Sample records for mercaptoethanol

  1. Stress lowers the detection threshold for foul-smelling 2-mercaptoethanol. (United States)

    Pacharra, Marlene; Schäper, Michael; Kleinbeck, Stefan; Blaszkewicz, Meinolf; Wolf, Oliver T; van Thriel, Christoph


    Previous studies have reported enhanced vigilance for threat-related information in response to acute stress. While it is known that acute stress modulates sensory systems in humans, its impact on olfaction and the olfactory detection of potential threats is less clear. Two psychophysical experiments examined, if acute stress lowers the detection threshold for foul-smelling 2-mercaptoethanol. Participants in Experiment 1 (N = 30) and Experiment 2 (N = 32) were randomly allocated to a control group or a stress group. Participants in the stress group underwent a purely psychosocial stressor (public mental arithmetic) in Experiment 1 and a stressor that combined a physically demanding task with social-evaluative threat in Experiment 2 (socially evaluated cold-pressor test). In both experiments, olfactory detection thresholds were repeatedly assessed by means of dynamic dilution olfactometry. Each threshold measurement consisted of three trials conducted using an ascending method of limits. Participants in the stress groups showed the expected changes in heart rate, salivary cortisol, and mood measures in response to stress. About 20 min after the stressor, participants in the stress groups could detect 2-mercaptoethanol at a lower concentration than participants in the corresponding control groups. Our results show that acute stress lowers the detection threshold for a malodor.

  2. Iodine-catalyzed addition of 2-mercaptoethanol to chalcone derivatives: Synthesis of the novel β-mercapto carbonyl compounds

    Directory of Open Access Journals (Sweden)

    Gürkan Yerli


    Full Text Available In this study, a series of novel β-mercapto carbonyl derivatives (3-(2-hydroxyethylthio-1,3-diarylpropan-1-one (5a-i were prepared by addition of 2-mercaptoethanol (4 to chalcones (3a-i in the presence of catalytic amount of iodine (10 mol % in CH 2Cl 2.

  3. Self assembled film based on hexane-1,6-diamine and 2-mercapto-ethanol on copper (United States)

    Tüken, Tunç; Kıcır, Nur; Elalan, N. Tuğba; Sığırcık, Gökmen; Erbil, Mehmet


    The inhibition efficiencies of hexane-1,6-diamine (HMDA), 2-mercapto-ethanol (ME) and a mixture of these two compounds have been investigated against copper corrosion, in acidic chloride solution. Electrochemical impedance spectroscopy (EIS), scanning electron microscopy (SEM), energy-dispersive X-ray (EDX), atomic force microscopy (AFM), Fourier transformed infrared (FT-IR) and atomic absorption spectroscopy (AAS) measurements were utilized to study the inhibition mechanism. ME was shown to have higher efficiency than HMDA, also their mixture could exhibit extra ordinary protection efficiency. It was shown that this excellent efficiency is a result of self-assembled film on the surface. The surface analysis showed that the film has high surface roughness and its thickness can get higher than 0.50 μm. After 7 days exposure time to corrosive environment, the corrosion current (icorr) values were calculated and it was shown that self assembled film had 98.22% protection efficiency.

  4. Comparison of Brucella immunoglobulin M and G flow assays with serum agglutination and 2-mercaptoethanol tests in the diagnosis of brucellosis

    NARCIS (Netherlands)

    Zeytinoğlu, Ayşin; Turhan, Ajda; Altuğlu, Imre; Bilgiç, Altinay; Abdoel, Theresia H.; Smits, Henk L.


    The diagnostic value of Brucella IgM/IgG flow assays was evaluated in comparison with serum agglutination and 2-mercaptoethanol tests by testing a selection of serum samples submitted to the laboratory because of clinical suspicion of brucellosis. All 39 admission and 11 follow-up samples that

  5. Synthesis, Structural, Viscosimetric, And Rheological Study, of A New Trifunctional Phosphorus Epoxyde Prepolymer , Tri-Glycidyl Ether Tri-Mercaptoethanol Of Phosphore (TGETMEP

    Directory of Open Access Journals (Sweden)

    Atiqa Bekhta


    Full Text Available The aim of our work is to synthesize a new phosphorus tri-functional epoxy resin tri-glycidyl ether tri-mercaptoethanol of phosphorus (TGETMEP in two stages. In the first stage, we got the precursor molecule of the epoxy matrix tri-mercaptoethanol phosphate (METR. The second step led us to the synthesis of tri-functional resin TMEP condensation with epichlorohydrin. The standard TGETMEP resin was characterized by the Fourier infrared transformation (FTIR and nuclear magnetic resonance (NMR, on one hand. The viscosimetric analysis was investigated by the Hebbelod capillary viscometer and rheometer Rheomat 01, on the other hand. The cross-linked resins and tertiary formulated composites have been studied by the rheometer 01 which was observed by means of SEM.

  6. A novel regiospecific cascade synthesis of sulfonamide derivatives from N-(2-polychloroethyl)sulfonamides via chloroaziridine intermediates in the presence of mercaptoethanol. (United States)

    Rozentsveig, Igor B; Popov, Aleksandr V; Rozentsveig, Gulnur N; Serykh, Valeriy Yu; Chernyshev, Kirill A; Krivdin, Leonid B; Levkovskaya, Galina G


    N-(1-Aryl-2-polychloroethyl)arenesulfonamides obtained on the basis of N,N-dichlorosulfoamides and polychloroethenes or phenylacetylene undergo a reaction cascade in the presence of mercaptoethanol. The reaction cascade opens a new route to the series of cyclic or open-chain sulfonamide derivatives. The process includes cyclization to aziridine intermediates, their further recyclization, and isomerization to imidoylchlorides or chloroimines, followed by substitution or reduction under the action of mercaptoethanol or hydrolysis. The final sulfonamide structures depend on the starting N-(polychloroethyl)sulfonamides. N-(2,2-Dichloroethyl)sulfonamides were transformed into sulfonamide-containing 1,4-oxathians while N-(2,2,2-trichloroethyl)sulfonamides were converted to N-(2-arylacetyl)arenesulfonamides. N-(2-Phenyl-2,2-dichloroethyl)sulfonamides form enamide derivatives that were transformed into aromatic ketones.

  7. Bare eye detection of Hg(II) ions based on enzyme inhibition and using mercaptoethanol as a reagent to improve selectivity. (United States)

    He, Liuying; Lu, Yuexiang; Wang, Feiyang; Gao, Xinxin; Chen, Ying; Liu, Yueying


    The authors describe a colorimetric method for the determination of Hg 2+ ions based on the inhibition of the activity of the enzyme urease. The pH value of solution increases when urease hydrolyzes urea, which can be visualized by adding a pH indicator such as Phenol Red (PhR). Mercaptoethanol as a typical thiol is added to the system to improve selectivity because it binds metal ions and then - unlike the Hg 2+ mercaptoethanol complex - does not inhibit urease. Hence, the color of the pH indicator PhR turns from yellow to pink as the solution becomes alkaline. The Hg 2+ mercaptoethanol complex, in contrast, strongly inhibits urease and the color of the solution remains yellow. The findings were used to design a photometric assay based on the measurement of the ratio of absorptions of PhR at 558 nm and 430 nm. It has a linear response over the 25 to 40 nM Hg 2+ concentration range and a 5 nM detection limit. This is well below the guideline values of Hg 2+ specified by the US Environmental Protection Agency and the World Health Organization for drinking water (10 nM and 30 nM, respectively). The method was employed to the determination of Hg 2+ in water samples spiked with 10 nM levels of Hg 2+ where color changes still can be observed visually. Graphical Abstract Schematic presentation of a colorimetric method for the ultrasensitive detection of Hg 2+ based on the inhibition of urease activity. Mercaptoethanol is used to improve the selectivity. Even at Hg 2+ concentrations as low as 5 nM, the color change still can be easily observed by bare eyes.

  8. The Effect of Insulin-like Growth Factor-1, Cysteamine and β-Mercaptoethanol on the In Vitro Maturation of Immature Mice Oocytes

    Directory of Open Access Journals (Sweden)

    A Dehghan Manshadi


    Full Text Available Background & aim: In vitro maturation of oocytes is a promising technique for reducing the costs and complications of ovarian stimulation by gonadotropins. The aim of this study was to investigate the effects of combination of insulin-like growth factor-1 and antioxidant cysteamine and &beta-Mercaptoethanol on maturation and fertilization of immature oocytes. Methods: in this experimental study, following 48 hrs injection of 7.5 IU PMSG to immature female mice, the germinal vesicle oocytes from ovaries were removed and transferred to TCM199 culture medium containing 50 ng /ml insulin-like growth factor-1 and 100 &mumol Cysteamine and &beta -Mercaptoethanol. After 24 hrs of culture, the oocytes of MII in IVF were fertilized and embryonic development to the two cells was studied under an inverted microscope. Data analysis was performed by using ANOVA and Post hoc Tukey test. Results: The results showed that the rate of maturation, fertilization and 2-cell embryo formation in GV oocytes with cumulus cells in TCM199 medium containing insulin-like growth factor-1, Cysteamine and BME were 92.10, 93.30, 80.60% and in the GV oocytes without Cumulus cells were cultured in the same medium were 65.80, 64.00, 58.60% respectively which were statistically significant (P <0.001. Conclusion: In the present study, the simultaneous combination of insulin-like growth factor-1, &beta-Mercaptoethanol and CYS increased maturation, fertilization and developmental rate to 2-cells stage with cumulus cells more than the oocyte without cumulus cells to a greater extent. This represented the need of adding supplemental growth factors and antioxidants to the medium and is associated with cumulus cells.

  9. Porous hydrogel of wool keratin prepared by a novel method: An extraction with guanidine/2-mercaptoethanol solution followed by a dialysis

    Energy Technology Data Exchange (ETDEWEB)

    Ozaki, Yuki; Takagi, Yusuke; Mori, Hideki; Hara, Masayuki, E-mail:


    In this study, we show a novel simple method to prepare a sponge-like porous keratin hydrogel through the extraction of wool keratin in a solution containing guanidine hydrochloride and 2-mercaptoethanol followed by dialysis for both aggregation of keratin and recrosslink. The gel had a highly porous structure and a fast-swelling property in rehydration after freeze-drying. It had also high mechanical strength both in the tensile test and the measurement of dynamic viscoelasticity. Three types of animal cells, PC12 cells, HOS cells and murine embryonic fibroblasts, well attached and grew on the surface of the porous hydrogel. - Graphical abstract: We show a novel simple method to prepare a sponge-like porous keratin hydrogel (A, B) through the extraction of wool keratin in a solution containing guanidine hydrochloride and 2-mercaptoethanol followed by dialysis for both aggregation of keratin and recrosslink. The gel had a highly porous structure (B) and a fast-swelling property in rehydration after freeze-drying. It had also high mechanical strength both in the tensile test (C) and the measurement of dynamic viscoelasticity (D). Three types of animal cells, PC12 cells (E), HOS cells (F) and murine embryonic fibroblasts (MEFs) (G), well attached and grew on the surface of the porous hydrogel. - Highlights: • We prepared a sponge-like porous keratin hydrogel by a novel method. • We used guanidine with 2-mercaptoethanol to extract keratin from wool fiber. • Extracted keratin was recrosslinked to form a porous keratin hydrogel in dialysis. • The keratin hydrogel had a high mechanical strength. • Three types of cells attached on the keratin hydrogel proliferated well.

  10. A microculture system for the measurement of antigen-induced murine lymphocyte proliferation: advantages of 5% horse serum and 5 X 10(-5) M mercaptoethanol. (United States)

    Brummer, E; Vris, T W; Lawrence, H S


    Short term microculture systems which measure murine lymphocyte proliferative responses to mitogens are well established. We demonstrate here that these microculture methods are not suitable for antigen-induced responses because of the high levels of murine lymphocyte proliferation in control cultures associated with the use of fetal calf serum or human serum. We also show that this problem can be eliminated with the use of a combination of 5% horse serum and 5 X 10(-5) M mercaptoethanol. We describe an antigen-induced murine lymphocyte proliferation microculture system in which good stimulation indices are achieved and the lymphocyte proliferation in control cultures remain at a low level throughout the 7 day culture period.

  11. Mercaptoethanol capped CdSe quantum dots and CdSe/ZnS core/shell: synthesis, characterization and cytotoxicity evaluation. (United States)

    Painuly, Diksha; Bhatt, Anugya; Krishnan, V Kalliyana


    CdSe Quantum dots (Q-dots) and CdSe/ZnS core/shell have been synthesized by wet chemical route using mercaptoethanol (ME) as cappant. The synthesized Q-dots and core/shell were characterized using X-ray diffraction (XRD), Transmission electron microscopy (TEM), Energy dispersive X-ray analysis (EDS), Dynamic Light Scattering (DLS), Optical absorption and luminescence spectroscopy. The core/shell formation was confirmed by both XRD and TEM analysis. The luminescence was shown to be considerably enhanced in the core/shell sample. Effect of dialysis process on the optical properties of the Q-dots and core/shell has also been discussed. Cytotoxicity studies have been carried out for Q-dots and core/shell. CdSe/ZnS core/shell was found to be non-cytotoxic as compared to CdSe Q-dots up to a certain concentration range. Polyethylene glycol (PEG) coating enhances the non-cytotoxic nature of CdSe/ZnS core/shell when compared with bare core/shell.

  12. Effect of B-mercaptoethanol on the viability of IVM/IVF/IVC bovine embryos during long-distance transportation in plastic straws. (United States)

    Takahashi, H; Kuwayama, M; Hamano, S; Takahashi, M; Okano, A; Kadokawa, H; Kariya, T; Nagai, T


    Experiments were conducted to assess the effect of beta-mercaptoethanol (beta-ME) on the quality and viability of bovine blastocysts derived from in-vitro culture (IVC) of in-vitro matured and fertilized (TVM-IVF) oocytes during their transport between 2 distant places. Follicular oocytes were collected from ovaries obtained at a slaughterhouse and were cultured for 20 to 21 h in modified TCM-199. The IVM oocytes were fertilized in vitro with frozen-thawed spermatozoa. Fertilized oocytes were cultured for 7 d, and embryos that developed to the blastocyst stage were used for the experiments. The blastocysts, packed in straws with transportation medium that consisted of modified TCM-199 with HEPES equilibrated in air and supplemented with 20 % calf serum and 0, 10, 50, 100 or 150 microM beta-ME, were transported at 37 degrees C from Tokyo to Sapporo by air (18.3 h). The quality of blastocysts was assessed and ranked as excellent (A), good (B), fair (C) or poor (D) after transportation. The percentages of blastocysts ranked as A or B were significantly higher (P plastic straws for several hours without control of CO2 and that the concentration of beta-ME used in this experiment is not detrimental to the blastocysts.

  13. Effect of beta-mercaptoethanol or epidermal growth factor supplementation on in vitro maturation of canine oocytes collected from dogs with different stages of the estrus cycle. (United States)

    Kim, Min Kyu; Fibrianto, Yuda Heru; Oh, Hyun Ju; Jang, Goo; Kim, Hye Jin; Lee, Kyu Seung; Kang, Sung Keun; Lee, Byeong Chun; Hwang, Woo Suk


    Supplementation of beta-mercaptoethanol (beta-ME) in in vitro maturation (IVM) medium was shown to improve embryo development and quality in several species. Epidermal growth factor (EGF) was also shown to improve IVM of human oocyte and embryo development after in vitro fertilization (IVF). The effect of these two compounds were suggested to be mediated through the synthesis of glutathione (GSH) which is known to play an important role in protecting the cell or embryos from oxidative damage. Thus, it is suggested that supplementation of canine IVM medium with beta-ME or EGF may be of benefit due to its positive role in IVM of various mammalian oocytes and embryo development, including cattle, pigs, rodents and humans. This study investigates the effect of ovarian estrus stage on canine oocyte quality and supplementation of medium with beta-ME or EGF on IVM of canine oocytes. As results, a significantly higher percentage of oocytes progressed to metaphase II (MII) stage in 50 or 100 microM of beta-ME supplemented oocytes collected from the follicular stage. The maturation rate to metaphase I (MI) stage was also significantly higher in oocytes collected from follicular stage and cultured with 25 or 100 microM compared to other experimental groups. After IVM culture, oocytes recovered from dogs with the follicular stage and matured in TCM-199 supplemented with 20 ng/ml EGF yielded better oocyte maturation to MII phase compared to other groups. Taken together, supplementation of beta-ME (50 or 100 microM) or EGF (20 ng/ml) improved IVM of canine oocytes to MII stage.

  14. A Comparative Study of the Radical-scavenging Activity of the Phenolcarboxylic Acids Caffeic Acid, p-Coumaric Acid, Chlorogenic Acid and Ferulic Acid, With or Without 2-Mercaptoethanol, a Thiol, Using the Induction Period Method

    Directory of Open Access Journals (Sweden)

    Seiichiro Fujisawa


    Full Text Available Phenolcarboxylic acid antioxidants do not act in vivo as radical-scavengers in isolation, but rather together with GSH (glutathione, a coantioxidant, they constitute an intricate antioxidant network. Caffeic acid, p-coumaric acid, ferulic acid and chlorogenic acid with or without 2-mercaptoethanol (ME, as a substitute for GSH, was investigated by the induction period (IP method for polymerization of methyl methacrylate (MMA initiated by thermal decomposition of 2,2'-azobisisobutyronitrile (AIBN, a source of alkyl radicals, R. and benzoyl peroxide (BPO, a source of peroxy radicals, PhCOO. using differential scanning calorimetry (DSC. Upon PhCOO. radical scavenging, the stoichiometric factors (n, number of free radical trapped by one mole of antioxidant for caffeic acid, ferulic acid, p-coumaric acid and chlorogenic acid were 2.4, 1.8, 1.7 and 0.9, whereas upon R. radical scavenging, the corresponding values were 1.3, 1.2, 1.0 and 0.8, respectively. Antioxidants with n values close to 2 suggest the stepwise formation of semiquinone radicals and quinones. By contrast, those with n values close to 1 suggest the formation of dimers after single-electron oxidation, possibly due to recombination of corresponding aryloxy radicals. The ratio of the rate constant of inhibition to that of propagation (kinh/kp declined in the order chlorogenic acid > p-coumaric acid > ferulic acid > caffeic acid. The ratio of the observed IP for the phenolcarboxylic acid/2-mercapto-ethanol (ME mixture (1:1 molar ratio (A to the calculated IP (the simple sum of phenol acid antioxidant and ME (B was investigated. Upon R. scavenging, the caffeic acid or p-coumaric acid/ME mixture was A/B > 1, particularly the former was 1.2, suggesting a synergic effect. By contrast, upon PhCOO. scavenging, the corresponding mixture was A/B < 1, particularly the latter was 0.7, suggesting an antagonistic effect. Upon both radicals scavenging, the A/B for the ferulic acid or chlorogenic acid

  15. A new vapor generation system for mercury species based on the UV irradiation of mercaptoethanol used in the determination of total and methyl mercury in environmental and biological samples by atomic fluorescence spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Yin, Yanmin; Qiu, Jianhua; Yang, Limin [College of Chemistry and Chemical Engineering, Xiamen University, Department of Chemistry and the MOE Key Laboratory of Analytical Sciences, Xiamen (China); Wang, Qiuquan [College of Chemistry and Chemical Engineering, Xiamen University, Department of Chemistry and the MOE Key Laboratory of Analytical Sciences, Xiamen (China); Xiamen University, State Key Laboratory of Marine Environmental Science, Xiamen (China)


    A new vapor generation system for mercury (Hg) species based on the irradiation of mercaptoethanol (ME) with UV was developed to provide an effective sample introduction unit for atomic fluorescence spectrometry (AFS). Preliminary investigations of the mechanism of this novel vapor generation system were based on GC-MS and FT-IR studies. Under optimum conditions, the limits of determination for inorganic divalence mercury and methyl mercury were 60 and 50 pg mL{sup -1}, respectively. Certified reference materials (BCR 463 tuna fish and BCR 580 estuarine sediment) were used to validate this new method, and the results agreed well with certified values. This new system provides an attractive alternative method of chemical vapor generation (CVG) of mercury species compared to other developed CVG systems (for example, the traditional KBH{sub 4}/NaOH-acid system). To our knowledge, this is the first systematic report on UV/ME-based Hg species vapor generation and the determination of total and methyl Hg in environmental and biological samples using UV/ME-AFS. (orig.)

  16. Toxic material advisory report - 2-mercaptoethanol

    Energy Technology Data Exchange (ETDEWEB)

    Bernholc, N. M.; White, O. Jr.; Baloyi, R. S.; Silverstein, B. D.


    A review of the animal toxicity data for 2-ME is presented. The results revealed that chronic inhalation exposures at a concentration of 6 mg/m/sup 3/ produced decreased oxygen consumption, lymphopenia, and neutrophilia. Comparison of acute toxicity data for 2-ME with data of structurally similar compounds suggests that 2-ME may be 2.3 times more toxic than butanethiol (TLV = 0.5 ppM), 6.5 times more toxic than ethanethiol, and 6 times more toxic than propanethiol (TLV = 0.5 ppM) via oral administration but may be comparable to propanethiol and less toxic than butanethiol and ethanethiol by the inhalation route of exposure. The TLVs for ethanethiol, methanethiol, and butanethiol were based on discomfort to human volunteers rather than toxicity. Since 2-ME has many effects similar to those of the thiols discussed and its odor threshold falls in the range of other thiols, by analogy the exposure limit for 2-ME should be comparable to the TLVs for butanethiol and ethanethiol. An interim exposure limit (IEL) of 0.5 ppM for a time-weighted average concentration during an 8-hour work shift is recommended. As with other thiols, a nuisance problem due to 2-ME odors and complaints of odor may serve as a primary reason for controlling workplace concentrations.

  17. The use of new surface-modified poly(2-hydroxyethyl methacrylate) hydrogels in tissue engineering: Treatment of the surface with fibronectin subunits versus Ac-CGGASIKVAVS-OH, cysteine, and 2-mercaptoethanol modification

    Czech Academy of Sciences Publication Activity Database

    Kubinová, Šárka; Horák, Daniel; Vaněček, Václav; Plichta, Zdeněk; Proks, Vladimír; Syková, Eva


    Roč. 102, č. 7 (2014), s. 2315-2323 ISSN 1549-3296 R&D Projects: GA ČR GAP304/11/0731; GA ČR GAP304/11/0653; GA ČR GA13-00939S Institutional support: RVO:68378041 ; RVO:61389013 Keywords : 2-hydroxyethyl methacrylate * IKVAV (Ile-Lys-Val-Ala-Val) peptide * fibronectin Subject RIV: FH - Neurology; CD - Macromolecular Chemistry (UMCH-V) Impact factor: 3.369, year: 2014

  18. Stabilization of solutions of feather keratins by sodium dodecyl sulphate

    NARCIS (Netherlands)

    Schrooyen, P.M.M.; Dijkstra, Pieter J.; Oberthür, Radulf C.; Bantjes, A.; Bantjes, Adriaan; Feijen, Jan


    Feather keratins were extracted from chicken feathers with aqueous solutions of urea and 2-mercaptoethanol. After filtration of the insoluble residue, a feather keratin solution was obtained. Removal of 2-mercaptoethanol and urea by dialysis resulted in aggregation of the keratin polypeptide chains

  19. Spatial Position Control of CdS Nanoclusters using a Self-Assembled Diblock Copolymer Template

    National Research Council Canada - National Science Library

    Yeh, Siao-Wei; Wu, Tsung-Lun; Wei, Kung-Hwa


    ...s. For controlling the spatial position of nanoparticles with block copolymers, it was found that the mercapto-ethanol modified CdS nanoparticles have a preferential binding to the poly(ethylene oxide...


