concentrations of E2, DHT, and DDE. XTT/LDH ratio, representative of mitochondria number per cell, was calculated. E2, DHT, and DDE all cause “U”-shaped growth curves, as measured by LDH levels. ASD B cells show less growth depression compared to siblings and controls (P<0.01. They also have reduced XTT/LDH ratios (P<0.01 when compared to external controls, whereas siblings had values of XTT/LDH between ASD and external controls. B-lymphocytes from people with ASD exhibit a differential response to E2, DHT, and hormone disruptors in regard to cell growth and mitochondrial upregulation when compared to non-ASD siblings and external controls. Specifically, ASD B-lymphocytes show significantly less growth depression and less mitochondrial upregulation when exposed to these effectors. A mitochondrial deficit in ASD individuals is implied.

  16. Cytotoxicity and Apoptotic Effects of Polyphenols from Sugar Beet Molasses on Colon Carcinoma Cells in Vitro

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    Mingshun Chen

    2016-06-01

    Full Text Available Three polyphenols were isolated and purified from sugar beet molasses by ultrasonic-aid extraction and various chromatographic techniques, and their structures were elucidated by spectral analysis. Cytotoxicity and the molecular mechanism were measured by methyl thiazolyl tetrazolium (MTT assay, flow cytometry, caspase-3 activity assay and Western blot assay. The results showed that gallic acid, cyanidin-3-O-glucoside chloride and epicatechin have cytotoxicity to the human colon, hepatocellular and breast cancer cells. Cyanidin-3-O-glucoside chloride showed its cytotoxicity against various tumor cell lines, particularly against colon cancer Caco-2 cells with half maximal inhibitory concentration (IC50 value of 23.21 ± 0.14 μg/mL in vitro. Cyanidin-3-O-glucoside chloride may be a potential candidate for the treatment of colon cancer. In the mechanism study, cyanidin-3-O-glucoside chloride increased the ratio of cell cycle at G0/G1 phase and reduced cyclin D1 expression on Caco-2 cells. Cyanidin-3-O-glucoside chloride decreased mutant p21 expression, and increased the ratio of Bax/Bcl-2 and the activation of caspase-3 to induce apoptosis.

  17. Cytotoxicity and morphological effects induced by carvacrol and thymol on the human cell line Caco-2.

    Science.gov (United States)

    Llana-Ruiz-Cabello, María; Gutiérrez-Praena, Daniel; Pichardo, Silvia; Moreno, F Javier; Bermúdez, José María; Aucejo, Susana; Cameán, Ana María

    2014-02-01

    Essential oils used as additives in the food industry due to its flavour, antimicrobial and antioxidant properties. Therefore, human can be exposed orally to these compounds through the ingestion of foods. In this sense, the present work aims to assess toxicological effects of oregano essential oil on the digestive tract. In concrete, the cytotoxic effects of two components of the oregano essential oils, carvacrol and thymol, and their mixture, on the intestinal cells line Caco-2 after 24 and 48 h of exposure are studied. The basal cytotoxicity endpoints assayed (total protein content, neutral red uptake and the tetrazolium salt reduction) and the annexin/propidium iodide staining indicated that carvacrol and the mixture carvacrol/thymol induced toxic effects. Moreover, a morphological study was performed in order to determine the ultrastructural cellular damages caused by these substances. The main morphological alterations were vacuolated cytoplasm, altered organelles and finally cell death. In addition, although no cytotoxic effects were recorded for thymol at any concentration and time of exposure, ultrastructural changes evidenced cellular damage such as lipid degeneration, mitochondrial damage, nucleolar segregation and apoptosis. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. Compounds used to produce cloned animals are genotoxic and mutagenic in mammalian assays in vitro and in vivo

    International Nuclear Information System (INIS)

    Oliveira, R.J.; Mantovani, M.S.; Silva, A.F. da; Pesarini, J.R.; Mauro, M.O.; Ribeiro, L.R.

    2014-01-01

    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

  19. Photosensitizing anthraquinones from Heterophyllaea lycioides (Rubiaceae).

    Science.gov (United States)

    Dimmer, Jesica A; Núñez Montoya, Susana C; Mendoza, Caterine S; Cabrera, José L

    2017-05-01

    Seven anthraquinones were isolated from aerial parts of Heterophyllaea lycioides (Rusby) Sandwith (Rubiaceae), including three derivatives that have not been described before: a hetero-bianthraquinone identified as (R)-2-hydroxymethyl-2'methyl-1,1',6,6'-tetrahydroxy-5,5' bianthraquinone (lycionine), and two mono-chlorinated derivatives related to soranjidiol. One of them is a homo-bianthraquinone: (R)-7-chloro-2,2'-dimethyl-1,1',6,6'-tetrahydroxy-5,5' bianthraquinone (7-chlorobisoranjidiol), whereas the second halogenated derivative corresponds to a monomeric structure: 5-chloro-1,6-dihydroxy-2-methyl anthraquinone (5-chlorosoranjidiol). The four known compounds were already isolated from another species of this genus, H. pustulata, and they were identified as 5,5'-bisoranjidiol, soranjidiol, pustuline and heterophylline. Structural elucidation was performed by means of an extensive spectroscopic analysis, including 1D and 2D NMR data as well as by HRMS analysis. Chemical structures of 7-chlorobisoranjidiol and 5-chlorosoranjidiol were confirmed by their synthesis from 5,5'-bisoranjidiol and soranjidiol, respectively. Type I photosensitizing properties (superoxide anion radical generation, O 2 - ) were assessed by using the nitroblue tetrazolium assay. When lycionine and chlorinated derivatives were irradiated, they enhanced the O 2 - production with respect to the control; 7-chlorobisoranjidiol stood out by generating an increase of 20%, whereas the other anthraquinones only produced a slight increase of 7%. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. The Marine Metabolite SZ-685C Induces Apoptosis in Primary Human Nonfunctioning Pituitary Adenoma Cells by Inhibition of the Akt Pathway in Vitro

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    Xin Wang

    2015-03-01

    Full Text Available Nonfunctioning pituitary adenoma (NFPA is one of the most common types of pituitary adenoma. The marine anthraquinone derivative SZ-685C has been isolated from the secondary metabolites of the mangrove endophytic fungus Halorosellinia sp. (No. 1403 which is found in the South China Sea. Recent research has shown that SZ-685C possesses anticancer and tumor suppressive effects. The tetrazolium-based colorimetric assay (MTT assay to investigate the different effect of the marine compound SZ-685C on the proliferation of primary human NFPA cells, rat normal pituitary cells (RPCs and rat prolactinoma MMQ cell lines. Hoechst 33342 dye/propidium iodide (PI double staining and fluorescein isothiocyanate-conjugated Annexin V/PI (Annexin V-FITC/PI apoptosis assays detected an enhanced rate of apoptosis in cells treated with SZ-685C. Enhanced expression levels of caspase 3 and phosphate and tensin homolog (PTEN were determined by Western blotting. Notably, the protein expression levels of Akt were decreased when the primary human NFPA cells were treated with SZ-685C. Here, we show that SZ-685C induces apoptosis of human NFPA cells through inhibition of the Akt pathway in vitro. The understanding of apoptosis has provided the basis for novel targeted therapies that can induce death in cancer cells or sensitize them to established cytotoxic agents and radiation therapy.