    Directory of Open Access Journals (Sweden)

    J. Mitra


    Full Text Available Antibodies against Brucella canis was detected in 5 out of 10 Labrador bitches tested for brucellasis by 2-mercapto-ethanol rapid slide agglutination Test and lateral flow immunechromatographic assay at Kolkata, West Bengal, India.

  1. Extraordinarily stable disulfide-linked homodimer of human growth hormone


    Grigorian, Alexei L.; Bustamante, Juan J.; Hernandez, Peter; Martinez, Andrew O.; Haro, Luis S.


    Although a 22-kDa human growth hormone (hGH) is the predicted protein product of the hGH-N gene, a pleiotropic collection of uncharacterized molecular weight and charge isoforms is also produced. Using chromatography and preparative SDS-PAGE under reducing conditions we isolated an unusually stable mercaptoethanol-resistant (MER) 45-kDa hGH. A 5-h incubation at 100°C in the presence of 2-mercaptoethanol was required to convert approximately 90% of MER-45-kDa hGH into a 22-kDa hGH. Other reduc...

  2. C.U. INY A G~ G. Grewe and H. Bisswanger3 Department of Food ...

    African Journals Online (AJOL)


    imponant enzymes employed in various biolechnological processes like food and detergent industries as well as in medicine. Their involvement in the life cycle of disease-causing organisms .... SDS-PAGE method was used. The purified enzyme protein was treated with 1% SOS and 1% 2- mercaptoethanol and subjected ...

  3. A microculture technique for rat lymphocyte transformation. (United States)

    Lindsay, V J; Allardyce, R A


    We report the development of an economical microculture technique suitable for measuring rat lymphocyte response to mitogens and in mixed lymphocyte reactions. The effects of varying culture conditions, i.e. source of serum, addition and concentration of 2-mercaptoethanol, mitogen concentrations, culture incubation times, absorption of serum, lymphocyte numbers and microtitre plate well shape are described.

  4. Inhibitory activity of a water-soluble morin derivative on phosphatase ...

    African Journals Online (AJOL)



    pH 7.5), 200 mM NaCl, 5% (v/v) glycerol, and 0.04%. (v/v) β-mercaptoethanol. After cell lysis by sonication, the His- tagged protein was ... (v/v) Triton X-100, and 4 µM 6,8-difluoro-4-methy-lumbelliferyl phosphate (DiFMUP).

  5. High-quality total RNA isolation from melon (Cucumis melo L. fruits rich in polysaccharides

    Directory of Open Access Journals (Sweden)

    Gabrielle Silveira de Campos


    Full Text Available Melon, a member of the family Cucurbitaceae, is the fourth most important fruit in the world market and, on a volume basis, is Brazil’s main fresh fruit export. Many molecular techniques used to understand the maturation of these fruits require high concentrations of highly purified RNA. However, melons are rich in polyphenolic compounds and polysaccharides, which interfere with RNA extraction. This study aimed to determine the most appropriate method for total RNA extraction from melon fruits. Six extraction buffers were tested: T1 guanidine thiocyanate/phenol/chloroform; T2 sodium azide/?-mercaptoethanol; T3 phenol/guanidine thiocyanate; T4 CTAB/PVP/?-mercaptoethanol; T5 SDS/sodium perchlorate/PVP/?-mercaptoethanol, and T6 sarkosyl/PVP/guanidine thiocyanate, using the AxyPrepTM Multisource Total RNA Miniprep Kit. The best method for extracting RNA from both mature and green fruit was based on the SDS/PVP/?-mercaptoethanol buffer, because it rapidly generated a high quality and quantity of material. In general, higher amounts of RNA were obtained from green than mature fruits, probably due to the lower concentration of polysaccharides and water. The purified material can be used as a template in molecular techniques, such as microarrays, RT-PCR, and in the construction of cDNA and RNA-seq data.

  6. Synthesis, characterization, photocatalytic and reusability studies of ...

    Indian Academy of Sciences (India)

    This paper presents results of a study on the structural and morphological properties of 2-mercaptoethanol (2-ME) capped ZnS nanoparticles (NPs). The photocatalytic and reusability study of the synthesized NPs to degrade dyes was also done. ZnS semiconductor NPs were synthesized via chemical precipitation route ...

  7. Improved stability of halohydrin dehalogenase from Agrobacterium radiobacter AD1 by replacement of cysteine residues

    NARCIS (Netherlands)

    Tang, Lixia; van Hylckama Vlieg, Johan E.T.; Lutje Spelberg, Jeffrey H.; Fraaije, Marco W.; Janssen, DB


    Halohydrin dehalogenase from Agrobacterium radiobacter AD1 is a homo-tetrameric protein containing three cysteines per 28 kDa subunit. Under oxidizing conditions the enzyme was found to be susceptible to inactivation which could be prevented by the addition of beta-mercaptoethanol or glycerol. Gel

  8. Genomic DNA isolation from Artemisia species grown in cold desert ...

    African Journals Online (AJOL)

    ... protocol to extract pure genomic DNA from different Artemisia species was tailored. The protocol was based on the CTAB method with slight modifications. In the study, 1.6 M NaCl, 2% cetyltrimethyl ammonium bromide (CTAB), 3% polyvinylpyrrolidone (PVP) and 0.5% β-mercaptoethanol was used in the extraction buffer.

  9. A simple, rapid and efficient method for the extraction of genomic ...

    African Journals Online (AJOL)

    Litchi chinensis Sonn.) was developed. This modified CTAB protocol include the use of 2 M NaCl, PVP, 5% mercaptoethanol and 80% ethanol in the extraction as well as reducing the centrifugation times during the separation and precipitation of ...

  10. to view fulltext PDF

    Indian Academy of Sciences (India)


    Jan 27, 2014 ... basic fibroblast growth factor; BM, bone marrow; BME, β-mercaptoethanol; ChAT, choline acetyltransferase; CTNF, ciliaryneurotrophic factor;. DA, dopaminergic; EGF, epithelium growth factor; ... ence and developmental biology to describe the cellular and molecular mechanisms by which complex nervous ...

  11. Evaluation of Brucella dipstick assay for the diagnosis of acute brucellosis

    NARCIS (Netherlands)

    Altuglu, Imre; Zeytinoğlu, Aysin; Bilgic, Altinay; Kamcioglu, Saniye; Karakartal, Guney; Smits, Henk


    The diagnostic value of the dipstick assay was evaluated by comparison with Rose Bengal (RB), serum aglutination tests (SAT) and 2 mercaptoethanol test (2-ME) on consecutive serum samples submitted because of suspicion of brucellosis. Serum samples of 232 patients with suspected brucellosis that

  12. The effects of counter-ion condensation and co-ion depletion upon the rates of chemical repair of poly(U) radicals by thiols

    International Nuclear Information System (INIS)

    Fahey, R.C.; Vojnovic, B.; Michael, B.D.


    Bimolecular rate constants for reactions of poly(U) radicals with a series of thiols of varying net charge (Z) were measured by pulse radiolysis with conductivity detection at low ionic strength. At pH 7 and 18 o C the values of k 2 (M -1 s -1 ) were: reduced glutathione (Z = -1), 3 ; 2-mercaptoethanol (Z =0), 1.8 x 10 5 ; cysteine (Z=0), 2.0 x 10 5 ; cysteamine (Z = +1), 4.1 x 10 7 . Values determined at pH4 were: 2-mercaptoethanol, 6.1 x 10 5 ; cysteamine 2.2 x 10 8 ; N-(2-mercaptoethyl)-1,3-diaminopropane (WR-1065, Z = +2), 4.6 x 10 8 . The variation in rate with structure could not reasonably be attributed to inherent reactivity differences in the thiols and was ascribed to inhomogeneous distributions of the thiols in solution resulting from electrostatic interactions. (Author)

  13. Technetium-99m pertechnetate - a tracer for radiolabelling antibody for inflammation detection

    International Nuclear Information System (INIS)

    Shaharuddin Mohd; Wan Hamirul Bahrin Wan Kamal; Shahrin A Hamid; Ang Woan Tze; Rosnani Hashim


    The polyclonal antibody, Human Immunoglobulin G (HlgG) was reduced by using 2-mercaptoethanol with molar ratio of 1000:1 (i.e. mercaptoethanol:antibody). The reduction of the antibody, was carried out for 30 minutes at room temperature. The reduced antibody was purified by using Sephadex G-25 fine column. The antibody kit for the detection of inflammation was prepared aseptically in Class 1 Laminar Flow cabinet. The kit passed the sterility test. Upon reconstitution of the antibody kit with sodium pertechnetate-99m ( 99m Tc) solution, the labelling efficiency obtained was more than 95%. This preparation was stable up to 24-hour stored at room temperature. Gamma camera scans showed the accumulation of technetium-99m labelled antibody ( 99m Tc-HIgG) at the turpentine-induced inflammation of female Sprague-Dawley rats. This indicated the possibility of using 99m Tc-HIgG for inflammation detection. (author)

  14. The influence of antioxidants in the thiyl radical induced lipid peroxidation and geometrical isomerization in micelles of linoleic acid. (United States)

    Tartaro Bujak, Ivana; Mihaljević, Branka; Ferreri, Carla; Chatgilialoglu, Chryssostomos


    The biomimetic model of micelles of linoleic acid containing 2-mercaptoethanol and the antioxidant was examined under gamma irradiation up to 400 Gy in aerobic or deoxygenated conditions where thiyl radicals are the main reactive species. Lipid peroxidation was retarded by ascorbic acid and α-tocopherol, whereas this process was strongly inhibited by resveratrol as effectively as the ascorbic acid/α-tocopherol mixture. Furthermore, antioxidants have a much stronger inhibitory effect on the peroxidation in the presence of 2-mercaptoethanol, and at the same time show protective properties of the double bond, decreasing the cis-trans isomerization. Under anaerobic conditions, cis-trans isomerization occurred and antioxidants efficiency increased along the series: resveratrol isomerizing activity and protection of the natural lipid geometry.

  15. Membrane-bound dd-carboxypeptidases from Bacillus megaterium KM. General properties, substrate specificity and sensitivity to penicillins, cephalosporins and peptide inhibitors of the activity at pH5 (United States)

    Diaz-Mauriño, Teresa; Nieto, Manuel; Perkins, Harold R.


    1. The membrane from Bacillus megaterium KM contained a dd-carboxypeptidase with optimum activity under the following conditions: pH5.2, bivalent cation, 3mm; ionic strength, 40mm; temperature, 35°C. It was inactivated by treatment with p-chloromercuribenzoate but was fairly insensitive to 2-mercaptoethanol. 2. The enzyme was inhibited by penicillins and cephalosporins. The inhibition of this enzyme was partially reversed on dialysis but 0.2m-2-mercaptoethanol could neither prevent nor reverse the inhibition. 3. The enzyme was extremely sensitive to changes in the configuration and size of the side chain of the C-terminal dipeptide of the substrate. An aliphatic side chain of a well-defined length and polarity was required in the residue that precedes the C-terminal dipeptide. 4. The enzyme was inhibited by a wide range of analogues of the peptidic portion of the natural substrate. PMID:4218954

  16. Prevalence of Brucella spp in humans

    Directory of Open Access Journals (Sweden)

    Catharina de Paula Oliveira Cavalcanti Soares


    Full Text Available Objective: to determine the seroprevalence of Brucella spp in humans.Method: this is an observational study, developed with 455 individuals between 18 and 64 years old, who use the Estratégia de Saúde da Família (Brazil's family health strategy. The serum samples of volunteers underwent buffered acid antigen tests, such as screening, agar gel immunodiffusion and slow seroagglutination test in tubes and 2-Mercaptoethanol.Results: among the samples, 1.98% has responded to buffered-acid antigen, 2.85% to agar gel immunodiffusion test and 1.54% to the slow seroagglutination tests on tubes/2-Mercaptoethanol. The prevalence of Brucella spp was 4.4%, represented by the last two tests.Conclusion: the results of this research suggest that the studied population is exposed to Brucella spp infection.

  17. Development of genomic tools for verification of hybrids and selfed ...

    African Journals Online (AJOL)

    ajl yemi


    Nov 30, 2011 ... Increased attention would go to the development of more efficient markers such as single nucleotide polymorphisms (SNPs) and other gene-based markers using .... [pH 8], 500 mM NaCl, 50mM EDTA, 1.0% SDS, 2% PVP, 1% β- mercaptoethanol, and 0.05 mg/ml Proteinase K. Fresh young leaves were ...

  18. Anti-t B toxopla Balsam asma g mocitru gondii us cam activit merune ...

    African Journals Online (AJOL)


    uninfected and infected non-treated cells. The plates were centrifuged at 500 g for 5 min. The cells were lysed in HEPES 100. mM pH 8, MgSO4 1 mM, Triton X 100 1 %, DTT 5 mM buffer for 1 h at 50°C. Lysis was observed by microscopy. The reaction buffer. (phosphate buffer 100 mM pH 7.3, β -mercaptoethanol 102 mM,.

  19. Isolation and partial characterization of a lectin from Bauhinia pentandra (bong) vog. Ex. Steua.




    Bauhinia pentandra (Bong) Vog. ex. Steua seeds were investigated with respect to phenologic aspects (size, mass, hilum and length) and with respect to their chemical composition. The total nitrogen content of the seed flour was determined, and the flour was extracted in different pH values. A lectin was isolated from the seeds by Sepharose-4B affinity chromatography. The homogeneity of the lectin was demonstrated by SDS-PAGE in the presence of beta-mercaptoethanol. Only one protein band with ...

  20. Inhibition of HIV-1 Integrase gene expression by 10-23 DNAzyme

    Indian Academy of Sciences (India)


    Jun 25, 2012 ... L-glutamine/mL, and 0.2% of sodium bicarbonate. For the growth of jurkat 50 μM β-mercaptoethanol was also added and all the cells were maintained at 37°C in 5% CO2 at 85–. 90% relative humidity. 2.6 Transient transfection of pIN-EGFP plasmid. The transient transfection of the constructed plasmid was.

  1. Stemcell Information: SKIP000628 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available Mercaptoethanol+5ng/ml human bFGF ... 5% Negative ... RIKEN BioResource Center 理化学研究所バイオリソ...ースセンター RIKEN BioResource Center 理化学研究所バイオリソースセンター Available RIKEN BioResource Center 理化学

  2. Stemcell Information: SKIP000238 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available 2-Mercaptoethanol+5ng/mL human bFGF ... Riken BioResource Center 理化学研究所バイオリソースセンター ...Riken BioResource Center 理化学研究所バイオリソースセンター Available Riken BioResource Center 理化学

  3. Labelling polypeptide with 99mTc and bioactivity get back

    International Nuclear Information System (INIS)

    Li Shaolin; Jiang Xiaofei; Pang Hua; Liu Fangxin; Shi Qiong


    A method for labelling polypeptide (insulin) with technetium-99 ( 99m Tc) was established without marked loss of biological activity. Following reduction of intrinsic disulfide bonds by mercaptoethanol and purification on a Sephadex G50 column, the polypeptide was labelled with 99m Tc by trans-chelation from methylene diphosphonate (MDP). 99m Tc labelled insulin was identified by thin layer chromatography (TLC) and the change of blood sugar of mice injected, their hypo-glycemic shock symptom was also observed. Six hours after labelling, the dissociation of labelled insulin was only 3%, From then on to 24 h, there was no more dissociation. The blood sugar concentration of mice injected with the mercaptoethanol-reduced insulin was (5.0 +- 3.2) μmol·L -1 , while those injected with the original insulin was (1.4 +- 1.2) μmol·L -1 , the difference was significant (Q test, p -1 for the labelled insulin, and was about the same with that for the original insulin. The labelling efficiency was 74.31% for the labelled insulin, whereas the original insulin cannot be labelled with 99m Tc. The result suggests that while disulfide bonds of polypeptide were reduced by mercaptoethanol, it became free sulfhydryl group, and its bioactivity descended. Then free sulfhydryl group was chelated with 99m Tc under mild condition, re-establishing the disulfide bond, therefore, the bioactivity came back. The 99m Tc-labelled insulin was stable during 24 h

  4. Novel ELISA for the detection of raw and processed egg using extraction buffer containing a surfactant and a reducing agent. (United States)

    Watanabe, Yumiko; Aburatani, Kenichi; Mizumura, Tasuku; Sakai, Masatoshi; Muraoka, Shiroo; Mamegosi, Shinichi; Honjoh, Tsutomu


    Enzyme-linked immunosorbent assay (ELISA) has been considered extremely useful for the detection of markers of allergenic substances in food, because it is simple, offers a suitable sensitivity, and is useful in providing quantitative results. Allergenic protein present in processed food can be denatured or altered, hindering therefore their possibility to be extracted and detected. This paper reports the development of an ELISA method that can be used for the determination of allergenic proteins in buffer solutions containing SDS, a surfactant, and 2-mercaptoethanol, a reducing agent. Measurement by ELISA in solutions containing 1% SDS and 7% 2-mercaptoethanol has been made possible by using an antibody prepared through immunization with an antigen denatured with SDS and 2-mercaptoethanol. This ELISA technique can be used to measure proteins in food that have been denatured by various manufacturing processes. An example is egg white albumin, which is susceptible to heat denaturation and has been difficult to recover from food in the past. Its recovery was improved 10- to 100-fold by the new ELISA method as compared with previous methods. This means that allergenic substances in food can now be detected quantitatively. This method can be very useful in allergy prevention and control strategies.

  5. Omeprazole, a specific inhibitor of gastric (H+-K+)-ATPase, is a H+-activated oxidizing agent of sulfhydryl groups

    International Nuclear Information System (INIS)

    Im, W.B.; Sih, J.C.; Blakeman, D.P.; McGrath, J.P.


    Omeprazole (5-methoxy-2-[[(4-methoxy-3,5- dimethylpyridinyl)methyl]sulfinyl]-1H-benzimidazole) appeared to inhibit gastric (H + -K + )-ATPase by oxidizing its essential sulfhydryl groups, since the gastric ATPase inactivated by the drug in vivo or in vitro recovered its K+-dependent ATP hydrolyzing activity upon incubation with mercaptoethanol. Biological reducing agents like cysteine or glutathione, however, were unable to reverse the inhibitory effect of omeprazole. Moreover, acidic environments enhanced the potency of omeprazole. The chemical reactivity of omeprazole with mercaptans is also consistent with the biological action of omeprazole. The N-sulfenylated compound reacted at neutral pH with another stoichiometric amount of ethyl mercaptan to produce omeprazole sulfide quantitatively. The gastric polypeptides of 100 kilodaltons representing (H + -K + )-ATPase in the rat gastric mucosa or isolated hog gastric membranes were covalently labeled with [ 14 C]omeprazole. The radioactive label bound to the ATPase, however, could not be displaced by mercaptoethanol under the identical conditions where the ATPase activity was fully restored. These observations suggest that the essential sulfhydryl groups which reacted with omeprazole did not form a stable covalent bond with the drug, but rather that they further reacted with adjacent sulfhydryl groups to form disulfides which could be reduced by mercaptoethanol

  6. Omeprazole, a specific inhibitor of gastric (H/sup +/-K/sup +/)-ATPase, is a H/sup +/-activated oxidizing agent of sulfhydryl groups

    Energy Technology Data Exchange (ETDEWEB)

    Im, W.B.; Sih, J.C.; Blakeman, D.P.; McGrath, J.P.


    Omeprazole (5-methoxy-2-(((4-methoxy-3,5- dimethylpyridinyl)methyl)sulfinyl)-1H-benzimidazole) appeared to inhibit gastric (H/sup +/-K/sup +/)-ATPase by oxidizing its essential sulfhydryl groups, since the gastric ATPase inactivated by the drug in vivo or in vitro recovered its K+-dependent ATP hydrolyzing activity upon incubation with mercaptoethanol. Biological reducing agents like cysteine or glutathione, however, were unable to reverse the inhibitory effect of omeprazole. Moreover, acidic environments enhanced the potency of omeprazole. The chemical reactivity of omeprazole with mercaptans is also consistent with the biological action of omeprazole. The N-sulfenylated compound reacted at neutral pH with another stoichiometric amount of ethyl mercaptan to produce omeprazole sulfide quantitatively. The gastric polypeptides of 100 kilodaltons representing (H/sup +/-K/sup +/)-ATPase in the rat gastric mucosa or isolated hog gastric membranes were covalently labeled with (/sup 14/C)omeprazole. The radioactive label bound to the ATPase, however, could not be displaced by mercaptoethanol under the identical conditions where the ATPase activity was fully restored. These observations suggest that the essential sulfhydryl groups which reacted with omeprazole did not form a stable covalent bond with the drug, but rather that they further reacted with adjacent sulfhydryl groups to form disulfides which could be reduced by mercaptoethanol.

  7. Effect of external conditions on the stability of enzymically synthesized carthamin

    Directory of Open Access Journals (Sweden)

    Koshi Saito


    Full Text Available Enzymically synthesized carthamin was subjected to testing of its stability under various conditions. It easily turned to unknown reddish-yellow or orange-yellow substances in water. This instability was further accelerated by the presence of a sulfhydryl reagent and or at higher temperature. In a typical result, the rate of the discolouration was calculated to be 21 nmol carthamin h-1 at 20 °C in water and rose to 44.0 nmol carthamin h-1 at 60 °C in water containing 1.0 mM 2-mercaptoethanol. Organic solvents such as methanol, ethanol and acetone showed little effect on carthamin discolouration, while the stability of carthamin in these solvents was gradually decreased by rising temperature. In various buffers, in the acidic range. carthamin was relatively stable, whereas, it turned unstable on the basic side. All of the thiol reagents tested were unfavourable for retaining stability of carthamin in solutions. 2-Mercaptoethanol, reduced glutathione and thioglycollic acid exhibited the strongest degrading effect on the pigment. At the 1.0 mM level, 2-mercaptoethanol reduced the absorption peak at 517 nm by 0.02 min-1, which corresponded to 0.12 nmol carthamin cm-3 min-1 L-Methionine, L-ascorbic and D-araboascorbic acids were found to be strong bathochromic effectors For example, L-methionine rised AA 517 nm up to 0.01 min-1, whose value equaled to 0-1 nmol carthamin cm-3 min-1 at 30 °C Carthamin was absorbed and adsorbed and stabilized by many macro-molecular Substances. Among the compounds tested, cellulose was found to be the most effective stabilizer. The stabilization effect of cellulose was specific for carthamin and the term "SAITO EFFECT" is proposed for it. This effect was reduced by hydrolysis of the carthamin-adsorbed cellulose with Asperqillus cellulase in an incubation system

  8. Expression analysis of four flower-specific promoters of Brassica spp ...

    African Journals Online (AJOL)



    Oct 19, 2009 ... protein extraction buffer (50 mM NaH2PO4, pH 7.0, 0.01% SDS,. 0.01 M EDTA, pH 8.0, 20% methanol, 0.1% Triton X-100 and 10. mM β-mercaptoethanol) and vortexed to homogeneity. The homogenate was centrifuged at 11,000 g for 15 min at 4oC and the supernatant was used for protein quantification ...

  9. Use of [75Se]selenomethionine in immunoglobulin biosynthetic studies

    International Nuclear Information System (INIS)

    Gutman, G.A.; Warner, N.L.; Harris, A.W.; Bowles, A.


    The gamma-emitting amino acid analog, [ 75 Se] selenomethionine, has been used as a biosynthetic label for immunoglobulins secreted by plasmacytomas in tissue culture. The secreted products are structurally intact with respect to their antibody combining sites and their class and allotype antigenic specificities. A component of [ 75 Se] selenomethionine preparations was found to bind to fetal calf serum proteins, in a manner releasable by mercaptoethanol, but not by sodium dodecyl sulfate and urea. Methods for circumventing the problems caused by this binding are described. (Auth.)

  10. Effect of Molar Concentration on Optical Absorption Spectra of ZnS:Mn Nanoparticles

    Directory of Open Access Journals (Sweden)

    Ravi Sharma


    Full Text Available The present paper reports the synthesis and characterization of luminescent nanocrystals of manganese doped zinc sulphide. Nanocrystals of zinc sulphide were prepared by chemical precipitation method using the solution of zinc chloride, sodium sulphide, manganese chloride and mercaptoethanol was used as the capping agent. It was found that change in the molar concentration changes the particle size. The particle size of such nanocrystals was measured using XRD pattern and it is found to be in between 3 nm – 5 nm. The blue-shift in absorption spectra was found with reducing size of the nanoparticles

  11. Milk protein polymorphisms in Brazilian Zebu cattle


    Silva, Ivana Tramontina da; Del Lama, Marco Antonio


    Five bovine milk protein polymorphisms were studied in Zebuine cattle raised in Brazil, through horizontal electrophoresis on starch gel containing urea and 2-mercaptoethanol, using basic and acidic buffer systems. Allelic frequencies for a-La, b-Lg, aS1-Cn, b-Cn and k-Cn loci were estimated in six Gyr herds (N = 283), six Guzerat herds (N = 205), one Nelore herd (N = 17) and one Sindi herd (N = 22), all from São Paulo or Minas Gerais State, Brazil. Genotypic frequencies observed for each loc...

  12. PIG-B: a homemade monophasic cocktail for the extraction of RNA. (United States)

    Weber, K; Bolander, M E; Sarkar, G


    An inexpensive monophasic reagent has been developed for the extraction of total RNA from cells or tissues. The main ingredients of the reagent are Phenol, Isoamyl alcohol, Guanidinium isothiocyanate, and Beta-mercaptoethanol (PIG-B). The quality and yield of RNA obtained by this reagent is at par with that obtained by TRIzol, an expensive but widely used monophasic reagent available commercially. The complete composition and method of preparation of PIG-B is provided to aid preparation of the reagent in the laboratory.

  13. The effect of solar irradiated Vibrio cholera on the secretion of pro-inflammatory cytokines and chemokines by the JAWS II dendritic cell line in vitro

    CSIR Research Space (South Africa)

    Ssemakalu, CC


    Full Text Available results from infection with pathogenic members of the species of a motile Gram-negative bacterium called Vibrio cholerae. Vibrio cholerae naturally exists within the aquatic environment [5]. The consumption of untreated environmental water contaminated... scientific (Waltham, MA); the rough form lipopoly- saccharide (LPS) from E. coli serotype J5 and the cholera toxin beta-subunit (CTB) were pur- chased from ENZO Life Sciences (Farmingdale, NY); 2-mercaptoethanol (2-ME) and 0.25% Trypsin-0.02% EDTA were...

  14. Improve T Cell Therapy in Neuroblastoma (United States)


    Walkersville, MD, USA), penicillin- streptomycin (BioWhittaker), and b-mercaptoethanol (50 mM) (Invitrogen, Carlsbad, CA, USA) and then activated with anti-CD3...acting hydrolase elements that mediate a ribosomal skip between 2A-linked genes,15 resulting in stoichiometric protein production16 that can improve...culture system shows that the viral- derived 2A-TAV and 2A-ERAV ribosomal skip sequences, and adjacent fusion protein regions exhibit very low (n¼ 5) to

  15. On the reaction of thiols with primary radiation lesions in bacteriophage

    International Nuclear Information System (INIS)

    Korystov, Yu.N.; Veksler, A.M.; Ehjdus, L.Kh.


    In order to answer the question in which form, RSH of RS-, thiols react with the target responsible for reproductive cell death a study was made of the pH dependence (4.7-8.2) of the radioprotective effect of mercaptoethanol and cysteamine on the bacteriophage T 4 under the anoxic conditions. It was shown that the protective effect does not depend upon pH. Since the concentration of RSH, within the studied range of pH values, remains virtually invariable, and RS - concentration sharply changes, the obtained results indicate that the RSH is the form in which thiols react with primary radiation damages to the phage [ru

  16. Chitinase production by Streptomyces viridificans: its potential in fungal cell wall lysis. (United States)

    Gupta, R; Saxena, R K; Chaturvedi, P; Virdi, J S


    Streptomyces viridificans was found to be a good chitinase producer among nine species of Streptomyces screened. Minimum levels of constitutive enzyme were observed with both simple and complex carbon substrate. Arabinose doubled the enzyme production amongst the various pentoses and hexoses used with chitin. However, with glucose end-product inhibition and catabolite repression were observed. The enzyme tolerated a wide range of temperature (30-55 degrees C) and pH (3-7.5). Among various divalent cations Mn2+ and Hg2+ completely inhibited the purified enzyme while beta-mercaptoethanol stimulated its activity. Crude and purified enzyme had potential for cell wall lysis of many fungal pathogens tested.

  17. Fluorimetric method of analysis for D-norpseudoephedrine hydrochloride, glycine and L-glutamic acid by reversed-phase high-performance liquid chromatography. (United States)

    Swanepoel, E; de Villiers, M M; du Preez, J L


    The determination of D-norpseudoephedrine HCl, an appetite suppressant, and glycine and L-glutamic acid, both dietary supplements, in pharmaceutical formulations and dissolution media using reversed-phase high-performance liquid chromatography (HPLC) combined with fluorimetric detection is reported. A reagent solution containing omicron-phthalaldehyde and a reducing agent, mercaptoethanol, appeared to be the most favourable reagent for derivatising the three compounds. The use of this HPLC method allowed for selective and quantitatively accurate analysis and was sufficiently specific, precise and sensitive for analytical characterisation.

  18. The influence of reducing agent and 1,10-phenanthroline concentration on DNA cleavage by phenanthroline + copper.


    Veal, J M; Merchant, K; Rill, R L


    Copper in the presence of excess 1,10-phenanthroline, a reducing agent, and molecular oxygen causes cleavage of DNA with a preference for T-3',5'-A-steps, particularly in TAT triplets. The active molecular species is commonly thought to be the bis-(1,10-phenanthroline)Cu(I) complex, (Phen)2Cu(I), regardless of the reducing agent type. We have found that (Phen)2Cu(I) is not the predominant copper complex when 3-mercaptopropionic acid (MPA) or 2-mercaptoethanol are used as the reducing agents, ...

  19. Download this PDF file

    African Journals Online (AJOL)


    buffers of different pH values were used for the pH determination: 5 mM citrate buffer (pH 6.0-6.5), 5 mM phosphate (pH 6.5-8.0), 5 mM Tris-HCl buffer (pH 8.0-. 11.0). Figure 7: Effect of 2-mercaptoethanol on the activity of thiaminase from gut of Z. variegatus. DISCUSSION. Edible insects constitute an important part of the.

  20. Aglutininas anti-Brucella abortus no soro e em secreção de bursite cervical em eqüinos

    Directory of Open Access Journals (Sweden)

    Ribeiro M.G.


    Full Text Available Fistulous wither secretions from three horses were tested by the plate agglutination (PAT, tube agglutination (SAT, buffered plate-Rose Bengal (RBPT and 2-mercaptoethanol (2-ME tests, comparatively with standard agglutination tests. In the modified tests, titers were increased in the PAT, SAT and 2-ME tests and positive reaction was observed in RBPT. Brucella abortus was isolated from the secretion of fistulous withers collected from one animal. These results suggest that the modified tests may be used as alternative tests to diagnose brucellosis in horses with fistulous withers.

  1. Stemcell Information: SKIP000221 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available mM 2-Mercaptoethanol+5ng/mL human bFGF ... Yes 46XY(20) ... Tsuyoshi Fujioka 藤岡 剛 RIKEN BRC 理化学...研究所バイオリソースセンター RIKEN BRC 理化学研究所バイオリソースセンター Yukio Nakamura 中村幸夫 Available RIKEN BRC 理化学研究所バイオリソースセンター http:/

  2. Stemcell Information: SKIP000223 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available 0.1mM NEAA+0.1mM 2-Mercaptoethanol+5ng/mL human bFGF ... 5% Negative ... RIKEN BioResource Center 理化学...研究所バイオリソースセンター RIKEN BioResource Center 理化学研究所バイオリソースセンター Available RIKEN BioResource Center 理化学研

  3. Growth hormone aggregates in the rat adenohypophysis (United States)

    Farrington, M.; Hymer, W. C.


    Although it has been known for some time that GH aggregates are contained within the rat anterior pituitary gland, the role that they might play in pituitary function is unknown. The present study examines this issue using the technique of Western blotting, which permitted visualization of 11 GH variants with apparent mol wt ranging from 14-88K. Electroelution of the higher mol wt variants from gels followed by their chemical reduction with beta-mercaptoethanol increased GH immunoassayability by about 5-fold. With the blot procedure we found 1) that GH aggregates greater than 44K were associated with a 40,000 x g sedimentable fraction; 2) that GH aggregates were not present in glands from thyroidectomized rats, but were in glands from the thyroidectomized rats injected with T4; 3) that GH aggregates were uniquely associated with a heavily granulated somatotroph subpopulation isolated by density gradient centrifugation; and 4) that high mol wt GH forms were released from the dense somatotrophs in culture, since treatment of the culture medium with beta-mercaptoethanol increased GH immunoassayability by about 5-fold. Taken together, the results show that high mol wt GH aggregates are contained in secretory granules of certain somatotrophs and are also released in aggregate form from these cells in vitro.

  4. The adenosine-5'-phosphosulfate sulfotransferase from spinach (Spinacea oleracea L.). Stabilization, partial purification, and properties. (United States)

    Schmidt, A


    Adenosine-5'-phosphosulfate (APS) sulfotransferase was purified 25-fold from spinach (Spinacea oleracea L.) leaves by Sephadex-G-200 gel filtration and chromatography on DEAE-cellulose. Enzyme activity was stabilized with 0.05 M Tris-HCl pH 8.0 containing 10 mM mercaptoethanol (ME), 10 mM MgCl2, and 30% glycerol. The molecular weight of the APS-sulfotransferase was estimated by gel filtration to be about 110,000 daltons. The enzyme is specific for the sulfonucleotide APS; PAPS is not a sulfur donor for this reaction. The apparent Km for APS was found to be 13 μM. The enzyme activity was determined with dithioerythritol (DTE) as acceptor, which has an apparent Km of 0.6 mM. Glutathione can substitute for DTE; other thiols such as mercaptoethanol and cysteine are less effective. The APS-sulfotransferase activity is inhibited by 5'-AMP, which increases the Km for APS but does not change Vmax, suggesting a competetive inhibition. Reduced methylviologen cannot substitute for a thiol in the spinach enzyme system. Thus it seems that assimilatory APS-sulfotransferase from spinach is different from the dissimilatory APS-reductase from Desulfovibrio or Thiobacillus, where methylviologen can be used as the electron donor.

  5. A computer program for quantification of SH groups generated after reduction of monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Escobar, Normando Iznaga; Morales, Alejo; Nunez, Gilda


    Reduction of disulfide bonds to sulfhydryl (SH) groups for direct radiolabeling of antibodies for immunoscintigraphic studies of colorectal and other cancers continues to be of considerable research interest. We have developed a general strategy and a versatile computer program for the quantification of the number of SH per molecule of antibody (Ab) generated after the treatment of monoclonal antibodies (MAbs) with reducing agents such as 2-mercaptoethanol (2-ME), stannous chloride (SnCl{sub 2}), dithiothreitol (DTT), dithioerythritol (DTE), ascorbic acid (AA), and the like. The program we describe here performs an unweighted least-squares regression analysis of the cysteine standard curve and interpolates the cysteine concentration of the samples. The number of SH groups per molecule of antibody in the 2-mercaptoethanol and in the other reducing agents was calculated from the cysteine standard curve using Ellman's reagent to develop the yellow color. The linear least-squares method fit the standard data with a high degree of accuracy and with the correlation coefficient r of 0.999. A program has been written for the IBM PC compatible computer utilizing a friendly menu to interact with the users. The package allows the user to change parameters of the assay, to calculate regression coefficients slope, intercept and its standard errors, to perform statistical analysis, together with detailed analysis of variance, and to produce an output of the results in a printed format.

  6. An inexpensive and rapid method for extracting papilionoid genomic DNA from herbarium specimens. (United States)

    Riahi, M; Zarre, S; Maassoumi, A A; Attar, F; Kazempour Osaloo, S


    Three DNA extraction protocols were compared for their ability to yield DNA from the leaves of herbarium specimens of nine species from nine genera of the Papilionoideae. We tested two protocols that use classic procedures for lysis and purification with cetyl trimethylammonium bromide (CTAB); a third protocol used a Nucleospin Plant kit. DNA obtained from all three procedures was quantified and tested by PCR. Test results indicated the superiority of one of the CTAB protocols. We made some modifications, developing a protocol that produced high-quality DNA from all nine species. The modification involved the use of a lower EDTA concentration (20 mM instead of 50 mM) and a higher beta-mercaptoethanol concentration (1% instead of 0.4%) in the extraction buffer. The modified protocol avoids the necessity for a second DNA precipitation step. This new CTAB protocol includes the use of 1.4 M NaCl, 20 mM EDTA and 1% beta-mercaptoethanol in the extraction; DNA precipitation time is reduced. A reduction in contaminating metabolites (such as PCR inhibitors) in the sample mixtures and lower costs for reagents are characteristics of this modified protocol; the cost of analysis per sample was lowered, compared to previous options. The quality of DNA was suitable for PCR amplification. This is a practical alternative to more difficult, time-consuming and expensive protocols.

  7. Immunochemical recognition of the binding of C-reactive protein to solid-phase phosphorylethanolamine. (United States)

    Coll, J M


    The influence of polyclonal and monoclonal antibodies, trypsin digestion and mercaptoethanol treatment of C-reactive protein (CRP) in the CRP binding to solid-phase phosphorylethanolamine (PE) has been investigated. Nine monoclonal antibodies reacting with CRP could be divided into at least 2 well-defined groups: one group of 6-7 monoclonals interfering with the binding of CRP to PE (mainly represented by monoclonal 2) and the not interfering with the binding of CRP to PE (mainly represented by monoclonal 5). Trypsin digestion resulted in sequence identified CRP fragments still able to bind to PE and detectable by monoclonal 5 but not by monoclonal 2. On the other hand, binding of CRP to PE was abolished by mercaptoethanol treatment. These results, together with the estimation of the extent of the antigenicity of the PE binding site and the characteristics of the hydrophobicity profile of CRP, suggest that most of the hydrophilic sequences contribute to the PE binding region except a non-overlapping region defined by monoclonal 5. Most probably, some of these sequences are located inside or around the internal bisulphide bridge of each monomer of the pentameric CRP.

  8. Promoting effect of small molecules in cardiomyogenic and neurogenic differentiation of rat bone marrow-derived mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Khanabdali R


    Full Text Available Ramin Khanabdali,1 Anbarieh Saadat,1 Maizatul Fazilah,1 Khairul Fidaa’ Khairul Bazli,1 Rida-e-Maria Qazi,2 Ramla Sana Khalid,2 Durriyyah Sharifah Hasan Adli,1 Soheil Zorofchian Moghadamtousi,1 Nadia Naeem,2 Irfan Khan,2 Asmat Salim,2 ShamsulAzlin Ahmad Shamsuddin,1 Gokula Mohan1 1Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia; 2Dr Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan Abstract: Small molecules, growth factors, and cytokines have been used to induce differentiation of stem cells into different lineages. Similarly, demethylating agents can trigger differentiation in adult stem cells. Here, we investigated the in vitro differentiation of rat bone marrow mesenchymal stem cells (MSCs into cardiomyocytes by a demethylating agent, zebularine, as well as neuronal-like cells by β-mercaptoethanol in a growth factor or cytokines-free media. Isolated bone marrow-derived MSCs cultured in Dulbecco’s Modified Eagle’s Medium exhibited a fibroblast-like morphology. These cells expressed positive markers for CD29, CD44, and CD117 and were negative for CD34 and CD45. After treatment with 1 µM zebularine for 24 hours, the MSCs formed myotube-like structures after 10 days in culture. Expression of cardiac-specific genes showed that treated MSCs expressed significantly higher levels of cardiac troponin-T, Nkx2.5, and GATA-4 compared with untreated cells. Immunocytochemical analysis showed that differentiated cells also expressed cardiac proteins, GATA-4, Nkx 2.5, and cardiac troponin-T. For neuronal differentiation, MSCs were treated with 1 and 10 mM β-mercaptoethanol overnight for 3 hours in complete and serum-free Dulbecco’s Modified Eagle’s Medium, respectively. Following overnight treatment, neuron-like cells with axonal and dendritic-like projections originating from the

  9. Prevalence of antibodies to Japanese encephalitis virus among pigs in Bali and East Java, Indonesia, 2008. (United States)

    Yamanaka, Atsushi; Mulyatno, Kris Cahyo; Susilowati, Helen; Hendrianto, Eryk; Utsumi, Takako; Amin, Mochamad; Lusida, Maria Inge; Soegijanto, Soegeng; Konishi, Eiji


    Japanese encephalitis virus (JEV) is a fatal disease in Asia. Pigs are considered to be the effective amplifying host for JEV in the peridomestic environment. Bali Island and Java Island in Indonesia provide a model to assess the effect of pigs on JEV transmission, since the pig density is nearly 100-fold higher in Bali than Java, while the geographic and climatologic environments are equivalent in these areas. We surveyed antibodies to JEV among 123 pigs in Mengwi (Bali) and 96 pigs in Tulungagung (East Java) in 2008 by the hemagglutination-inhibition (HAI) test. Overall prevalences were 49% in Bali and 6% in Java, with a significant difference between them (P Java. In addition, 2-mercaptoethanol-sensitive antibodies were found only from Bali samples. Further, the average HAI antibody titer obtained from positive samples was significantly higher in Bali (1:52) than Java (1:10; P Java.

  10. Changes in protein characteristics during soybean storage under adverse conditions as related to tofu making. (United States)

    Kong, Fanbin; Chang, Sam K C


    Soybeans stored under adverse conditions decrease in protein recovery (content) in the soymilk and tofu yield. This study investigated how protein structural changes contributed to the decrease in tofu yield. Soymilks were produced from original soybeans (Proto and IA2032 cultivars) and adversely stored soybeans, respectively, and soymilk protein contents were adjusted to the same level before making into tofu. Tofu yield was compared with that made from soybeans without protein content adjustment. For understanding protein structural changes, soy proteins were extracted from Proto soybean by using different solvents, including distilled water, sodium dodecyl sulfate (SDS), and 2-mercaptoethanol. The proteins in the extracts were analyzed by using SDS-PAGE and gel filtration. Results showed that tofu yield was more significantly affected by protein structural characteristics than the protein content in soymilk. Different levels of aggregations among 7S and 11S proteins during adverse storage were responsible for decreasing protein recovery in the soymilk.

  11. End-to-end Structural Restriction of α-Synuclein and Its Influence on Amyloid Fibril Formation

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Chul Suk; Park, Jae Hyung; Choe, Young Jun; Paik, Seung R. [Seoul National University, Seoul (Korea, Republic of)


    Relationship between molecular freedom of amyloidogenic protein and its self-assembly into amyloid fibrils has been evaluated with α-synuclein, an intrinsically unfolded protein related to Parkinson's disease, by restricting its structural plasticity through an end-to-end disulfide bond formation between two newly introduced cysteine residues on the N- and C-termini. Although the resulting circular form of α-synuclein exhibited an impaired fibrillation propensity, the restriction did not completely block the protein's interactive core since co-incubation with wild-type α-synuclein dramatically facilitated the fibrillation by producing distinctive forms of amyloid fibrils. The suppressed fibrillation propensity was instantly restored as the structural restriction was unleashed with β-mercaptoethanol. Conformational flexibility of the accreting amyloidogenic protein to pre-existing seeds has been demonstrated to be critical for fibrillar extension process by exerting structural adjustment to a complementary structure for the assembly.

  12. End-to-end Structural Restriction of α-Synuclein and Its Influence on Amyloid Fibril Formation

    International Nuclear Information System (INIS)

    Hong, Chul Suk; Park, Jae Hyung; Choe, Young Jun; Paik, Seung R.


    Relationship between molecular freedom of amyloidogenic protein and its self-assembly into amyloid fibrils has been evaluated with α-synuclein, an intrinsically unfolded protein related to Parkinson's disease, by restricting its structural plasticity through an end-to-end disulfide bond formation between two newly introduced cysteine residues on the N- and C-termini. Although the resulting circular form of α-synuclein exhibited an impaired fibrillation propensity, the restriction did not completely block the protein's interactive core since co-incubation with wild-type α-synuclein dramatically facilitated the fibrillation by producing distinctive forms of amyloid fibrils. The suppressed fibrillation propensity was instantly restored as the structural restriction was unleashed with β-mercaptoethanol. Conformational flexibility of the accreting amyloidogenic protein to pre-existing seeds has been demonstrated to be critical for fibrillar extension process by exerting structural adjustment to a complementary structure for the assembly

  13. Acute energy deprivation in man: effect on serum immunoglobulins antibody response, complement factors 3 and 4, acute phase reactants and interferon-producing capacity of blood lymphocytes. (United States)

    Palmblad, J; Cantell, K; Holm, G; Norberg, R; Strander, H; Sunblad, L


    The effects of 10 days of total energy deprivation on serum levels of immunoglobulins, antibodies acute phase reactants and on interferon production were evaluated in fourteen healthy, normal-weight males. A significant depression was noted of the serum levels of complement factor 3, haptoglobin and orosomucoid. The titres of mercaptoethanol-sensitive specific antibodies to flagellin were higher in the subjects inoculated at the end of the starvation period than in controls and those inoculated at the start of the period. The serum levels of IgG, IgM, IgA, IgE, alpha-1-antitrypsin and complement factor 4, and the interferon-producing capacity of blood lymphocytes, were not changed. Thus, 10 days of total energy deprivation depresses the serum levels of several acute phase reactants and re-feeding may enhance antibody production. PMID:606438

  14. Development of radiolabelling techniques of anti-CEA monoclonal antibody

    International Nuclear Information System (INIS)

    Castiglia, S.G. de


    The purpose of this work was to label monoclonal and polyclonal antibodies with 99 Tc m such as the ior-CEA-1 antibody and polyclonal IgG using a direct method, to check the radiochemical and biological behavior of labelled products, to prepare it under sterile and apyrogenic conditions as a lyophilized kit and to employ it in clinical trials. In addition, a photoactivation method was used to label polyclonal IgG with 99 Tc m and to compare with the established method using mercaptoethanol (2-ME) as the reducing agent. Finally polyclonal IgG was labelled using an indirect method in which a chelator was covalently attached to the protein and the 99 Tc m added as glucoheptonate complex. The properties of 99 Tc m when labelled with monoclonal and polyclonal antibodies by different methods were assessed by in vitro and in vivo studies

  15. Conversion of Type III penumococcal polysaccharide low responders to high responders by immunization with a thymus-dependent form of antigen

    International Nuclear Information System (INIS)

    Braley-Mullen, H.; Sharp, G.C.


    C57BL/Ks mice immunized with 0.6 μg Type III pneumococcal polysaccharide (S3) or with 10 9 S3 conjugated sheep erythrocytes (S3-SRBC) produced 5 to 7 times fewer S3-specific plaque-forming cells than similarly immunized BALB/c mice. However, when mice were primed with the SRBC carrier prior to challenge with S3-SRBC the low responder C57BL/Ks mice responded as well as the high-responder BALBc strain. The cell activated by the carrier priming was shown to be a thymus-derived (T) cell and the antibody produced by primed mice was mercaptoethanol sensitive (presumably IgM). Nonspecific T cell activation by unrelated antigens did not enhance C57BL/Ks responses to the same degree as specific carrier priming. These findings are discussed in relation to the possible cellular basis for genetic control of the S3 immune response

  16. Metabolism of allylnitrile to cyanide: in vitro studies. (United States)

    Farooqui, M Y; Ybarra, B; Piper, J


    In liver fractions from male Sprague-Dawley rats, the metabolism of allylnitrile (ALN) to cyanide (CN-) was localized in the microsomal fraction and required NADPH and oxygen for maximal activity. The biotransformation of ALN to CN- was characterized with respect to time, microsomal protein concentration, pH and temperature. Metabolism of ALN was increased in microsomes obtained from phenobarbital-treated rats (160% of control) and decreased with cobaltous chloride and beta-diethyl aminoethyl-2,2-diphenyl pentanoate (SKF 525-A) treatments (48% of control). Addition of SKF 525-A to the incubation mixtures inhibited ALN metabolism to CN-. Addition of the epoxide hydrolase inhibitor, 1,1,1-trichloropropane 2,3-oxide, decreased the formation of CN- from ALN. Addition of glutathione, cysteine, D-penicillamine, and 2-mercaptoethanol enhanced the release of CN- from ALN. These findings indicate that ALN is metabolized to CN- via a cytochrome P-450-dependent mixed-function oxidase system.

  17. A microculture method for the generation of primary immune responses in vitro. (United States)

    Pike, B L


    A microculture method for the generation and study of the primary immune response of murine spleen cells to defined antigens in vitro is described. Many of the variable parameters which occur in culture systems have been studied in an attempt to define the optimal culture conditions for this system. Cultures of 10(6) CBA spleen cells consistently produced an immune response of 300-600 hapten-specific plaque-forming cells after 3 days of incubation with the T cell-independent antigens DNP-POL and NIP-POL. Cultures were set up in Microtest II tissue culture plates in a volume of 0.2 ml of medium containing 10(-4) M 2-mercaptoethanol. The system described has the advantages of being highly efficient and reproducible and utilises small amounts of cells, medium and antigen. It provides a simple, economic and reliable approach for the systematic study of the immune response in vitro.

  18. Detection of Brucella sp. infection through serological, microbiological, and molecular methods applied to buffaloes in Maranhão State, Brazil. (United States)

    Dos Santos, Larissa Sarmento; Sá, Joicy Cortez; Dos Santos Ribeiro, Diego Luiz; Chaves, Nancyleni Pinto; da Silva Mol, Juliana Pinto; Santos, Renato Lima; da Paixão, Tatiane Alves; de Carvalho Neta, Alcina Vieira


    The aim of the current study is to diagnose Brucella spp. infection using methods such as serology, bacterial isolation, and molecular analysis in buffaloes bred in Maranhão State. In order to do so, 390 samples of buffalo serum were subjected to serological tests, to Rose Bengal Plate Test (RBPT) and to 2-mercaptoethanol (2-ME) combined with slow agglutination test (SAT). Vaginal swabs were collected from seropositive animals and subjected to bacterial isolation and to generic PCR. According to the serological test, 16 animals had a positive reaction to the confirmatory test (2-ME/SAT). As for bacterial isolation, three samples resulted in the isolation of Brucella spp.-characteristic colonies, which were confirmed through PCR. These results confirmed Brucella spp. infection in the buffalo herd from Maranhão State.

  19. Field trial of a brucellosis competitive enzyme linked immunoabsorbent assay (ELISA)

    International Nuclear Information System (INIS)

    Samartino, L.E.; Gregoret, R.J.; Sigal, G.


    The purpose of this study was to evaluate the performance of a competitive ELISA system for the diagnosis of bovine brucellosis in comparison to conventional aerological tests routinely used in Argentina. A total of 2.500 serum samples, comprising Brucella-free herds, vaccinated cattle and naturally infected animals, was tested by the following tests: buffered plate agglutination, Rose Bengal, 2-mercaptoethanol, complement fixation, and indirect and competitive ELISAs. Specificity and relative sensitivity at each test were determined. The competitive ELISA was considered suitable for detection of vaccinated animals and had higher specificity than the other tests. The results point to the potential use of the test as a complementary assay in the brucellosis control programme in Argentina. (author)

  20. An Auto-Inductive Cascade for the Optical Sensing of Thiols in Aqueous Media: Application in the Detection of a VX Nerve Agent Mimic. (United States)

    Sun, Xiaolong; Anslyn, Eric V


    A new auto-inductive protocol employs a Meldrum's-acid-based conjugate acceptor (1) as a latent source of thiol for signal amplification, as well as optical detection of thiols. The auto-induction is initiated by a thiol-disulfide exchange that leads to the generation of β-mercaptoethanol, which in turn decouples the conjugate acceptor to release more thiols, resulting in a self-propagating cycle that continues until all the conjugate acceptor is consumed. Using 1 in a two-step integrated protocol yields a rapid, sensitive, and precise diagnostic assay for the ultratrace quantitation of a thiophosphate nerve agent surrogate. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.


    Directory of Open Access Journals (Sweden)

    Thomas Mata Hine


    Full Text Available The objective of the research is determining the ICM isolation method to produce ESC. Blastocyst stage of DDy mice embryos were used in this study. Zona pellucida of blastocysts were removed by 0.25% pronase, the ICM isolation were done by enzimatic or immunosurgery method, and then they were cultured in DMEM-high glucose supplemented with mercaptoethanol, gentamycin, fetal bovine serum, and cumulus cells as feeder layer. The result of the research indicated that immunosurgery method yielding attachment rate and number ESC colony 93.85% and 43.08%, respectively, higher (P<0.05 than enzimatic method that weree 79.63% and 18.52%, respectively, but the viability of ICM cells were equal (P >0.05 that are 93.59% in enzymatic method and 98.56% in immunosurgery method. This research concluded that immunosurgery more effective method for isolation of ICM and ESC production than enzymatic method.

  2. Characteristic properties of lipase from cowpea (vigna unguiculata) seedlings

    International Nuclear Information System (INIS)

    Pahoja, V.M.; Dahot, M.U.


    Lipase activity was assayed in 4 days old cowpea seedlings using olive oil emulsion stabilized with 10% gum acacia as a substrate. The maximum lipase activity was observed at pH 8.0 with Tris-HCI buffer and at 30 degree C. The pH stability was found in between 7.5-8.5. Lipase activity was fairly stable up to 60 degree C and retaining 80% activity whereas, 26% lipase activity was remaining at 100 degree C within 15 minutes. Lipase activity was slightly increased in the presence of MnC/sub 2/ and decreased by the addition of Triton X 100, Tween 80, ZnCI/sub 2/ and mercaptoethanol. (author)

  3. Y2O3: Eu,Zn nanocrystals as a fluorescent probe for the detection of biotin

    International Nuclear Information System (INIS)

    Ganjkhanlou, Y.; Hosseinnia, A.; Kazemzad, M.; Khanlarkhani, A.; Moghaddam, A.B.


    We report on the application of nanocrystals (NCs) of the type Y 2 O 3 : Eu,Zn as a probe for the fluorescent detection of biotin in aqueous solution. The NCs were dispersed in water in the presence of various surface modifiers including mercaptoethanol (ME), monoethanolamine and ethylene glycol. Both the absorbance of surfactant and the stability of the suspensions were investigated in order to optimize the experimental conditions. ME is found to be the most suitable surfactant for stabilization of the suspended NCs. Their photoluminescence intensity is found to be quenched by biotin. The Stern-Volmer constant for the quenching process is 7.6 x 10 3 M -1 . This NC probe can be applied to the detection of biotin in the 1-60 μM concentration range with detection limit of 1.89 μM. The possible mechanisms of quenching also are discussed. (author)

  4. Bolevenine, a toxic protein from the Japanese toadstool Boletus venenatus. (United States)

    Matsuura, Masanori; Yamada, Mina; Saikawa, Yoko; Miyairi, Kazuo; Okuno, Toshikatsu; Konno, Katsuhiro; Uenishi, Jun'ichi; Hashimoto, Kimiko; Nakata, Masaya


    A toxic protein, called bolevenine, was isolated from the toxic mushroom Boletus venenatus based on its lethal effects on mice. On SDS-PAGE, in either the presence or absence of 2-mercaptoethanol, this protein showed a single band of approximately 12 kDa. In contrast, based on gel filtration and MALDI-TOFMS, its relative molecular mass was estimated to be approximately 30 kDa and approximately 33 kDa, respectively, indicating that the protein consists of three identical subunits. This toxin exhibited its lethal activity following injection at 10mg/kg into mice. The N-terminal amino acid sequence was determined up to 18, and found to be similar to the previously reported bolesatine, a toxic compound isolated from Boletus satanas.

  5. Purification and multimer formation of allurin, a sperm chemoattractant from Xenopus laevis egg jelly. (United States)

    Sugiyama, Hitoshi; Burnett, Lindsey; Xiang, Xueyu; Olson, John; Willis, Shaun; Miao, Amy; Akema, Tatsuo; Bieber, Allan L; Chandler, Douglas E


    Allurin, a sperm chemoattractant isolated from Xenopus laevis egg jelly, can be purified in one step from an extract of diffusible jelly proteins ("egg water") using a FPLC or HPLC anion exchange column and a multi-step NaCl gradient. Allurin homomultimers were detected by Western blotting with antibodies prepared against the purified protein or peptides within the protein. Allurin multimers were stable and resisted dissociation by SDS and beta-mercaptoethanol. Alkylation of allurin provided evidence for two free sulfhydryl groups but did not eliminate multimer formation, suggesting that intermolecular disulfide bond formation is not required for allurin aggregation. Concentration of egg water was accompanied by a reduction of chemoattractant activity that could not be fully accounted for by homomultimer formation. Rather, the presence of a multiphasic dose-activity curve upon partial purification and formation of hetero-allurin complexes during concentration suggested that egg water may contain allurin-binding proteins that reduce multimer formation and activity.

  6. Transformation and electrophoretic karyotyping of Coniochaeta ligniaria NRRL30616. (United States)

    Nichols, Nancy N; Szynkarek, Matthew P; Skory, Christopher D; Gorsich, Steven W; López, Maria J; Guisado, Gema M; Nichols, Wade A


    Coniochaeta ligniaria NRRL30616 is an ascomycete that grows with yeast-like appearance in liquid culture. The strain has potential utility for conversion of fibrous biomass to fuels or chemicals. Furans and other inhibitory compounds in lignocellulosic biomass are metabolized by NRRL30616, facilitating subsequent microbial fermentation of biomass sugars. This study undertook initial characterization of the genetic system of C. ligniaria NRRL30616. Transformation using hygromycin as a dominant selectable marker was achieved using protoplasts generated by incubating cells in 1% (v/v) β-mercaptoethanol, followed by cell wall-digesting enzymes. Thirteen chromosomes with an estimated total size of 30.1 Mb were detected in C. ligniaria. The GC content of chromosomal DNA and of coding regions from cDNA sequences were 49.2 and 51.9%, respectively. This study is the first report of genome size, electrophoretic karyotype, and transformation system for a member of the Coniochaetales.

  7. Freezing of in vitro produced bovine embryos in animal protein-free medium containing vegetal peptones. (United States)

    George, F; Vrancken, M; Verhaeghe, B; Verhoeye, F; Schneider, Y-J; Massip, A; Donnay, I


    Successful cryopreservation is essential for a large-scale dispersal of bovine in vitro produced (IVP) embryos that have been shown to be more sensitive to cryopreservation than their in vivo counterparts. On the other hand, the use of animal proteins in freezing media increases sanitary risks. We first replaced animal proteins, such as bovine serum albumin (BSA) in the freezing medium by plant-derived peptides (vegetal peptones). A batch of wheat peptones was selected after a preliminary experiment showing the absence of toxicity of concentrationsanimal protein-free freezing medium containing 1.8 mg/mL peptones. No beneficial effect of adding 1 mg/mL sodium hyaluronate or 100 microM beta-mercaptoethanol was observed on embryo survival or quality. In conclusion, we have demonstrated that vegetal peptones can replace BSA in freezing media without affecting blastocyst survival and quality.

  8. Optimized and validated flow-injection spectrophotometric analysis of topiramate, piracetam and levetiracetam in pharmaceutical formulations. (United States)

    Hadad, Ghada M; Abdel-Salam, Randa A; Emara, Samy


    Application of a sensitive and rapid flow injection analysis (FIA) method for determination of topiramate, piracetam, and levetiracetam in pharmaceutical formulations has been investigated. The method is based on the reaction with ortho-phtalaldehyde and 2-mercaptoethanol in a basic buffer and measurement of absorbance at 295 nm under flow conditions. Variables affecting the determination such as sample injection volume, pH, ionic strength, reagent concentrations, flow rate of reagent and other FIA parameters were optimized to produce the most sensitive and reproducible results using a quarter-fraction factorial design, for five factors at two levels. Also, the method has been optimized and fully validated in terms of linearity and range, limit of detection and quantitation, precision, selectivity and accuracy. The method was successfully applied to the analysis of pharmaceutical preparations.

  9. ATP-independent DNA synthesis in Vaccinia-infected L cells

    International Nuclear Information System (INIS)

    Berger, N.A.; Kauff, R.A.; Sikorski, G.W.


    Mouse L cells can be made permeable to exogenous nucleotides by a cold shock in 0.01 M Tris . HCl pH 7.8, 0.25 M sucrose, 1 mM EDTA, 30 mM 2-mercaptoethanol and 4 mM MgCl 2 . DNA synthesis in permeabilized L cells requires ATP whereas DNA synthesis in permeabilized L cells that are infected with Vaccinia virus is ATP-independent. Permeabilized L cells that are infected with ultraviolet-irradiated virus show a marked suppression of DNA synthesis which is not corrected by an excess of deoxynucleoside triphosphates and ATP. The ATP-dependent and ATP-independent processes of DNA synthesis are inhibited to the same extent by Mal-Net, pHMB, ara CTP and phosphonoacetate. Concentrations of daunorubicin and cytembena, which cause marked inhibition of the ATP-dependent enzymes, only cause partial inhibition of the ATP-independent enzymes. (Auth.)

  10. Extraction of high quality of RNA and construction of a suppression subtractive hybridization (SSH) library from chestnut rose (Rosa roxburghii Tratt). (United States)

    Xu, Qiang; Wen, Xiaopeng; Tao, Nengguo; Hu, Zhiyong; Yue, Hailin; Deng, Xiuxin


    Chestnut rose (Rosa roxburghii Tratt) is a rare fruit crop of promising economical importance in fruit and ornamental exploitation in China. Isolation of high quality RNA from chestnut rose is difficult due to its high levels of polyphenols, polysaccharides and other compounds, but a modified CTAB extraction procedure without phenol gave satisfactory results. High concentrations of PVP (2%, w/v), CTAB (2%, w/v) and beta-mercaptoethanol (4%, v/v) were used in the extraction buffer to improve RNA quality. The average yield was about 200 microg RNA g(-1) fresh leaves. The isolated RNA was of sufficient quality for construction of suppression subtraction hybridization (SSH) library, which allowed the isolation of several pathogen-induced defense genes.

  11. Investigations of prevalence of antibodies to B.canis in stray dogs in territory of Belgrade

    Directory of Open Access Journals (Sweden)

    Radojičić Sonja


    Full Text Available The paper covers investigations of stray dogs in the territory of the city of Belgrade. A total of 184 blood serum samples were examined for the presence of antibodies specific to Brucella canis. The method of slow agglutination in a test tube with 2- mercaptoethanol was used in the diagnostic procedure. Of the 184 examined serums, 49 (26.63% had a titer of 1/50, 25 serums had a titer of 1/100 (13.58%, while 20 serums had a titer equal to or bigger than 1/200 (10.87%. Furthermore, 15 samples of full blood from serologiclly negative animals were also presented for isolation. The bacteriological finding for these samples was negative. The obtained results indicate that the number of seropositive stray dogs in the territory of Belgrade is extremely high and that 10.87% of the testes animals are definitely infected with Brucella canis.

  12. Ultraviolet enhancement of DNA base release by bleomycin

    International Nuclear Information System (INIS)

    Kakinuma, J.; Tanabe, M.; Orii, H.


    The effect of UV irradiation on base-releasing activity of bleomycin was studied on bleomycin A 2 -DNA reaction mixture in the presence of Fe(II) and 2-mercaptoethanol. This effect was measured by the release of free bases from calf thymus DNA with high-performance liquid chromatography. UV irradiation enhanced DNA base-releasing activity of bleomycin and simultaneously caused disappearance of fluorescence emission maximum at 355 nm assigned to bithiazole rings and increase in the intensity of a peak at 400 nm. UV irradiation at 295 nm, the UV absorption maximum of bleomycin, is the most effective in releasing free bases and in changing fluorescence emission patterns. From these results, we suggest that some alterations in the bithiazole group of bleomycin molecule were initiated by UV irradiation and contributed to increased base-releasing activity of bleomycin through a yet unexplained mechanism, presumably through bleomycin dimer formation. (orig.)

  13. Sero-prevalence of brucellosis among blood donors in Ahvaz, Southwest Iran

    Directory of Open Access Journals (Sweden)

    Abdolhussein Shakurnia


    Full Text Available Objective: To identify sero-prevalence of brucellosis among blood donors in Ahvaz city, Southwest Iran. Methods: A total number of 1 450 serum samples from blood donation were collected and were screened for the presence of brucella antibody. Rose Bengal Plate Test, Standard Agglutination Test (SAT, and 2-mercaptoethanol (2ME agglutination were tested in the sample. SAT dilution ≥1/80 and 2ME agglutination ≥1/40 were considered positive. Results: Sero-prevalence of brucellosis among the blood donors was 0.70%, 0.34%, and 0.20% by Rose Bengal Plate Test, SAT, and 2ME respectively. Conclusions: Considering the 1/80 titer of SAT as the criteria of contamination with brucella, routine screening of sero-prevalence of brucella in blood donors is not recommended in this area.

  14. Brucellosis as a neglected disease in a neglected population: a seroepidemiological study of migratory nomads in the Fars province of Iran. (United States)

    Honarvar, B; Moghadami, M; Lankarani, K B; Davarpanah, M A; Ataolahi, M; Farbod, A; Eskandari, E; Panahi, M; Ghorbani, A; Zahiri, Z; Tabrizi, R; Pourjafar, M; Heidari, S M M


    This study assessed the seroprevalence of brucellosis and its risk factors in migratory nomads in the Fars province of Iran. Active brucellosis was defined as the combination of clinical symptoms, including fever, chills, night sweats, headache, low back pain, arthralgia, or myalgia, and positive laboratory testing, including either a serum agglutination test (SAT) ⩾1:80 with a 2-mercaptoethanol (2-ME) test ⩾1:40, or a SAT brucellosis was detected in 54 cases, indicating a prevalence of 10% (95% confidence interval 8-12). In conclusion, we determined that brucellosis is a prevalent yet neglected disease in this nomadic population. Brucellosis control is not possible as long as these high-risk populations remain neglected.

  15. Labelling of. beta. -endorphin (. beta. -END) and. beta. -lipotropin (. beta. -LPH) by /sup 125/I

    Energy Technology Data Exchange (ETDEWEB)

    Deby-Dupont, G.; Joris, J.; Franchimont, P. (Universite de Liege (Belgique)); Reuter, A.M.; Vrindts-Gevaert, Y. (Institut des Radioelements, Fleurus (Belgique))


    5 of human ..beta..-endorphin were labelled with 2 mCi /sup 125/I by the chloramine T technique. After two gel filtrations on Sephadex G-15 and on Sephadex G-50 in phosphate buffer with EDTA, Trasylol and mercapto-ethanol, a pure tracer was obtained with a specific activity about 150 at + 4/sup 0/C, the tracer remained utilizable for 30 days without loss of immunoreactivity. The labelling with lactoperoxydase and the use of another gel filtration method (filtration on Aca 202) gave a /sup 125/I ..beta..-END tracer with the same immunoreactivity. The binding of this tracer to the antibody of an anti-..beta..-END antiserum diluted at 1/8000 was 32% with a non specific binding of 2%. 5 of human ..beta..-lipotropin were labelled with 0.5 mCi /sup 125/I by the lactoperoxydase method. After two gel filtrations on Sephadex G-25 and on Sephadex G-75 in phosphate buffer with EDTA, Trasylol and mercapto-ethanol, a pure tracer with a specific activity of 140 was obtained. It remained utilizable for 30 days when kept at + 4/sup 0/C. Gel filtration on Aca 202 did not give good purification, while gel filtration on Aca 54 was good but slower than on Sephadex G-75. The binding to antibody in absence of unlabelled ..beta..-LPH was 32% for an anti-..beta..-LPH antiserum diluted at 1/4000. The non specific binding was 2.5%.

  16. Mercuric Chloride Effects on Root Water Transport in Aspen Seedlings. (United States)

    Wan; Zwiazek


    HgCl(2) (0.1 mM) reduced pressure-induced water flux and root hydraulic conductivity in the roots of 1-year-old aspen (Populus tremuloides Michx.) seedlings by about 50%. The inhibition was reversed with 50 mM mercaptoethanol. Mercurial treatment reduced the activation energy of water transport in the roots from 10.82 +/- 0.700 kcal mol(-1) to 6.67 +/- 0.193 kcal mol(-1) when measured over the 4 degrees C to 25 degrees C temperature range. An increase in rhodamine B concentration in the xylem sap of mercury-treated roots suggested a decrease in the symplastic transport of water. However, the apoplastic pathway in both control and mercury-treated roots constituted only a small fraction of the total root water transport. Electrical conductivity and osmotic potentials of the expressed xylem sap suggested that 0.1 mM HgCl(2) and temperature changes over the 4 degrees C to 25 degrees C range did not induce cell membrane leakage. The 0.1 mM HgCl(2) solution applied as a root drench severely reduced stomatal conductance in intact plants, and this reduction was partly reversed by 50 mM mercaptoethanol. In excised shoots, 0.1 mM HgCl(2) did not affect stomatal conductance, suggesting that the signal that triggered stomatal closure originated in the roots. We suggest that mercury-sensitive processes in aspen roots play a significant role in regulating plant water balance by their effects on root hydraulic conductivity.

  17. Systematic Evaluation of Protein Reduction and Alkylation Reveals Massive Unspecific Side Effects by Iodine-containing Reagents. (United States)

    Müller, Torsten; Winter, Dominic


    Reduction and alkylation of cysteine residues is part of virtually any proteomics workflow. Despite its frequent use, up to date no systematic investigation of the impact of different conditions on the outcome of proteomics studies has been performed. In this study, we compared common reduction reagents (dithiothreitol, tris-(2-carboxyethyl)-phosphine, and β-mercaptoethanol) and alkylation reagents (iodoacetamide, iodoacetic acid, acrylamide, and chloroacetamide). Using in-gel digests as well as SAX fractionated in-solution digests of cytosolic fractions of HeLa cells, we evaluated 13 different reduction and alkylation conditions resulting in considerably varying identification rates. We observed strong differences in offsite alkylation reactions at 7 amino acids as well as at the peptide N terminus, identifying single and double adducts of all reagents. Using dimethyl labeling, mass tolerant searches, and synthetic peptide experiments, we identified alkylation of methionine residues by iodine-containing alkylation reagents as one of the major factors for the differences. We observed differences of more than 9-fold in numbers of identified methionine-containing peptide spectral matches for in-gel digested samples between iodine- and noniodine-containing alkylation reagents. This was because of formation of carbamidomethylated and carboxymethylated methionine side chains and a resulting prominent neutral loss during ESI ionization or in MS/MS fragmentation, strongly decreasing identification rates of methionine-containing peptides. We achieved best results with acrylamide as alkylation reagent, whereas the highest numbers of peptide spectral matches were obtained when reducing with dithiothreitol and β-mercaptoethanol for the in-solution and the in-gel digested samples, respectively. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Characterization of esterase activity in the Bianchetta trevigiana grape variety under reducing conditions

    Directory of Open Access Journals (Sweden)

    Lomolino G


    Full Text Available Giovanna Lomolino, Anna LanteDepartment of Agronomy Food Natural Resources Animals and Environment, Agripolis, Università di Padova Viale dell'Università, Padova, ItalyBackground and methods: While extensive research has been carried out on the enzymes responsible for ester synthesis and hydrolysis by wine strains of Saccharomyces cerevisiae, grape esterase activity is limited. In this study, the autochthonous grape variety, Bianchetta trevigiana, widespread in the Prosecco wine production area of Treviso, Conegliano, and Asolo, Italy, was characterized according to its esterase activity. Because grape skin is very rich in compounds which impart qualitative characteristics to wine, the study of esterase was carried out on this part of the fruit.Results: During enzyme extraction from grape skin, the presence of the reducing agent, β-mercaptoethanol, allowed a better protein yield but reduced esterase activity. Further addition of increasing doses of reducing agents to grape skin protein extract, such as of K2S2O5 (used in winemaking and DTT, reduced or inhibited esterase activity. Even though the zymographic profiles of the extracts obtained with and without β-mercaptoethanol were qualitatively equal, the intensity of enzymatic bands, measured by densitometry, was different.Conclusion: The presence of reducing agents affected the activity of grape skin esterase, and given that this enzyme is involved in the hydrolysis and synthesis of esters, which are important compounds responsible for the flavor of wine, addition of reducing agents could affect the aromatic profile of wine.Keywords: esterase, grape, reducing agent, wine

  19. Characteristics of purified cows' milk xanthine oxidase and its submolecular characteristics. (United States)

    Cheng, S G; Koch, U; Brunner, J R


    Xanthine oxidase (EC was purified from fresh cows' milk by differential centrifugation and hydroxylapatite chromatography in the absence of reducing agents and proteases. The purified isolate possessed an absorbance at 280 nm:absorbance at 450 nm ratio of 4.84; an absorbance (1 cm at 280 nm 1%) of 11.9; an activity:absorbance at 450 nm of 141, a specific activity of 3.59 units/mg; and detectable dehydrogenase activity. The enzyme preparation was obtained in a reversible oxidase form that could be partially converted to xanthine dehydrogenase in the presence of 10mM dithiothreitol or 1% mercaptoethanol. Amino acid analyses revealed that the enzyme was hydrophobic in nature and that lysine constituted its N-terminal residue. The protein contained 22 disulfide and 38 sulfhydryl groups, four of which were detectable in the undenatured protein complex. Discontinuous PAGE in the presence of selected dissociation agents did not result in further resolution. Sodium dodecyl sulfate-PAGE of the purified enzyme revealed a sharp zone with a molecular weight of 151,000 +/- 4000 (i.e., monomer). The purified enzyme exhibited oxidase activity in the presence of 6 M urea and following limited proteolysis by trypsin, chymotrypsin, plasmin, pancreatin, pepsin, and papain. Proteolyzed xanthine oxidase migrated as a single zone in polyacrylamide gels in the presence and absence of dissociating agents such as 1% mercaptoethanol and 6 M urea. Restricted digestion of xanthine oxidase by proteases was indicated by the presence of three major zones with molecular weights ranging from 85,000 to 100,000, 30,000 to 35,000, and 18,000 to 20,000 commonly observed in SDS gels. Amino acid profiles of the principal peptidyl fragments of trypsin-cleaved xanthine oxidase indicated their hydrophobic nature and lysine as the N-terminal residue for all fragments.

  20. Roles of cysteines Cys115 and Cys201 in the assembly and thermostability of grouper betanodavirus particles (United States)

    Wang, Chun-Hsiung; Hsu, Chi-Hsin; Wu, Yi-Min; Luo, Yu-Chun; Tu, Mei-Hui; Chang, Wei-hau; Cheng, R. Holland


    The virus-like particle (VLP) assembled from capsid subunits of the dragon grouper nervous necrosis virus (DGNNV) is very similar to its native T = 3 virion. In order to investigate the effects of four cysteine residues in the capsid polypeptide on the assembly/dissociation pathways of DGNNV virions, we recombinantly cloned mutant VLPs by mutating each cysteine to destroy the specific disulfide linkage as compared with thiol reduction to destroy all S–S bonds. The mutant VLPs of C187A and C331A mutations were similar to wild-type VLPs (WT-VLPs); hence, the effects of Cys187 and Cys331 on the particle formation and thermostability were presumably negligible. Electron microscopy showed that either C115A or C201A mutation disrupted de novo VLP formation significantly. As shown in micrographs and thermal decay curves, β-mercaptoethanol-treated WT-VLPs remained intact, merely resulting in lower tolerance to thermal disruption than native WT-VLPs. This thiol reduction broke disulfide linkages inside the pre-fabricated VLPs, but it did not disrupt the appearance of icosahedrons. Small dissociated capsomers from EGTA-treated VLPs were able to reassemble back to icosahedrons in the presence of calcium ions, but additional treatment with β-mercaptoethanol during EGTA dissociation resulted in inability of the capsomers to reassemble into the icosahedral form. These results indicated that Cys115 and Cys201 were essential for capsid formation of DGNNV icosahedron structure in de novo assembly and reassembly pathways, as well as for the thermal stability of pre-fabricated particles. PMID:20446029

  1. Aryldithioethyloxycarbonyl (Ardec): a new family of amine protecting groups removable under mild reducing conditions and their applications to peptide synthesis. (United States)

    Lapeyre, Milaine; Leprince, Jérôme; Massonneau, Marc; Oulyadi, Hassan; Renard, Pierre-Yves; Romieu, Anthony; Turcatti, Gerardo; Vaudry, Hubert


    The development of phenyldithioethyloxycarbonyl (Phdec) and 2-pyridyldithioethyloxycarbonyl (Pydec) protecting groups, which are thiol-labile urethanes, is described. These new disulfide-based protecting groups were introduced onto the epsilon-amino group of L-lysine; the resulting amino acid derivatives were easily converted into N alpha-Fmoc building blocks suitable for both solid- and solution-phase peptide synthesis. Model dipeptide(Ardec)s were prepared by using classical peptide couplings followed by standard deprotection protocols. They were used to optimize the conditions for complete thiolytic removal of the Ardec groups both in aqueous and organic media. Phdec and Pydec were found to be cleaved within 15 to 30 min under mild reducing conditions: i) by treatment with dithiothreitol or beta-mercaptoethanol in Tris.HCl buffer (pH 8.5-9.0) for deprotection in water and ii) by treatment with beta-mercaptoethanol and 1,8-diazobicyclo[5.4.0]undec-7-ene (DBU) in N-methylpyrrolidinone for deprotection in an organic medium. Successful solid-phase synthesis of hexapeptides Ac-Lys-Asp-Glu-Val-Asp-Lys(Ardec)-NH2 has clearly demonstrated the full orthogonality of these new amino protecting groups with Fmoc and Boc protections. The utility of the Ardec orthogonal deprotection strategy for site-specific chemical modification of peptides bearing several amino groups was illustrated firstly by the preparation of a fluorogenic substrate for caspase-3 protease containing the cyanine dyes Cy 3.0 and Cy 5.0 as FRET donor/acceptor pair, and by solid-phase synthesis of an hexapeptide bearing a single biotin reporter group.

  2. An investigation into the inter-relationships of sulphur xeno-biotransformation pathways in Parkinson's and motor neurone diseases. (United States)

    Steventon, Glyn B; Waring, Rosemary H; Williams, Adrian C


    The role of defective 'sulphur xenobiotic' biotransformations in the aetiology of Parkinson's and motor neurone diseases has been in the literature for over a decade. Problems in the S-oxidation of aliphatic thioethers, sulphation of phenolic compounds and the S-methylation of aliphatic sulphydryl groups have all been reported. These reports have also been consistent in observing that only a 'significant minority' of patients express these problems in sulphur biotransformation pathways. However, no investigation has yet reported on the incidence of these three defective pathways in control invididuals and in patients with Parkinson's and motor neurone disease. This investigation has found that: 1. Forty percent of patients with Parkinson's and motor neurone disease have a defect in the S-oxidation of S-carboxymethyl-L-cysteine compared to 4% of controls. 2. 35-40% of patients with Parkinson's and motor neurone disease have a defect in the sulphation of paracetamol compared to 4% of controls. 3. 60% of patients with motor neurone disease have a high capacity for the S-methylation of 2-mercaptoethanol compared to 4% of controls. 4. 38% of patients with Parkinson's disease have a low capacity for the S-methylation of 2-mercaptoethanol compared to 4% of controls. 5. There is no correlation between the S-oxidation phenotype, low paracetamol sulphation phenotype and low or high S-methylation phenotype in controls or patients with Parkinson's or motor neurone disease. 6. The number of controls that expressed one of the aberrant phenotypes was 4% compared to 38% of the patients with Parkinson's disease and 47% of the patients with motor neurone disease. 7. The number of controls that expressed two of the aberrant phenotypes was 0% compared to 18% of the patients with Parkinson's disease and 19% of those with motor neurone disease. 8. No controls or patients with Parkinson's disease or motor neurone disease expressed all three of the aberrant phenotypes. The results

  3. Investigation of glutathione peroxidase activity in chicken meat under different experimental conditions

    Directory of Open Access Journals (Sweden)

    Alexandre José Cichoski


    Full Text Available Due to the fact that previous studies on the enzymatic activity of Glutathione peroxidase (GSH-Px diverge widely in their methodology and results, this study aimed to investigate the influence of different analytical conditions on GSH-Px activity in chicken thighs from broilers that were fed different diets with different sources and concentrations of selenium. GSH-Px activity was evaluated six hours after slaughter and 120 days after frozen storage at -18 ºC. The different analytical conditions included time of pre-incubation (0, 10 and 30 minutes, reaction medium, types of substrate (H2O2 (0.72 mM, 7.2 mM, and 72 mM and Terc-butil hydroperoxide 15 mM, and different buffer concentrations (buffer 1, potassium phosphate 50 mM pH 7.0 + EDTA 1 mM + mercaptoethanol 1 mM, and buffer 2, tris-HCl 50 mM pH 7.6 + EDTA 1 mM + mercapthanol 5 mM. The results show that the highest GSH-Px activity was observed when enzyme and substrate were in contact at 22 ºC without any pre-incubation, and that, when used at concentrations above 0.72 mM, hydrogen peroxide saturated the GSH-Px enzyme and inhibited its activity. The enzyme presented higher affinity to hydrogen peroxide when compared to terc-butil peroxide, and the addition of a buffer containing mercaptoethanol did not increase GSH-Px enzymatic activity. The activity of GSH-Px was not influenced by the source and concentration of selenium in the diet either. The obtained results allowed the determination of the best temperature of contact between the enzyme and substrate (22 ºC, the optimum concentration, and the type of substrate and buffer to be used. This information is extremely useful for future studies on GSH-Px activity in meat due to the divergence and little information found in the literature.

  4. 99mTc-Labeling of Monoclonal Antibody to Carcinoembryonic Antigen and Biodistribution

    International Nuclear Information System (INIS)

    Moon, Dae Hyuk; Chung, June Key; Lee, Myu ng Chul; Koh, Chang Soon; Chung, Hong Keun; Park, Jae Gahb


    This study was designed to evaluate a direct method of 99m Tc labeling using β-mercaptoethanol as a reducing agent, and to investigate whether 99m Tc labeled specific monoclonal antibody against carcinoembryonic antigen (CEA-92) can be used for the scintigraphic localization of human colon cancer xenograft. Purified CEA-92 IgG was fragmented into F(ab') 2 and then labeled with 99m Tc by transchelation method using glucarate as a chelator. Labeling efficiency, immunological reactivity and in vitro stability of 99m Tc CEA-92 F(ab') 2 were measured and then injected intravenously into nude mice bearing human colon cancer (SNU-C4). Scintigrams were obtained at 24 hour after injection. Then nude mice were sacrificed and the radioactivity was measured. Labeling efficiency of injected 99m Tc CEA-92 F(ab') 2 , immunoreactive fraction and in vitro stability at 24 hour of injected 99m Tc CEA-92 F(ab') 2 was 45.2%, 32.8% and 57.4%, respectively. At 24 hour after injection, %ID/g in kidney (46.77) showed high uptake, but %ID/g in tumor (1.65) was significantly higher than spleen (0.69), muscle (0.16), intestine (0.45), stomach (0.75), heart (0.48) and blood(0.45). There was no significant difference between tumor and liver (1.81). Tumor contrast as quantitated by tumor to blood ratio of 99m Tc CEA-92 F(ab') 2 was increased significantly (p 131 I-CEA-92 F(ab') 2 . The scintigram demonstrated localization of radioactivity over transplanted tumor, but significant background radioactivity was also noted over kidney and abdomen. It is concluded that CEA-92 F(ab') 2 can be labeled with 99m Tc by a direct transchelation method using β-mercaptoethanol as a reducing agent and 99m Tc labeled CEA-92 F(ab') 2 can be used for the scintigraphic localization of human colon cancer xenograft in nude mice model.

  5. Comparison of different methodologies for DNA extraction from Aegla longirostri

    Directory of Open Access Journals (Sweden)

    João Vitor Trindade Bitencourt


    Full Text Available The aim of this study was to compare some DNA extraction methodologies for Aegla longirostri. The protocols were based on the traditional phenol-chloroform DNA extraction methodology and using a commercial kit for DNA extraction. They differed in tissues used, the addition - or not - of beta-mercaptoethanol to the lysis buffer, times and methods for the animal's conservation (frozen, in ethanol or fresh. Individuals stored at -20°C for a long time supplied lower molecular weight DNA than those stored for a short time. The best yield for the specimens preserved in ethanol was obtained for 15 days storage in 95% ethanol. The kit resulted in a low quantity of high molecular weight DNA. The best protocol for DNA extraction from Aeglidae, and probably for other crustaceans should, therefore, utilize fresh specimens, with addition of beta-mercaptoethanol to the lysis buffer.Marcadores moleculares são ferramentas úteis para esclarecer dúvidas a respeito dos Aeglidae, único grupo de crustáceos Anomura de água doce. Essas técnicas dependem da obtenção de DNA de boa qualidade e sem contaminantes. O objetivo deste estudo foi comparar algumas metodologias de extração de DNA de Aegla longirostri. Quatorze protocolos foram analisados, baseados na metodologia tradicional de extração de DNA com fenol-clorofórmio, exceto o protocolo K no qual se utilizou um Kit. Os procedimentos diferiram quanto aos tecidos utilizados e a adição de beta-mercaptoetanol ao tampão de lise. Avaliaram-se também diferentes tempos e maneiras de conservação. Indivíduos congelados apresentaram maior degradação do material obtido conforme o tempo em que ficaram congelados. Para os indivíduos conservados em álcool, aqueles mantidos em etanol 95% forneceram material de melhor qualidade. A utilização do Mini Kit resultou em uma quantidade muito pequena de DNA de alto peso molecular. O melhor protocolo para extração de DNA de Aeglidae utilizou músculos e br

  6. Amino acids profile of sugar cane spirit (cachaça), rum, and whisky. (United States)

    Aquino, Francisco W B; Boso, Lisangela M; Cardoso, Daniel R; Franco, Douglas W


    An analytical procedure for the separation and quantification of 20 amino acids in cachaças has been developed involving C18 solid phase cleanup, derivatization with o-phthalaldehyde/2-mercaptoethanol, and reverse phase liquid chromatography with fluorescence detection. The detection limit was between 0.0050 (Cys) and 0.25 (Ser)mgL(-1), whereas the recovery index varies from 69.5 (Lys) to 100 (Tyr)%. Relative standard deviations vary from 1.39 (Trp) to 13.4 (Glu)% and from 3.08 (Glu) to 13.5 (His) for the repeatability and intermediate precision, respectively. From the quantitative profile of amino acids in 41 cachaças, 5 rums, and 12 whisky samples, the following order of amino acids in significant quantities is observed: Gly=Ser

  7. Purification and partial characterization of a tripeptidase from Pediococcus pentosaceus K9.2. (United States)

    Simitsopoulou, M; Vafopoulou, A; Choli-Papadopoulou, T; Alichanidis, E


    A tripeptidase was purified from the cytoplasm of Pediococcus pentosaceus K9.2 by anion-exchange chromatography, gel filtration chromatography, and high-performance liquid chromatography. The molecular mass of the enzyme was estimated by gel filtration at 100,000 Da. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified peptidase showed one protein band of 45,000 Da. Optimal enzyme activity was obtained at pH 7.0 and at 50 degrees C. The peptidase hydrolyzed all tripeptides tested. Cleavage was not observed with dipeptides, oligopeptides, or amino acid-p-nitroanilide derivatives. Strong inhibition of activity was caused by EDTA, 1,10-phenanthroline, dithiothreitol, and beta-mercaptoethanol, whereas phenylmethylsulfonyl fluoride and sulfur-reactive reagents had no effect on peptidase activity. Mg2+, Mn2+, and Ca2+ stimulated the hydrolyzing activity of the enzyme. The 20 N-terminal amino acids of the tripeptidase from P. pentosaceus had 84% identity with those from the corresponding N-terminal region of the tripeptidase from Lactococcus lactis subsp. cremoris Wg2.

  8. An efficient spectrofluorimetric method adopts doxazosin, terazosin and alfuzosin coupling with orthophthalaldehyde: Application in human plasma (United States)

    Omar, Mahmoud A.; Mohamed, Abdel-Maaboud I.; Derayea, Sayed M.; Hammad, Mohamed A.; Mohamed, Abobakr A.


    A new, selective and sensitive spectrofluorimetric method was designed for the quantitation of doxazosin (DOX), terazosin (TER) and alfuzosin (ALF) in their dosage forms and human plasma. The method adopts efficient derivatization of the studied drugs with ortho-phthalaldehyde (OPA), in the presence of 2-mercaptoethanol in borate buffer (pH 9.7) to generate a highly fluorescent isoindole derivatives, which can strongly enhance the fluorescence intensities of the studied drugs, allowing their sensitive determination at 430 nm after excitation at 337 nm. The fluorescence-concentration plots were rectilinear over the ranges (10.0-400.0) ng/mL. Detection and quantification limits were found to be (0.52-3.88) and (1.59-11.76) ng/mL, respectively. The proposed method was validated according to ICH guidelines, and successfully applied for the determination of pharmaceutical preparations of the studied drugs. Moreover, the high sensitivity of the proposed method permits its successful application to the analysis of the studied drugs in spiked human plasma with % recovery (96.12 ± 1.34-100.66 ± 0.57, n = 3). A proposal for the reaction mechanism was presented.

  9. Isolation of cellulase-producing bacteria and characterization of the cellulase from the isolated bacterium Cellulomonas sp. YJ5. (United States)

    Yin, Li-Jung; Huang, Po-Shin; Lin, Hsin-Hung


    A cellulase-producing bacterium was isolated from soil and identified as Cellulomonas sp. YJ5. Maximal cellulase activity was obtained after 48 h of incubation at 30 degrees C in a medium containing 1.0% carboxymethyl cellulose (CMC), 1.0% algae powder, 1.0% peptone, 0.24% (NH4)2SO4, 0.20% K2HPO4, and 0.03% MgSO(4).7H2O. The cellulase was purified after Sephacryl S-100 chromatography twice with a recovery of 27.9% and purification fold of 17.5. It was, with N-terminal amino acids of AGTKTPVAK, stable at pH 7.5-10.5 and 20-50 degrees C with optimal pH and temperature of 7.0 and 60 degrees C, respectively. Cu2+, Fe2+, Hg2+, Cr3+, and SDS highly inhibited, but cysteine and beta-mercaptoethanol activated, its activity. Substrate specificity indicated it to be an endo-beta-1,4-glucanase.

  10. Synthesis of polyisobutylene-polycaprolactone block copolymers using enzyme catalysis

    Directory of Open Access Journals (Sweden)

    M. Castano


    Full Text Available The synthesis of poly(isobutylene-b-ε-caprolactone diblock and poly(ε-caprolactone-b-isobutylene-b-ε-caprolactone triblock copolymers was accomplished using a combination of living carbocationic polymerization of isobutylene (IB with the ring-opening polymerization (ROP of ε-caprolactone (ε-CL. OH-PIB-allyl was prepared by living carbocationic polymerization of IB initiated with 1,2-propylene oxide/TiCl4 followed by termination with allyltrimethylsilane. Hydroxyl telechelic HO-PIB-OH was obtained by living IB polymerization initiated by 2,4,4,6-tetramethyl-heptane-2,6-diol/TiCl4, termination with allyltrimethylsilane and subsequent thiol-ene click reaction with mercaptoethanol. The structure of the hydroxyl PIBs was confirmed by 1H NMR (proton Nuclear Magnetic Resonance spectroscopy. OH-PIB-allyl and HO-PIB-OH were then successfully used as macroinitiators for the polymerization of ε-CL catalyzed by Candida antarctica Lipase B (CALB, yielding poly(ε-caprolactone-b-isobutylene diblock and poly(ε-caprolactone-b-isobutylene-b-ε-caprolactone triblock copolymers, respectively. Differential Scanning Calorimetry (DSC, Transition Electron Microscopy (TEM and Atomic Force Microscopy (AFM demonstrated that the amorphous PIB and the semicrystalline PCL block segments phase separated, creating nanostructured phase morphology.

  11. Investigation of cadmium-induced alterations in renal glomerular function

    International Nuclear Information System (INIS)

    Long, T.J.


    This research was designed to test the hypothesis that certain aspects of cadmium-induced renal dysfunction are the result of glomerular, rather than classic tubular, injury. To determine whether cadmium-induced proteinuria was due to altered glomerular function, cadmium was administered chronically at a concentration of 185 ppm in the drinking water. This protocol resulted in the production of proteinuria which when analyzed by high pressure liquid chromatography and radioimmunoassay was indistinguishable from that occurring in control rats. Glomerular filtration rate, renal blood flow, and filtration fraction were all significantly depressed after 20-30 weeks of exposure. In order to further investigate these alterations in glomerular function, an acute exposure model was developed. It was found that a single i.p. injection of cadmium in mercaptoethanol resulted in the onset of acute renal failure. The clinical picture was characterized by a reduction in glomerular filtrate rate of 50-90% within 24 hours, with partial to total recovery occurring by day 7 post-exposure. Histological evidence indicated that to a large extent the reduction in GFR was due to tubular blockade and/or backleak of filtrate across damaged tubules

  12. Liquid chromatographic determination of fumonisins B1 and B2 in corn samples after reusable immunoaffinity column clean-up

    Directory of Open Access Journals (Sweden)



    Full Text Available The possibility of the liquid chromatographic determination of fumonisins B1 (FB1 andB2 (FB2 in corn samples with a reused immunoaffinity column (IMA for the clean-up of the samples was investigated. After optimization of the chromatographic determination of FB1 and FB2 derivatized with o-phthaldialdehyde- 2-mercaptoethanol, the efficiency of the clean-up of spiked corn extracts with reuse of the IMA columns was studied, both with and without column regeneration. It was found that the IMAcolumn, designated for single-use only, can be used at least five times without regeneration and additional five times after regeneration. Regeneration consists of leaving the phosphate buffer saline solution on the column for one day at 4 ºC. The efficiency of the columns was tested by determining the recovery of FB1 and FB2, as well as the reproducibility of the determinations. The mean recoveries of FB1 and FB2 from corn spiked with FB1 at 1.0 mg/g and with FB2 at 0.5 mg/g (on the basis of 10 measurements were 88.7%(RSD 10.2% and 90.5%(RSD 6.1 %, respectively.

  13. The effects of dietary administration with chemical treated Saccharomyces cerevisiae strain YG3-1 on the growth of aquatic invertebrates in Artemia

    Directory of Open Access Journals (Sweden)

    Behnam Shekarchi


    Full Text Available Objective: To investigate the biological effects of β-glucans in cell wall of new identified strain Saccharomyces cerevisiae strain YG3-1 on the growth of aquatic invertebrates, in Artemia as model organism. Methods: All yeasts used in the present study were isolated from Rainbow trout intestine and then cultured in yeast extract-peptone-glycerol medium. Activation of β-glucan in yeasts was performed by chemical treatment with 2-mercaptoethanol (2ME (3.5% v/v. Then nauplii and larvae individuals of Artemia urmiana and Artemia franciscana (two different species of Artemia as test organisms were fed with 2ME-treated yeasts during the culture. At the end of experiment, after feeding individual length (total length and growth rate in adult individuals of Artemia was measured. Results: Following this administration, growth in both species of Artemia was improved (P < 0.05. So, the results showed that Artemia urmiana adults individuals that fed with 2MEtreated yeasts had the highest growth and total length. These results were confirmed with growth measurement in adult individuals of Artemia. Conclusions: This study suggested that 2ME-treated Saccharomyces cerevisiae strain YG3-1 yeasts can be used for enhancing the growth of other aquatic invertebrates like shrimps as probiotic supplement and growth promoter.

  14. Synthesis, characterization and thermoluminescence studies of Mn-doped ZnS nanoparticles. (United States)

    Chandrakar, Raju Kumar; Baghel, R N; Chandra, B P


    ZnS:Mn nanoparticles were prepared by a chemical precipitation method and characterized by X-ray diffraction (XRD), field emission gun scanning electron microscope (FEGSEM), and high resolution transmission electron microscopy (HRTEM). Capping agent (mercaptoethanol) concentrations used were 0 M, 0.005 M, 0.01 M, 0.015 M, 0.025 M, 0.040 M, and 0.060 M, and resulted in nanoparticles sizes of 2.98 nm, 2.9 nm, 2.8 nm, 2.7 nm, 2.61 nm, 2.2 nm and 2.1 nm, respectively. The thermoluminescence (TL) glow curve was recorded by heating the sample exposed to UV-radiation, at a fixed heating rate 1°C sec(-1). The TL intensity initially increased with temperature, attained a peak value Im for a particular temperature, and then decreased with further increase in temperature. The peak TL intensity increased with decreasing nanoparticle size, whereas the temperature corresponding to the peak TL intensity decreased slightly with reducing nanocrystal size. As a consequence of increase in surface-to-volume ratio and increased carrier recombination rates, the TL intensity increased with decreasing nanoparticle size. It was found that, whereas activation energy slightly decreased with decreasing nanoparticle size, the frequency factor decreased significantly with reduction in nanoparticle size. Copyright © 2015 John Wiley & Sons, Ltd.

  15. Subunit structure of karatasin, the proteinase isolated from Bromelia plumieri (karatas). (United States)

    Montes, C; Amador, M; Cuevas, D; Cordoba, F


    Close to 15% of the karatasin proteinase activity in the fruit juice of Bromelia plumieri (karatas) is present outside dialysis Visking tubing in 7 days in 0.2 M acetate buffer (pH) 3.5 or 6.5) containing phenyl mercuric acetate. The small proteinase(s), distinct from the 85% activity in juice due to nondialysable karatasin with a reported Mr of 24,868, separates across Spectrapore (13 kDa) membranes but not across Spectrapore with 3.5 kDa average pore diameter. The dialyzed proteinase is named karatasin-D (K-D). Purified non-Dialysable karatasin can be dissociated to what seems to be K-D by incubation in a buffer solution, containing SDS and 2-mercaptoethanol with phenyl mercuric acetate, in dialysis experiments for 8 days at room temperature using Spectrapore 13 kDa tubing. Thus, native karatasin in B. plumieri fruit juice seem to be the result of association of 2 small molecular mass K-D subunits, linked together by disulfide bonds and electrostatic forces, in equilibrium with small amounts of free K-D molecules. The amino acid composition and partial sequence of karatasin up to the 14th position from the amino terminus have discrete analogies with papain and with stem bromelain.


    Directory of Open Access Journals (Sweden)

    Wagih El-Shouny


    Full Text Available The bacteriocin bound to the cells and that secreted into the culture filtrate of Lactobacillus plantarum SR18 were precipitated by 75% ammomium sulphate, dialysed and further purified by Gel filtration on Sephadex G-100. Bacteriocins were purified from proteins bound to the cell of L. plantarum SR18 (plantarcin SR18 a and culture filtrate proteins (plantarcin SR18 b, respectively. The SDS-PAGE of partially purified Plantarcin SR18a showed a molecular weight of 3.5 KDa. While, plantarcin SR18 b had a molecular weight of 10.3 KDa. The antibacterial activity of the tested plantarcin SR18 preparations suffered no measurable loss after 45 min at 80ºC. Whereas, At 100ºC, significant decrease in the activity of bacteriocin preparations (60- 80 % took place by the end of 45 min. At pH ranged from 5-8, the activity of the plantarcin SR18 preparations suffered no measurable loss. Dissociating agents significantly affected the bacteriocin activity. Thus, tween 80 and mercaptoethanol increased the activity of bacteriocin preparations to 1.2-1.4 fold. Sodium dodecyl sulphate (SDS increased the activity of the tested bacteriocin preparations by about 20%.The lowest residual activity (60% was recorded after treatment with Triton X100 for 45 min. Protease completely inhibited the activities of all forms of plantarcin SR18 after 45 min at 37ºC.

  17. Purification and characterization of thiol dependent, oxidation-stable serine alkaline protease from thermophilic Bacillus sp.

    Directory of Open Access Journals (Sweden)

    Aysha Kamran


    Full Text Available Alkaline serine protease was purified to homogeneity from culture supernatant of a thermophilic, alkaliphilic Bacillus sp. by 80% ammonium sulphate precipitation followed by CM-cellulose and DEAE-cellulose ion exchange column chromatography. The enzyme was purified up to 16.5-fold with 6900 U/mg activity. The protease exhibited maximum activity towards casein at pH 8.0 and at 80 °C. The enzyme was stable at pH 8.0 and 80 °C temperature up to 2 h. The Ca2+ and Mn2+ enhanced the proteolytic activity up to 44% and 36% as compared to control, respectively. However, Zn2+, K+, Ba2+, Co2+, Hg2+ and Cu2+ significantly reduced the enzyme activity. PMSF (phenyl methyl sulphonyl fluoride completely inhibited the protease activity, whereas the activity of protease was stimulated up to two folds in the presence of 5 mM 2-mercaptoethanol. The enzyme was also stable in surfactant (Tween-80 and other commercial detergents (SDS, Triton X-100.

  18. Lytic and nonlytic mechanism of inactivation of gram-positive bacteria by lysozyme under atmospheric and high hydrostatic pressure. (United States)

    Masschalck, Barbara; Deckers, Daphne; Michiels, Chris W


    A different behavior was observed in three gram-positive bacteria exposed to hen egg white lysozyme by plate counts and phase-contrast microscopy. The inactivation of Lactobacillus johnsonii was accompanied by spheroplast formation, which is an indication of peptidoglycan hydrolysis. Staphylococcus aureus was resistant to lysozyme and showed no signs of peptidoglycan hydrolysis, and Listeria innocua was inactivated and showed indications of cell leakage but not of peptidoglycan hydrolysis. Under high hydrostatic pressure, S. aureus also became sensitive to lysozyme but did not form spheroplasts and was not lysed. These results suggested the existence of a nonlytic mechanism of bactericidal action of lysozyme on the latter two bacteria, and this mechanism was further studied in L. innocua. Elimination of the enzymic activity of lysozyme by heat denaturation or reduction with beta-mercaptoethanol eliminated this bactericidal mechanism. By means of a LIVE/DEAD viability stain based on a membrane-impermeant fluorescent dye, the nonlytic mechanism was shown to involve membrane perturbation. In the absence of lysozyme, high-pressure treatment was shown to induce autolytic activity in S. aureus and L. innocua.

  19. Toxic reagents and expensive equipment: are they really necessary for the extraction of good quality fungal DNA? (United States)

    Rodrigues, P; Venâncio, A; Lima, N


    The aim of this work was to evaluate a fungal DNA extraction procedure with the lowest inputs in terms of time as well as of expensive and toxic chemicals, but able to consistently produce genomic DNA of good quality for PCR purposes. Two types of fungal biological material were tested - mycelium and conidia - combined with two protocols for DNA extraction using Sodium Dodecyl Sulphate (SDS) and Cetyl Trimethyl Ammonium Bromide as extraction buffers and glass beads for mechanical disruption of cell walls. Our results showed that conidia and SDS buffer was the combination that lead to the best DNA quality and yield, with the lowest variation between samples. This study clearly demonstrates that it is possible to obtain high yield and pure DNA from pigmented conidia without the use of strong cell disrupting procedures and of toxic reagents. There are numerous methods for DNA extraction from fungi. Some rely on expensive commercial kits and/or equipments, unavailable for many laboratories, or make use of toxic chemicals such as chloroform, phenol and mercaptoethanol. This study clearly demonstrates that it is possible to obtain high yields of pure DNA from pigmented conidia without the use of strong and expensive cell disrupting procedures and of toxic reagents. The method herein described is simultaneously inexpensive and adequate to DNA extraction from several different types of fungi. © 2017 The Society for Applied Microbiology.

  20. Purification and characterization of vanilla bean (Vanilla planifolia Andrews) beta-D-glucosidase. (United States)

    Odoux, Eric; Chauwin, Audrey; Brillouet, Jean-Marc


    Vanilla bean beta-D-glucosidase was purified to apparent homogeneity by successive anion exchange, hydrophobic interaction, and size-exclusion chromatography. The enzyme is a tetramer (201 kDa) made up of four identical subunits (50 kDa). The optimum pH was 6.5, and the optimum temperature was 40 degrees C at pH 7.0. K(m) values for p-nitrophenyl-beta-D-glucopyranoside and glucovanillin were 1.1 and 20.0 mM, respectively; V(max) values were 4.5 and 5.0 The beta-D-glucosidase was competitively inhibited by glucono-delta-lactone and 1-deoxynojirimycin, with respective K(i) values of 670 and 152 microM, and not inhibited by 2 M glucose. The beta-D-glucosidase was not inhibited by N-ethylmaleimide and DTNB and fully inhibited by 1.5-2 M 2-mercaptoethanol and 1,4-dithiothreitol. The enzyme showed decreasing activity on p-nitrophenyl-beta-D-fucopyranoside, p-nitrophenyl-beta-D-glucopyranoside, p-nitrophenyl-beta-D-galactopyranoside, and p-nitrophenyl-beta-D-xylopyranoside. The enzyme was also active on prunasin, esculin, and salicin and inactive on cellobiose, gentiobiose, amygdalin, phloridzin, indoxyl-beta-D-glucopyranoside, and quercetin-3-beta-D-glucopyranoside.

  1. Degradation of bradykinin by isolated perfused rat lung

    International Nuclear Information System (INIS)

    Churchill, M.; Orawski, A.T.; AchutaMurthy, P.N.; Simmons, W.H.


    Several studies have suggested that the essentially complete degradation of circulating bradykinin (BK) in lung is mediated in part by peptidase(s) other than the well-characterized angiotensin converting enzyme (ACE). The authors report here that the isolated perfused rat lung can inactivate BK by sequential N-terminal cleavage. [ 3 H-2, 3-Pro] BK was perfused through the lung and the products in the perfusate identified by HPLC. In the absence of inhibitors, BK was 89-100% degraded with 3 H-Pro 2 -Pro 3 and 3 H-Pro as the major products. The dipeptidylaminopeptidase IV (DAP IV) inhibitor, diprotein A (Ile-Pro-Ile), greatly reduced the Pro-Pro and Pro peaks and produced a prominent BK/sub 2-7/ peak (or BK/sub 2-9/ peak if the ACE inhibitor, captopril, was also present). 2-Mercapto-ethanol, a rather specific inhibitor of aminopeptidase P (AP-P), prevented the release of Arg 1 , producing major BK and/or BK/sub 1-7/ peaks. The neutral metalloendopeptidase inhibitor, phosphoramidon, had no effect on the pattern of degradation of BK by the perfused rat lung by the release of Arg 1 by AP-P followed by release of Pro 2 -Pro 3 by DAP IV

  2. Solubilization of proteins in extracted oil bodies by SDS: a simple and efficient protein sample preparation method for Tricine-SDS-PAGE. (United States)

    Ying, Yusang; Zhao, Luping; Kong, Lingzhi; Kong, Xiangzhen; Hua, Yufei; Chen, Yeming


    A simple and efficient method for preparing Tricine-SDS-PAGE protein sample of extracted oil bodies (OBs) was supplied: OB suspension was vortexed with SDS buffer (pH 6.8) for 2 min at room temperature with SDS/protein of 1.52/1(w/w), which could be analyzed by Tricine-SDS-PAGE after simple treatments (dilution and 2-mercaptoethanol). At SDS/protein of 1.52/1, about 95% of proteins in soybean OB suspension were solubilized, whereas residual 5% of proteins were weakly bound to SDS-destroyed OBs; proteins in destroyed OBs might be further solubilized by SDS in the gel and cathode buffer of Tricine-SDS-PAGE, causing about 99% of proteins in soybean OB suspension recover on Tricine-SDS-PAGE gel, which was better than acetone (89%) and diethyl ether (96%) harvested protein samples. Higher or lower SDS/protein was unbeneficial for protein solubilization from OBs. Additionally, the above method was also better than organic solvent method for peanut, sesame, and rapeseed OB suspensions. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Interaction of insulin with colloidal ZnS quantum dots functionalized by various surface capping agents. (United States)

    Hosseinzadeh, Ghader; Maghari, Ali; Farniya, Seyed Morteza Famil; Keihan, Amir Homayoun; Moosavi-Movahedi, Ali A


    Interaction of quantum dots (QDs) and proteins strongly influenced by the surface characteristics of the QDs at the protein-QD interface. For a precise control of these surface-related interactions, it is necessary to improve our understanding in this field. In this regard, in the present work, the interaction between the insulin and differently functionalized ZnS quantum dots (QDs) were studied. The ZnS QDs were functionalized with various functional groups of hydroxyl (OH), carboxyl (COOH), amine (NH 2 ), and amino acid (COOH and NH 2 ). The effect of surface hydrophobicity was also studied by changing the alkyl-chain lengths of mercaptocarboxylic acid capping agents. The interaction between insulin and the ZnS QDs were investigated by fluorescence quenching, synchronous fluorescence, circular dichroism (CD), and thermal aggregation techniques. The results reveal that among the studied QDs, mercaptosuccinic acid functionalized QDs has the strongest interaction (∆G ° =-51.50kJ/mol at 310K) with insulin, mercaptoethanol functionalized QDs destabilize insulin by increasing the beta-sheet contents, and only cysteine functionalized QDs improves the insulin stability by increasing the alpha-helix contents of the protein, and. Our results also indicate that by increasing the alkyl-chain length of capping agents, due to an increase in hydrophobicity of the QDs surface, the beta-sheet contents of insulin increase which results in the enhancement of insulin instability. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Purification and Characterization of a New Thermostable, Haloalkaline, Solvent Stable, and Detergent Compatible Serine Protease from Geobacillus toebii Strain LBT 77

    Directory of Open Access Journals (Sweden)

    Wajdi Thebti


    Full Text Available A new thermostable, haloalkaline, solvent stable SDS-induced serine protease was purified and characterized from a thermophilic Geobacillus toebii LBT 77 newly isolated from a Tunisian hot spring. This study reveals the potential of the protease from Geobacillus toebii LBT 77 as an additive to detergent with spectacular proprieties described for the first time. The protease was purified to homogeneity by ammonium sulfate precipitation followed by Sephadex G-75 and DEAE-Cellulose chromatography. It was a monomeric enzyme with molecular weight of 30 kDa. The optimum pH, temperature, and NaCl for maximum protease activity were 13.0, 95°C, and 30%, respectively. Activity was stimulated by Ca2+, Mg2+, DTNB, β-mercaptoethanol, and SDS. The protease was extremely stable even at pH 13.25, 90°C, and 30% NaCl and in the presence of hydrophilic, hydrophobic solvents at high concentrations. The high compatibility with ionic, nonionic, and commercial detergents confirms the utility as an additive to cleaning products. Kinetic and thermodynamic characterization of protease revealed Km=1 mg mL−1,  Vmax=217.5 U mL−1, Kcat/Km=99 mg mL−1 S−1, Ea=51.5 kJ mol−1, and ΔG⁎=56.5 kJ mol−1.

  5. Rapid affinity-purification and physicochemical characterization of pumpkin (Cucurbita maxima) phloem exudate lectin. (United States)

    Narahari, Akkaladevi; Swamy, Musti J


    The chito-oligosaccharide-specific lectin from pumpkin (Cucurbita maxima) phloem exudate has been purified to homogeneity by affinity chromatography on chitin. After SDS/PAGE in the presence of 2-mercaptoethanol, the pumpkin phloem lectin yielded a single band corresponding to a molecular mass of 23.7 kDa, whereas ESI-MS (electrospray ionization MS) gave the molecular masses of the subunit as 24645 Da. Analysis of the CD spectrum of the protein indicated that the secondary structure of the lectin consists of 9.7% alpha-helix, 35.8% beta-sheet, 22.5% beta-turn and 32.3% unordered structure. Saccharide binding did not significantly affect the secondary and tertiary structures of the protein. The haemagglutinating activity of pumpkin phloem lectin was mostly unaffected in the temperature range 4-70 degrees C, but a sharp decrease was seen between 75 and 85 degrees C. Differential scanning calorimetric and CD spectroscopic studies suggest that the lectin undergoes a co-operative thermal unfolding process centred at approx. 81.5 degrees C, indicating that it is a relatively stable protein.

  6. Tryptophan exposure and accessibility in the chitooligosaccharide-specific phloem exudate lectin from pumpkin (Cucurbita maxima). A fluorescence study. (United States)

    Narahari, Akkaladevi; Swamy, Musti J


    The exposure and accessibility of the tryptophan residues in the chitooligosaccharide-specific pumpkin (Cucurbita maxima) phloem exudate lectin (PPL) have been investigated by fluorescence spectroscopy. The emission lambda(max) of native PPL, seen at 338nm was red-shifted to 348nm upon denaturation by 6M Gdn.HCl in the presence of 10mM beta-mercaptoethanol, indicating near complete exposure of the tryptophan residues to the aqueous medium, whereas a blue-shift to 335nm was observed in the presence of saturating concentrations of chitotriose, suggesting that ligand binding leads to a decrease in the solvent exposure of the tryptophan residues. The extent of quenching was maximum with the neutral molecule, acrylamide whereas the ionic species, iodide and Cs(+) led to significantly lower quenching, which could be attributed to the presence of charged amino acid residues in close proximity to some of the tryptophan residues. The Stern-Volmer plot for acrylamide was linear for native PPL and upon ligand binding, but became upward curving upon denaturation, indicating that the quenching occurs via a combination of static and dynamic mechanisms. In time-resolved fluorescence experiments, the decay curves could be best fit to biexponential patterns, for native protein, in the presence of ligand and upon denaturation. In each case both lifetimes systematically decreased with increasing acrylamide concentrations, indicating that quenching occurs predominantly via a dynamic process.

  7. Purification and characterization of cell suspensions peroxidase from cotton (Gossypium hirsutum L.). (United States)

    Kouakou, Tanoh Hilaire; Dué, Edmond Ahipo; Kouadio, N'guessan Eugène Jean Parfait; Niamké, Sébastien; Kouadio, Yatty Justin; Mérillon, Jean-Michel


    Two peroxidases, cPOD-I and rPOD-II, have been isolated and purified from cotton cell suspension and their biochemical characteristics studied. rPOD-II from R405-2000, a non-embryogenic cultivar, has higher activity than cPOD-I derived from Coker 312, which developed an embryogenic structure. The cPOD-I and rPOD-II had molecular mass of 39.1 and 64 kDa respectively, as determined by SDS-PAGE. Both enzymes showed high efficiency of interaction with the guaiacol at 25 mM. The optimal pH for cPOD-I and rPOD-II activity was 5.0 and 6.0, respectively. The enzyme had an optimum temperature of 25 degrees C and was relatively stable at 20-30 degrees C. The isoenzymes were highly inhibited by ascorbic acid, dithiothreitol, sodium metabisulfite, and beta-mercaptoethanol. Their activities were highly enhanced by Al(3+), Fe(3+), Ca(2+), and Ni(2+), but they were moderately inhibited by Mn(2+) and K(+). The enzyme lost 50% to 62% of its activity in the presence of Zn(2+) and Hg(2+).

  8. The putative cocaine receptor in striatum is a glycoprotein with thiol function

    International Nuclear Information System (INIS)

    Cao, C.J.; Young, M.M.; Wang, J.B.; Mahran, L.; Eldefrawi, M.E.


    Dopamine transporters of bovine and rat striata are identified by their specific [ 3 H] cocaine binding and cocaine-sensitive [ 3 H] dopamine ([ 3 H]DA) uptake. Both binding and uptake functions of bovine striatal transporters were potentiated by lectins. Concanavalin A (Con A) increased the velocity but did not change the affinity of the transporter for DA. On the other hand, ConA increased its affinity for cocaine without changing the number of binding sites. The data suggest that the DA transporter is a glycoprotein. Inorganic and organic mercury reagents inhibited both [ 3 H] cocaine binding, though they were all more potent inhibitors of the former. N-ethylmaleimide inhibited [ 3 H]DA uptake totally but [ 3 H]cocaine binding only partially. Also, N-pyrenemaleimide had different effects on uptake and binding, inhibiting uptake and potentiating binding. [ 3 H]DA uptake was not affected by mercaptoethanol up to 100 mM whereas [ 3 H]cocaine binding was inhibited by concentration above 10 mM. On the other hand, both uptake and binding were fairly sensitive to dimercaprol ( 10 mM). Loss of activity after treatment with the dithio reagents may be a result of reduction of a disulfide bond, which may affect the transporter conformation

  9. Purification and properties of a soluble reduced nicotinamide–adenine dinucleotide (phosphate) dehydrogenase from the hepatopancreas of Octopus vulgaris (United States)

    Di Prisco, G.; Casola, L.; Giuditta, A.


    1. The oxidation of NADH and NADPH catalysed by the soluble supernatant from the hepatopancreas of Octopus vulgaris is due to a single enzyme, which has been purified approximately 100-fold. The enzyme reacts rapidly with potassium ferricyanide, and more slowly with 2,6-dichlorophenol-indophenol. No activity is obtained with oxygen, cytochrome c, lipoic acid, vitamin K1, vitamin K3, ubiquinone-30, p-benzoquinone, 2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium chloride or methylene blue. 2. GSH, cysteine and mercaptoethanol stimulate the enzymic activity up to fivefold. GSSG is without any apparent effect. When stimulated by GSH the enzyme becomes sensitive to dicoumarol, which produces an inhibition competitive with respect to the activator. 3. The purified enzyme contains an acid-removable flavine component, which has been identified as FMN by spectrofluorimetry and chromatography in three solvent systems. After acid ammonium sulphate treatment the enzymic activity is lost, but it can be almost fully restored by incubation with FMN. FAD produces only a partial reactivation. PMID:4171422

  10. Biological and biochemical properties of human chorionic gonadotropin from urine of patients with hydatidiform mole and its radioimmunoassay

    International Nuclear Information System (INIS)

    Nishimura, Ryuichiro; Hamamoto, Tamotsu; Tanabe, Keizo; Takemori, Masayuki; Ashitaka, Yoshihiko


    Human chorionic gonadotropin (hCG) was extracted and purified from the urine of four patients with hydatidiform mole. The immunological activities of the hCG-hydatidiform mole by hCG radioimmunoassay (RIA) ranged from 9,380 to 9,700 IU/mg, and the biological activities measured by the immature rat ovarian weight method ranged from 7,250 to 7,780 IU/mg. The results of the amino acid compositions of all the hCG-hydatidiform moles were practically identical with those of hCG-normal pregnancies. The carbohydrate moiety of the hCG-hydatidiform mole was also suspected to be almost similar to that of hCG-normal pregnancies by the results of their in vitro and in vivo biological activities. It was demonstrated that hCG-hydatidiform mole was composed of α and β subunits (similar to a hCG-normal pregnancy) when hCG-hydatidiform mole was separated into subunits by SDS disc electrophoresis after treatment with mercaptoethanol. The RIA system of hCG-hydatidiform mole can be established. The concentrations of hCG in sera of normal pregnant women and patients with trophoblastic diseases assayed by hCG-hydatidiform mole RIA were equivalent to those obtained by a standard hCG RIA. Hence, a standard hCG-immunoassay method used in the management of trophoblastic diseases is considered reasonable so far as the immunoantigenecity of hCG is concerned. (author)

  11. A unique uracil-DNA binding protein of the uracil DNA glycosylase superfamily. (United States)

    Sang, Pau Biak; Srinath, Thiruneelakantan; Patil, Aravind Goud; Woo, Eui-Jeon; Varshney, Umesh


    Uracil DNA glycosylases (UDGs) are an important group of DNA repair enzymes, which pioneer the base excision repair pathway by recognizing and excising uracil from DNA. Based on two short conserved sequences (motifs A and B), UDGs have been classified into six families. Here we report a novel UDG, UdgX, from Mycobacterium smegmatis and other organisms. UdgX specifically recognizes uracil in DNA, forms a tight complex stable to sodium dodecyl sulphate, 2-mercaptoethanol, urea and heat treatment, and shows no detectable uracil excision. UdgX shares highest homology to family 4 UDGs possessing Fe-S cluster. UdgX possesses a conserved sequence, KRRIH, which forms a flexible loop playing an important role in its activity. Mutations of H in the KRRIH sequence to S, G, A or Q lead to gain of uracil excision activity in MsmUdgX, establishing it as a novel member of the UDG superfamily. Our observations suggest that UdgX marks the uracil-DNA for its repair by a RecA dependent process. Finally, we observed that the tight binding activity of UdgX is useful in detecting uracils in the genomes. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. Radiolabelling of antibodies with technetium-99m and their clinical evaluation

    International Nuclear Information System (INIS)

    Varvarigou, A.D.


    Our study referred mainly to the comparative evaluation of two different methods, one direct and the other indirect, for the labelling of MoAbs with 99 Tc m . With the direct method, disulphide bridges were cleaved by the sue of 2-mercaptoethanol (2-ME), as reductant, while with the indirect method, the antibody was coupled to 2-iminothiolane (2-IM). The latter reacts with the amine groups of the protein's lysine molecules, thus creating free thiol groups, capable of binding reduced 99 Tc m . In both cases a preformed intermediate chelate was used for the 99 Tc m exchange. The 99 Tc m labelled species were analyzed by Instant Thin Layer Chromatography (ITLC), High Performance Liquid Chromatography (HPLC), and Polyacrylamide Gel Electrophoresis (PAGE). We investigated the influence of the labelling systems on MoAbs' integrity, as well as the ability of the labelled species to tag on human cancer cells. The biodistribution of the labelled derivatives was comparatively evaluated in normal male Swiss mice and the stability of labelling was measured by cysteine challenge. The in vitro stability was comparatively studied at different temperatures and by incubation with human serum for 24 h

  13. Cell wall proteinaceous components in isolates of Candida albicans and non-albicans species from HIV-infected patients with oropharyngeal candidiasis. (United States)

    López-Ribot, J L; Kirkpatrick, W R; McAtee, R K; Revankar, S G; Patterson, T F


    Oropharyngeal candidiasis (OPC) remains a common opportunistic infection in HIV-infected patients. Candida albicans is the most frequent causative agent of OPC. However, non-albicans spp. are being increasingly isolated. Candidal cell wall proteins and mannoproteins play important roles in the biology and patogenesis of candidiasis. In the present study, we have analyzed the proteinaceous components associated with cell wall extracts from C. albicans, Candida tropicalis, Candida pseudotropicalis, Candida krusei, Candida glabrata, Candida parapsilosis, Candida guilliermondii and Candida rugosa obtained from HIV-infected patients with recurrent OPC. Cell wall proteinaceous components were extracted with beta-mercaptoethanol and analyzed using electrophoresis, immunoblotting (with antisera generated against C. albicans cell wall components, and with serum samples and oral saline rinses from patients with OPC), and lectin-blotting (concanavalin A) techniques. Numerous molecular species were solubilized from the various isolates. Major qualitative and quantitative differences in the polypeptidic and antigenic profiles associated with the cell wall extracts from the different Candida spp. were discernible. Some of the antibody preparations generated against C. albicans cell wall components were able to recognize homologous materials present in the extracts from non-albicans spp. Information on cell wall antigens of Candida species may be important in the therapy and prevention of HIV-related OPC.

  14. Properties of Folate Binding Protein Purified from Cow’s Milk

    Directory of Open Access Journals (Sweden)



    Full Text Available Folic acid played an important role in the metabolism of the body. To measure the serum folic acid levels could use the folate binding protein (FBP from cow’s milk with a technique analogous to ELISA. The aims of this study were to identify characteristics of FBP from cow’s milk and binding capacity of FBP to folic acid and to purify FBP from other whey protein passed through DEAE-cellulose chromatography column. Each of DEAE-cellulose peaks was passed in affinity chromatography column. FBP was released from affinity column with sodium acetate buffer pH 3.5. The purity of obtained FBP was demonstrated by a single spot in SDS-PAGE analysis and the estimated molecular weight of FBP was around 31 kDa. Our study indicated that 1 mol FBP bound 1 mol folic acid. Alkylation with iodoacetic acid decreased the binding capacity of FBP which suggested the presence of a–SH or imidazol group in its active site. The importance of disulfide bridge was proven by decreasing of folate binding capacity of FBP after -mercaptoethanol treatment. In contrary, the folate binding didn need Ca2+ ion, as indicated by EDTA test which gave the same result as control.

  15. Comparison between immune responses and resistance induced in BALB/c mice vaccinated with RB51 and Rev. 1 vaccines and challenged with Brucella melitensis bv. 3. (United States)

    Hamdy, M E R; El-Gibaly, S M; Montasser, A M


    BALB/c mice were immunized with live rough Brucella abortus RB51 or smooth Brucella melitensis Rev. 1 vaccines and challenged with a B. melitensis field strain. Protection was assessed by a variety of serological tests and recovery of vaccinal and challenge strains by culture. Mice vaccinated with RB51 gave negative results in the conventional serological tests prior to challenge, namely; standard tube agglutination test (SAT), Rose Bengal plate test (RBPT), buffered acidified plate antigen test (BAPAT), and mercaptoethanol test (MET). Sero-conversion took place to a whole-cell bacterial buffered RB51 antigen after vaccination and persisted for 7 weeks post-vaccination. Mice challenged with B. melitensis were assessed for bacterial load and immune response for 12 weeks after challenge. Protection units were showed that Rev. 1 vaccine was superior to RB51 vaccine in protection of mice against B. melitensis. However, RB51 vaccine has the advantage that it would not elicit antibodies to standard serological tests based on the LPS O antigen. RB51 vaccine could therefore be used for control of B. melitensis infection and avoid confusion in the use of standard sero-diagnostic tests.

  16. Application of enzyme linked immunosorbent assays for the diagnosis of bovine brucellosis in Rio Grande do Sul, Brazil

    International Nuclear Information System (INIS)

    Poester, F.P.; Ramos, E.T.; Thiesen, S.V.


    The results of an indirect ELISA (I-ELISA) and a competitive ELISA (C-ELISA) for detection of antibody to Brucella abortus in cattle were compared with those of conventional serological tests. The sensitivity of I-Elisa using 230 sera from infected animals was 98.6% and the specificity in 720 sera from brucellosis-free animals was 98.2%. The C-ELISA when tested in 94 positive sera and 91 negative sera was 100% sensitive and specific. The relative sensitivity and specificity of I-ELISA compared to C-ELISA in 582 sera from groups of animals vaccinated with S19 by different routes and doses was respectively 64% and 100%. When comparing five serological tests on sera from vaccinated animals, the specificity was 94% for C-ELISA, 93% for 2-mercaptoethanol (2ME), 88% for Rose Bengal Plate test (RBPT), 84% for Slow Tube Agglutination test (SAT) and 34% for I-ELISA. It can be concluded from this study that the RBPT gave less false positive results than the I-ELISA in vaccinated animals and the C-ELISA is a good confirmatory test with the advantage of distinguishing the antibody response due to vaccination from that resulting from infection with Brucella abortus. (author)

  17. Establishment and characterization of a chicken mononuclear cell line. (United States)

    Qureshi, M A; Miller, L; Lillehoj, H S; Ficken, M D


    A new chicken mononuclear cell line (MQ-NCSU) has been established. The starting material used to initiate this cell line was a transformed spleen from a female Dekalb XL chicken which had been experimentally challenged with the JM/102W strain of the Marek's disease virus. After homogenization, a single cell suspension of splenic cells was cultured using L.M. Hahn medium supplemented with 10 microM 2-mercaptoethanol. Under these culture conditions, a rapidly proliferating cell was observed and then expanded after performing limiting dilution cultures. These cells were moderately adherent and phagocytic for sheep red blood cells and Salmonella typhimurium. When tested against a panel of monoclonal antibodies (mAb) using the flow cytometry, MQ-NCSU cells stained readily with anti-chicken monocyte specific (K-1) mAb but did not stain with mAb detecting T-helper, T-cytotoxic/suppressor, and NK cells. MQ-NCSU cells expressed very high levels of Ia antigens and transferrin receptors. In addition, cell-free supernatant obtained from MQ-NCSU culture contained a factor which exhibited cytolytic activity against tumor cell targets. Based on their cultural, morphological, and functional characteristics and mAb reactivity profile, we conclude that MQ-NCSU cell line represents a malignantly-transformed cell which shares features characteristic of cells of the mononuclear phagocyte lineage.

  18. Diagnostic Efficacy of Modified Coagglutination Test in the Diagnosis of Human Brucellosis

    Directory of Open Access Journals (Sweden)

    Mohite S.T


    Full Text Available Background: Laboratory help is must for thediagnosis of human brucellosis due to proteanclinical manifestations. As culture is hazardous,time consuming and less sensitive, serologicaltests are preferred for the diagnosis. Aggluti-nation tests like Rose Bengal PlateTest (RBPT, Serum Agglutination tests (SAT,2-Mercaptoethanol test (2-ME that are com-monly employed for the diagnosis either lacksensitivity or specificity. Coombs test andBrucellacapt though are sensitive and specific,workout costly. Therefore, modifiedcoagglutination test was developed and its di-agnostic efficacy was evaluated. Aims and Ob-jectives: To develop modified coagglutinationtest for the diagnosis of human brucellosis andcompare it with Coombs test. Materials andMethods: Serum samples collected from 191brucellosis patients and 100 controls were sub-jected to 2-ME, Coombs test and modifiedcoagglutination test (MCOAG. Blood culturewas performed by Castaneda’s method in all thepatients. Results: Significant difference in thepositivity rate was seen between MCOAG and2-ME. The results of MCOAG were compa-rable with Coombs test. Conclusions: Modi-fied coagglutination test is a better option toCoombs test for the serodiagnosis of brucel-losis in resource constrained countries as it issensitive, specific and cost effective.

  19. Microbial reclamation of shellfish wastes for the production of chitinases. (United States)

    Wang, S -L.; Hwang, J -R.


    Shrimp and crab shell powder (SCSP), prepared by treating shellfish processing waste with boiling and crushing, was used as a substrate for isolating chitinolytic microorganisms. Three potential strains (E1, J1, and J1-1) were isolated and identified as Bacillus cereus, B. alvei, and B. sphaericus, respectively. Three extracellular chitinases (FB1, FB2, and FB3) were purified from the culture supernatants of Bacillus cereus E1, B. alvei J1, and B. sphaericus J1-1, respectively. The molecular weights of FB1, FB2, and FB3 were 71,000, 71,000, and 65,000, respectively, by SDS-PAGE. The pIs for FB1, FB2, and FB3 were 7.1, 7.2, and 7.4, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of FB1 were pH 9, 50 degrees C, pH 7 to 10, and 70 degrees C; those of FB2 were pH 9, 60 degrees C, pH 5 to 9, and 70 degrees C; and those of FB3 were pH 7, 50 degrees C, pH 5 to 9, and 60 degrees C. The activities of all enzymes were strongly inhibited by Hg(2+) and completely inhibited by glutathione, dithiothreitol, and 2-mercaptoethanol.

  20. Production, Purification, and Characterization of a Major Penicillium glabrum Xylanase Using Brewer's Spent Grain as Substrate

    Directory of Open Access Journals (Sweden)

    Adriana Knob


    Full Text Available In recent decades, xylanases have been used in many processing industries. This study describes the xylanase production by Penicillium glabrum using brewer's spent grain as substrate. Additionally, this is the first work that reports the purification and characterization of a xylanase using this agroindustrial waste. Optimal production was obtained when P. glabrum was grown in liquid medium in pH 5.5, at 25 °C, under stationary condition for six days. The xylanase from P. glabrum was purified to homogeneity by a rapid and inexpensive procedure, using ammonium sulfate fractionation and molecular exclusion chromatography. SDS-PAGE analysis revealed one band with estimated molecular mass of 18.36 kDa. The optimum activity was observed at 60 °C, in pH 3.0. The enzyme was very stable at 50 °C, and high pH stability was verified from pH 2.5 to 5.0. The ion Mn2+ and the reducing agents β-mercaptoethanol and DTT enhanced xylanase activity, while the ions Hg2+, Zn2+, and Cu2+ as well as the detergent SDS were strong inhibitors of the enzyme. The use of brewer's spent grain as substrate for xylanase production cannot only add value and decrease the amount of this waste but also reduce the xylanase production cost.

  1. Isolation and characterization of horridum toxin with arginine ester hydrolase activity from Heloderma horridum (beaded lizard) venom. (United States)

    Nikai, T; Imai, K; Sugihara, H; Tu, A T


    A hemorrhagic toxin with lethal and arginine ester hydrolytic activities was isolated from Heloderma horridum (beaded lizard) venom by Sephadex G-75, DEAE-Sephacel, and Q-Sepharose column chromatography. The hemorrhagic toxin was shown to be homogeneous as demonstrated by a single band on acrylamide gel electrophoresis and immunodiffusion. Its molecular weight is approximately 31,000 with an isoelectric point of 3.9. Hemorrhagic, lethal, and benzoyl-L-arginine ethyl ester hydrolytic activities of this preparation were inhibited by diisopropyl fluorophosphate (DFP), N-bromosuccinimide, and beta-mercaptoethanol, suggesting that serine, tryptophan, and disulfide bonds are involved in these activities. Also there was an increase in creatine kinase activity in mice serum which is an indicator that the toxin is involved in muscle damage. This protein was stable to heat and pH ranges between 2 and 11. The Michaelis constant (Km), for benzoyl-L-arginine ethyl ester, and inhibition constant (Ki), for DFP, were found to be 6.9 X 10(-3) and 1.93 X 10(-4) M, respectively.

  2. Isolation and characterization of arginine ester hydrolase from Heloderma horridum (beaded lizard) venom. (United States)

    Nikai, T; Imai, K; Komori, Y; Sugihara, H


    1. An arginine ester hydrolase was isolated from Heloderma horridum (beaded lizard) venom by Sephadex G-75, DEAE-Sephacel and Q-Sepharose column chromatography, resulting in 5.4 mg of purified enzyme from 320.0 mg of crude venom. 2. The enzyme was shown to be homogeneous by both SDS and non-SDS disc electrophoresis on polyacrylamide gel at pH 8.3. 3. The enzyme possesses arginine ester hydrolase and transglutaminase-like activities, but did not exhibit clotting activity. 4. Molecular weight was determined to be ca 29 kDa, with an isoelectric point of 4.4. 5. The enzyme was stable to heat treatment (95 degrees C, 10 min) and to pH changes over the range 2-11. 6. The arginine ester hydrolase was inactivated by diisopropylfluorophosphate (DFP), beta-mercaptoethanol and N-bromosuccinimide, suggesting that serine, disulfide bonds and tryptophan are involved in enzymatic activity. 7. Amino terminal sequences were determined and appear to be similar to porcine pancreatic kallikrein.

  3. "Turn-on" fluorescence detection of lead ions based on accelerated leaching of gold nanoparticles on the surface of graphene. (United States)

    Fu, Xiuli; Lou, Tingting; Chen, Zhaopeng; Lin, Meng; Feng, Weiwei; Chen, Lingxin


    A novel platform for effective "turn-on" fluorescence sensing of lead ions (Pb(2+)) in aqueous solution was developed based on gold nanoparticle (AuNP)-functionalized graphene. The AuNP-functionalized graphene exhibited minimal background fluorescence because of the extraordinarily high quenching ability of AuNPs. Interestingly, the AuNP-functionalized graphene underwent fluorescence restoration as well as significant enhancement upon adding Pb(2+), which was attributed to the fact that Pb(2+) could accelerate the leaching rate of the AuNPs on graphene surfaces in the presence of both thiosulfate (S(2)O(3)(2-)) and 2-mercaptoethanol (2-ME). Consequently, this could be utilized as the basis for selective detection of Pb(2+). With the optimum conditions chosen, the relative fluorescence intensity showed good linearity versus logarithm concentration of Pb(2+) in the range of 50-1000 nM (R = 0.9982), and a detection limit of 10 nM. High selectivity over common coexistent metal ions was also demonstrated. The practical application had been carried out for determination of Pb(2+) in tap water and mineral water samples. The Pb(2+)-specific "turn-on" fluorescence sensor, based on Pb(2+) accelerated leaching of AuNPs on the surface of graphene, provided new opportunities for highly sensitive and selective Pb(2+) detection in aqueous media.

  4. Selective recovery of gold and other metal ions from an algal biomass

    Energy Technology Data Exchange (ETDEWEB)

    Darnall, D.W.; Greene, B.; Henzl, M.T.; Hosea, J.M.; McPherson, R.A.; Sneddon, J.; Alexander, M.D.


    The authors observed that the pH dependence of the binding of Au/sup 3 +/, Ag/sup +/, and Hg/sup 2 +/ to the algae Chlorella vulgaris is different than the binding of other metal ions. Between pH 5 and 7, a variety of metal ions bind strongly to the cell surface. Most of these algal-bound metal ions can be selectively desorbed by lowering the pH to 2; however, Au/sup 3 +/, Hg/sup 2 +/, and Ag/sup +/ are all bound strongly at pH 2. Addition of a strong ligand at different pHs is required to elute these ions from the algal surface. Algal-bound gold and mercury can be selectively eluted by using mercaptoethanol. An elution scheme is demonstrated for the binding and selective recovery of Cu/sup 2 +/, Zn/sup 2 +/, Au/sup 3 +/, and Hg/sup 2 +/ from an equimolar mixture. 20 references, 2 figures.

  5. Excretion and retention of cadmium, zinc, and mercury by rabbit kidney

    International Nuclear Information System (INIS)

    Foulkes, E.C.


    Mean renal artery-to-vein transit times (anti t) of 109 Cd, 65 Zn, or 203 Hg were compared to those of Evans blue (EB) and inulin in rabbits. All three elements were fully recovered in venous plasma, where they slightly preceded inulin, although anti t exceeded anti t/sub EB/. Competition for the injected metals is suggested between circulating plasma protein and fixed endothelial ligands. Upon addition of mercaptoethanol (ME) to the bolus injection, 109 Cd appeared in urine; total recovery in blood and urine dropped to two-thirds of the dose reaching the kidney. Renal retention of the remaining one-third was reduced by 30% after ureteral occlusion. Both luminal and peritubular cell membranes thus are involved in Cd uptake, a conclusion in agreement with the previous finding of lesions at those two sites. In the presence of ME, essentially all 203 Hg was retained in the kidney but renal handling of Zn was not affected. The stronger chelator EDTA led to recoveries of 109 Cd in blood and urine approaching those of inulin. These results bear on the role of metal ligands in determining renal excretion and accumulation of heavy metals. 17 references, 2 figures, 4 tables

  6. Effect of size and shell: Enhanced optical and surface properties of CdS, ZnS and CdS/ZnS quantum dots (United States)

    Kumar, Hitanshu; Barman, P. B.; Singh, Ragini Raj


    This study reports systematic structural, optical and surface studies on wurtzite CdS, ZnS and CdS/ZnS quantum dots where the effects of size and shell thickness were analysed. Size tunable, stable and luminescent quantum dots (QDs) and their core/shell structures were synthesized by wet chemical growth method at low temperature using 2-mercaptoethanol as a stabilizer. Formation of non-agglomerated wurtzite QDs with reduced particle sizes have been confirmed from x-ray diffraction and transmission electron microscopy studies. Size dependent blue shifts have been observed by absorbance spectroscopy and discussed on the basis of various theoretical models. Significantly enhanced luminescence and monochromaticity have been observed in QDs due to particle size reduction and on core/shell structure formation. Fourier transform infrared spectroscopy indicates that OH, CH2 and C-O functional groups are present on the QDs surfaces and for this reason these QDs can be used in various biological applications.

  7. Low-Temperature Growth of Inverted Hexagonal ZnS/CdS Quantum Dots: Functional and Luminescence Properties (United States)

    Kumar, Hitanshu; Barman, P. B.; Singh, Ragini Raj


    A novel low-temperature wet chemical method is proposed for direct growth of type-I inverted hexagonal ZnS/CdS quantum dots (QD). 2-Mercaptoethanol (2-ME) was used as a capping agent for confinement by passivation, and also helped to prevent agglomeration of the QD. The band gap calculated from optical absorption spectra was 2.63 eV for the smallest core/shell QD. Absorption edge onset and results from transmission electron microscopy revealed formation of inverted core/shell QD. X-ray diffraction studies revealed the ZnS/CdS had a stable hexagonal crystal structure at low temperature. The average diameter of the core/shell QD was 4.2 nm. Tunable luminescence with substantial tunability was revealed by study of the photoluminescence of the inverted ZnS/CdS quantum dots. Surface passivation of ZnS/CdS QD by 2-ME was confirmed by Fourier-transform infrared spectroscopy.

  8. Different effects of reducing agents on ω-conotoxin GVIA inhibition of [3H]-acetylcholine release from rat cortical slices and guinea-pig myenteric plexus (United States)

    Casali, T A A; Gomez, R S; Moraes-Santos, T; Romano-Silva, M A; Prado, M A M; Gomez, M V


    The effect of reducing reagents on ω-conotoxin GVIA (ω-CgTX) inhibition of the release of [3H]-acetylcholine ([3H]-ACh) induced by tityustoxin, K+ 50 mM and electrical stimulation was investigated in rat brain cortical slices.In cortical slices the inhibition of tityustoxin or electrically-stimulated [3H]-ACh release by ω-CgTX was dramatically increased by reducing reagents ascorbate or β-mercaptoethanol. Dehydroascorbic acid did not substitute for ascorbateDepolarization induced by K+ 50 mM caused [3H]-ACh release from cortical slices which was not inhibited by ω-CgTX, even in the presence of ascorbate.In the guinea-pig myenteric plexus, ω-CgTX inhibition of the tityustoxin induced release of [3H]-ACh was independent of ascorbate.It is suggested that N-type-like calcium channels in guinea-pig myenteric plexus may have pharmacological/biochemical diversity from similar channels of rat cerebral cortex. PMID:9117104

  9. 188Re-IorCEA1 as radioimmunotherapeutical agent: Preparation and evaluation

    International Nuclear Information System (INIS)

    Pimental, G.


    Effective immunotherapy of human malignancies requires the selective delivery of these agents to relevant antigen-expressing tumour cells. A variety of strategies have been investigated to achieve this objective. It has very clearly established that ior-CEA1 monoclonal antibody is an extremely good agent for immunoscintigraphy with a high sensitivity (94%) and specificity (87%) (OLIVA 94, OLIVA 95). Ior-CEA1 is hence a good candidate for development as a therapeutic agent. Moreover, it is an attractive novel protein, which conserves its biological properties, despite chemical treatment. Radiolabelling of ior-CEA1 is accomplished by a direct method using 2-mercaptoethanol as the reducing agent. Two different weak chelating agents were employed, which had been reported in other published works before. All experiments carried out with MDP showed low labelling yields and high precipitate formation, even while SnF 2 , which has better redox properties. The best labelling yields were attained with low citric acid concentration, as well as with 400 μg of SnF 2 at pH 3. The optimised method gave high labelling yields (∼ 95 %). The best labelling yields were attained with low citric acid concentration, which was used as a weak transchelation ligand. Ascorbic acid and Albumin could be used as the stabilising agents in order to achieve less than 3,5% dissociation

  10. Serological study of brucellosis in Argentine Creole sheep. (United States)

    López, Gustavo E; Peña, Sabrina; Escobar, Gabriela I; Hasan, Déborah B; Lucero, Nidia E


    Ovine cattle was introduced into America during the Spanish conquest with the second journey of Columbus to the Antilles and was disseminated throughout the region. In 1587, sheep were introduced into Argentina, later developing into the "Creole" breed. We selected 486 animals from different Argentine provinces with the aim of determining the serological status of brucellosis caused by Brucella melitensis and Brucella ovis. For the detection of antibodies against smooth Brucella spp., the Rose Bengal test (RBT) was performed as screening test while the serum agglutination test (SAT) and 2 mercapto-ethanol (2ME) were run as a confirmatory technique. Moreover, for the detection of antibodies against rough Brucella spp., we used the rapid slide agglutination test (RSAT) for screening and an indirect ELISA (IELISA) as confirmatory assay. This study showed that the total positive percentage of brucellosis due to B. ovis was 2.9%. Excluding the animals mixed with the Suffolk breed; seropositivity would be 0.6%. All animals tested negative for brucellosis caused by B. melitensis. Copyright © 2017 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  11. Effect of disulfide and sulfhydryl reagents on abortive and productive elongation catalyzed by ''Escheridia coli'' RNA polymerase

    International Nuclear Information System (INIS)

    Radlowski, M.; Job, D.


    The effect of disulfide and sulfhydryl reagents on the rate of abortive and productive elongation has been studied using ''Escherichia coli'' RNA polymerase holoenzyme and poly[d(A-T)] as template. In the presence of UTP as a single substrate and UpA as a primer, the enzyme catalyzed efficiently the synthesis of the trinucleotide product UpApU. Incubation of RNA polymerase with 1 mM 2-mercaptoethanol resulted in a 5-fold increase of the rate of UpApU synthesis. In contrast, incubation of the enzyme with 1 mM 5,5'-dithio-bis(2-nitrobenzoic) acid resulted in a 6-fold decrease of the rate of abortive elongation. Determination of the steady state kinetic constants associated with UpApU synthesis disclosed that the disulfide and sulfhydryl reagents mainly affected the rate of UpApU release from the ternary transcription complexes and therefore influenced the stability of such complexes. (author). 15 refs, 1 fig., 1 tab

  12. Cloning, Expression and 3D Structure Prediction of Chitinase from Chitinolyticbacter meiyuanensis SYBC-H1

    Directory of Open Access Journals (Sweden)

    Zhikui Hao


    Full Text Available Two CHI genes from Chitinolyticbacter meiyuanensis SYBC-H1 encoding chitinases were identified and their protein 3D structures were predicted. According to the amino acid sequence alignment, CHI1 gene encoding 166 aa had a structural domain similar to the GH18 type II chitinase, and CHI2 gene encoding 383 aa had the same catalytic domain as the glycoside hydrolase family 19 chitinase. In this study, CHI2 chitinase were expressed in Escherichia coli BL21 cells, and this protein was purified by ammonium sulfate precipitation, DEAE-cellulose, and Sephadex G-100 chromatography. Optimal activity of CHI2 chitinase occurred at a temperature of 40 °C and a pH of 6.5. The presence of metal ions Fe3+, Fe2+, and Zn2+ inhibited CHI2 chitinase activity, while Na+ and K+ promoted its activity. Furthermore, the presence of EGTA, EDTA, and β-mercaptoethanol significantly increased the stability of CHI2 chitinase. The CHI2 chitinase was active with p-NP-GlcNAc, with the Km and Vm values of 23.0 µmol/L and 9.1 mM/min at a temperature of 37 °C, respectively. Additionally, the CHI2 chitinase was characterized as an N-acetyl glucosaminidase based on the hydrolysate from chitin. Overall, our results demonstrated CHI2 chitinase with remarkable biochemical properties is suitable for bioconversion of chitin waste.

  13. High-quality RNA preparation from Rhodosporidium toruloides and cDNA library construction therewith. (United States)

    Yang, Fan; Tan, Haidong; Zhou, Yongjin; Lin, Xinping; Zhang, Sufang


    Oleaginous yeast Rhodosporidium toruloides is an excellent microbial lipid producer. Therefore, it is important to develop molecular biology tools to understand the basic mechanism for lipid accumulation and further manipulate the microorganism. High-quality RNA extraction from R. toruloides is particularly challenging due to high level of polysaccharides, lipids, and other secondary metabolites. To obtain an optimal protocol for RNA extraction from R. toruloides, four methods were evaluated. Large difference in RNA yield and quality among these protocols was found. The optimum method was modified RNAiso procedure, where RNA was isolated using liquid nitrogen-RNAiso method with salt precipitation and the addition of β-mercaptoethanol. This method consistently recovered RNA in good quality with high yield. Around 297 μg total RNA per gram of cells was obtained with an average purity measured as A₂₆₀/A₂₈₀ of 2.09. A titer of 10⁵ cfu/ml could be harvested to construct a full-length cDNA library with the RNA sample in this quality. Electrophoresis gel analysis indicated the fragments ranged from 200 bp to 4.0 kb, with the average size of 1000 bp. Randomly picked clones showed the recombination efficiency at 80%. These results showed that RNA of R. toruloides was successfully extracted for the first time using the modified RNAiso method, and the cDNA library was appropriate for screening the genes related to lipid accumulation.

  14. Purification of restriction endonuclease from Acetobacter aceti IFO 3281 (AatII) and its properties. (United States)

    Sato, H; Suzuki, T; Yamada, Y


    The restriction endonuclease AatII was purified from cell-free extracts of Acetobacter aceti IFO 3281 by streptomycin treatment, ammonium sulfate fractionation, combined column chromatographies on DEAE-Toyopearl 650S, heparin-Sepharose CL-6B and DEAE-Sepharose CL-6B and FPLC on Mono Q and on Superose 12 (gel filtration). The purified enzyme was homogeneous on SDS-polyacrylamide gel disk electrophoresis. The relative molecular mass of the purified enzyme was 190,000 daltons by gel filtration. The SDS-polyacrylamide gel disk electrophoresis gave the relative molecular mass of 47,500 daltons. These data indicated that the purified, native enzyme is a tetramer (190,000 daltons) composed of four 47,500-dalton subunits. The isoelectric point of the enzyme was 6.0. The purified enzyme was intensely activated by manganese ion (50-fold increase or more when compared with magnesium ion). The enzyme worked best at 37 degrees C and pH 8.5 in a reaction mixture (50 microliters) containing 1.0 micrograms lambda DNA, 10 mM Tris-HCl, 7 mM 2-mercaptoethanol, 7 mM MnCl2 and 50 mM NaCl. The enzyme recognizes the same palindromic hexanucleotide sequence 5'-GACGTC-3', cuts between T and C and produces a 3'-tetranucleotide extension in the presence of MnCl2, as it does in the presence of MgCl2.

  15. Disulfide bond within mu-calpain active site inhibits activity and autolysis. (United States)

    Lametsch, René; Lonergan, Steven; Huff-Lonergan, Elisabeth


    Oxidative processes have the ability to influence mu-calpain activity. In the present study the influence of oxidation on activity and autolysis of mu-calpain was examined. Furthermore, LC-MS/MS analysis was employed to identify and characterize protein modifications caused by oxidation. The results revealed that the activity of mu-calpain is diminished by oxidation with H2O2 in a reversible manner involving cysteine and that the rate of autolysis of mu-calpain concomitantly slowed. The LC-MS/MS analysis of the oxidized mu-calpain revealed that the amino acid residues 105-133 contained a disulfide bond between Cys(108) and Cys(115). The finding that the active site cysteine in mu-calpain is able to form a disulfide bond has, to our knowledge, not been reported before. This could be part of a unique oxidation mechanism for mu-calpain. The results also showed that the formation of the disulfide bond is limited in the control (no oxidant added), and further limited in a concentration-dependent manner when beta-mercaptoethanol is added. However, the disulfide bond is still present to some extent in all conditions indicating that the active site cysteine is potentially highly susceptible to the formation of this intramolecular disulfide bond.

  16. Simple, high-yield purification of xanthine oxidase from bovine milk. (United States)

    Ozer, N; Müftüoglu, M; Ataman, D; Ercan, A; Ogüs, I H


    Xanthine oxidase, a commercially important enzyme with a wide area of application, was extracted from fresh milk, without added preservatives, using toluene and heat. The short purification procedure, with high yield, consisted of extraction, ammonium sulfate fractionation, and DEAE-Sepharose (fast flow) column chromatography. Xanthine oxidase was eluted as a single activity peak from the column using a buffer gradient. The purification fold, specific activity and yield for the purified xanthine oxidase were 328, 10.161 U/mg and 69%, respectively. The enzyme was concentrated by ultrafiltration, although 31% of the activity was lost during concentration, no change in specific activity was observed. Activity and protein gave coincident staining bands on native polyacrylamide gels. The intensity and the number of bands were dependent on the oxidative state(s) of the enzyme; reduction by 2-mercaptoethanol decreased the intensity of the slow-moving bands and increased the intensity of the fastest-moving band. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two major bands (molecular masses of 152 and 131 kDa) were observed, accounting for > or = 95% of xanthine oxidase. Native- and SDS-PAGE showed that the purified xanthine oxidase becomes a heterodimer due to endogenous proteases.

  17. New procedure for the control of the treatment of industrial effluents to remove volatile organosulfur compounds. (United States)

    Boczkaj, Grzegorz; Makoś, Patrycja; Fernandes, André; Przyjazny, Andrzej


    We present a new procedure for the determination of volatile organosulfur compounds in samples of industrial effluents using dispersive liquid-liquid microextraction and gas chromatography with flame photometric detection. Initially, the extraction parameters were optimized. These included: type and volume of extraction solvent, volume of disperser solvent, salting out effect, pH, time and speed of centrifugation as well as extraction time. The procedure was validated for 30 compounds. The developed procedure has low detection limits of 0.0071-0.49 μg/L and a good precision (relative standard deviation values of 1.2-5.0 and 0.6-4.1% at concentrations of 1 and 10 μg/L, respectively). The procedure was used to determine the content of volatile organosulfur compounds in samples of effluents from the production of bitumens before and after chemical treatment, in which six compounds were identified, including 2-mercaptoethanol, thiophenol, thioanisole, dipropyl disulfide, 1-decanethiol, and phenyl isothiocyanate at concentrations ranging from 0.47 to 8.89 μg/L. Problems in the determination of organosulfur compounds related to considerable changes in composition of the effluents, increase in concentration of individual compounds and appearance of secondary pollutants during effluent treatment processes are also discussed. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Fatal toxoplasmosis in free-ranging endangered 'Alala from Hawaii (United States)

    Work, Thierry M.; Massey, J. Gregory; Rideout, Bruce A.; Gardiner, Chris H.; Ledig, David B.; Kwok, O.C.H.; Dubey, J.P.


    The ‘Alala (Corvus hawaiiensis) is the most endangered corvid in the world, and intensive efforts are being made to reintroduce it to its former native range in Hawaii. We diagnosed Toxoplasma gondii infection in five free-ranging ‘Alala. One ‘Alala, recaptured from the wild because it was underweight and depressed, was treated with diclazuril (10 mg/kg) orally for 10 days. Antibodies were measured before and after treatment by the modified agglutination test (MAT) using whole T. gondii tachyzoites fixed in formalin and mercaptoethanol. The MAT titer decreased four-fold from an initial titer of 1:1,600 with remarkable improvement in physical condition. Lesions of toxoplasmosis also were seen in two partially scavenged carcasses and in a third fresh intact carcass. Toxoplasma gondii was confirmed immunohistochemically by using anti-T. gondii specific serum. The organism was also cultured by bioassay in mice from tissues of one of these birds and the brain of a fifth ‘Alala that did not exhibit lesions. The life cycle of the parasite was experimentally completed in cats. This is the first record of toxoplasmosis in ‘Alala, and the parasite appears to pose a significant threat and management challenge to reintroduction programs for ‘Alala in Hawaii.

  19. Preparation of silver nanowires coated with TiO2 using chemical binder and their applications as photoanodes in dye sensitized solar cell (United States)

    Jang, Inseok; Kang, Taeho; Cho, Woohyung; Kang, Yong Soo; Oh, Seong-Geun; Im, Seung Soon


    In this study, the core-shell structured Ag@TiO2 wire was prepared for application to dye-sensitized solar cell (DSSC). The Ag nanowire, having an excellent electrical conductivity, was synthesized by using the facile microwave-assisted polyol reduction process. The diameter and length of Ag wires were 40-50 nm and 20-30 μm, respectively, and the face-centered cubic silver crystal structure was obtained. In the presence of 2-mercaptoethanol as a chemical binder, the entire surface of Ag wire was coated with the TiO2 shell, which has thickness of 20 nm, through solvothermal method. The crystalline structure of TiO2 shell was the anatase phase possessing an advantage to achieve the high efficiency in DSSC. The core-shell structured Ag@TiO2 wire exhibited the high thermal stability. The high conversion efficiency (5.56%) in fabricated device with Ag@TiO2 electrode, which is about 10% higher than reference cell, was achieved by enhancement of short-current density (Jsc) value. The core-shell structured Ag@TiO2 wire could effectively reduce the charge recombination through the contribution to electron shortcut for improvement in the electron transfer rate and lifetime.

  20. Partial Characterization of α-Galactosidic Activity from the Antarctic Bacterial Isolate, . LX-20 as a Potential Feed Enzyme Source

    Directory of Open Access Journals (Sweden)

    Inkyung Park


    Full Text Available An Antarctic bacterial isolate displaying extracellular α-galactosidic activity was named Paenibacillus sp. LX-20 based on 16S rRNA gene sequence analysis. Optimal activity for the LX-20 α-galactosidase occurred at pH 6.0–6.5 and 45°C. The enzyme immobilized on the smart polymer Eudragit L-100 retained 70% of its original activity after incubation for 30 min at 50°C, while the free enzyme retained 58% of activity. The enzyme had relatively high specificity for α-D-galactosides such as p-nitrophenyl-α-galactopyranoside, melibiose, raffinose and stachyose, and was resistant to some proteases such as trypsin, pancreatin and pronase. Enzyme activity was almost completely inhibited by Ag+, Hg2+, Cu2+, and sodium dodecyl sulfate, but activity was not affected by β-mercaptoethanol or EDTA. LX-20 α-galactosidase may be potentially useful as an additive for soybean processing in the feed industry.

  1. The effect of FF-MAS on porcine cumulus-oocyte complex maturation, fertilization and pronucleus formation in vitro

    DEFF Research Database (Denmark)

    Færge, Inger; Strejcek, Frantisek; Laurincik, Jozef


    Follicular fluid meiosis-activating sterol (FF-MAS) has been isolated from the follicular fluid (FF) of several species including man. FF-MAS increases the quality of in vitro oocyte maturation, and thus the developmental potential of oocytes exposed to FF-MAS during in vitro maturaion is improved....... The aim of the present study was to investigate the effects of FF-MAS on porcien oocyte maturation and pronucleus formation in vitro. Porcine cumulus-oocyte complexes (COCs) were isolated from abattoir ovaries and in vitro matured for 48 h in NCSU 37 medium supplemented with 1 mg/1 cysteine, 10 ng....../ml epidermal growth factor and 50µM 2-mercaptoethanol with or without 10% porcine follicular fluid (pFF). For the first 22 h, 1 mM db-cAMP and 10 I.E PMSG/hCG was added. The medium was supplemented with 1 µM, 3 µM, 10 µM, 30 µM or 100 µM FF-MAS dissolved in ethanol. After maturation the COCs were denuded...

  2. Isolation of high-quality DNA in 16 aromatic and medicinal Colombian species using silica-based extraction columns

    Directory of Open Access Journals (Sweden)

    Vega Vela Nelson Enrique


    Full Text Available

    Aromatic and medicinal plant species are a valuable resource for research and development of pharmaceutical, cosmetic, crop protection and nutritional agents, due to the high amount of bioactive phytochemicals that they contain. However, these compounds are a major obstacle in the isolation of high-quality DNA suitable for genetic analyses. In this paper, we report a protocol that optimizes the use of the cationic detergent CTAB and the reductant β-mercaptoethanol in cell lysis. The elimination of plant secondary metabolites such as polysaccharides and polyphenols, that typically co-isolate with DNA, was achieved using the chemical denaturing properties of the guanidinium cation, which together with the adsorbent chemical specificity of the silica, resulted in the purification of high-quality DNA suitable for digestion with restriction enzymes and optimal for PCR amplification of AFLP-type molecular markers. This protocol was evaluated on 16 Colombian aromatic and medicinal plant species promising for their essential oils. The results allow suggesting that this procedure might be appropriate for other species, tissues and sample types recalcitrant to DNA extraction.

  3. Analysis of serine proteases from marine sponges by 2-D zymography. (United States)

    Wilkesman, Jeff G; Schröder, Heinz C


    Proteolytic activities isolated from the marine demosponges Geodia cydonium and Suberites domuncula were analyzed by 2-D zymography, a technique that combines IEF and zymography. After purification, a 200 kDa proteolytically active protein band was obtained from G. cydonium when analyzed in gelatin copolymerized 1-D zymograms. The enzymatic activity was quantified using alpha-N-benzoyl-D-arginine p-nitroanilide (BAPNA) as a substrate and corresponded to a serine protease. The protease activity was resistant to urea and SDS. DTT and 2-mercaptoethanol (2-ME) did not significantly change the protease activity, but induced a shift in molecular mass of the proteolytic band to lower M(r) values as detected by zymography. Under mild denaturing conditions, lower M(r) bands (zymography, the protease from G. cydonium revealed a pI of 8.0 and an M(r) shift from 200 to 66 kDa. To contrast these results, a cytosolic sample from S. domuncula was analyzed. The proteolytic activity of this sponge after 2-D zymography corresponded to an M(r) of 40 kDa and a pI of 4.0. The biological function of both sponge proteases is not yet known. This study demonstrates that mild denaturing conditions required for IEF may alter the interpretation of the 2-D zymography, and care must be taken during sample preparation.

  4. Effect of gamma irradiation on rat thymus arginine-rich H3 histone in vitro

    International Nuclear Information System (INIS)

    Patil, M.S.; Narasimhan, Saroja; Sreenivasan, A.


    Physicochemical properties of rat thymus H3 histone have been studied following gamma radiation (25-90 krad) in 0.2 N HCl. Polyacrylamide gel electrophoretic pattern (PGE) of H3 histone indicated that aggregates were formed in the histone fraction following gamma irradiation. The PGE pattern of the irradiated-histone fraction remained unaltered even after it was treated with 8.0 M urea to eliminate noncovalent bonding. On the other hand, the irradiated sample treated with β-mercaptoethanol exhibited the PGE pattern which was essentially similar to that of unirradiated sample. These results indicate that the aggregates seen in the PGE pattern of irradiated-H3 histone may be formed through interpolypeptide chain disulphide linkeges rather than by noncovalent bonding. This contention is also supported by the fact that irradiated-H3 histone exhibited hyperchromic shift at 240-250 nm region as well as increased disulphide content. Other results revealed that DNA-dependent RNA synthesis in vitro was inhibited to a greater extent by irradiated-H3 histone than by unirradiated-H3 histone. (author)


    Directory of Open Access Journals (Sweden)

    Tatjana Jovanović


    Full Text Available S-nitrosothiols (RSNO are considered to be natural depo and a transport form of nitric oxide (NO; their physiological activity is realized by releasing NO, which stimulates vasodilatation, prevents platelet aggregation and damages originating from ischemia/reperfusion injury. The main reservoir of non-protein RSNO is S-nitrosoglutathione (GSNO created by S-nitrosylation of glutathione (GSH, most widespread endogenous non-protein thiol. The aim of this study was to set up an efficient GSNO synthesis and to examine the possibility of creating fluorescent derivatives of GSNO with o-phthalaldehyde (OPA, perform their spectroscopic characterization and behavior in the system of high performance liquid chromatography (HPLC. Synthesized GSNO and its OPA derivative were analyzed by HPLC system by using fluorescent (FLD and “diode array” (DAD detection. We developed the synthetic procedure by which GSNO was obtained in a solid, anhydrous form with a good yield. After UV characterization of the obtained GSNO, derivation of GSH and GSNO with OPA reagent, with or without mercaptoethanol (ME, was performed, and the derived products were analyzed by HPLC-FLD-DAD system. We optimized the reaction conditions that had lead to the formation of a stable and fluorescent tricyclic isoindole derivative of GSNO with OPA-ME reagent, which is essential for the development of HPLC method that would be used for the quantification of GSNO in real biological systems.

  6. Imaging rheumatoid arthritis specifically with technetium99m CD4-specific (T-helper lymphocytes) antibodies

    International Nuclear Information System (INIS)

    Becker, W.; Emmrich, F.; Horneff, G.; Burmester, G.; Seiler, F.; Schwarz, A.; Kalden, J.; Wolf, F.; Behringwerke AG, Frankfurt am Main


    CD 4 expressing T-lymphocytes are involved in the pathogenesis of rheumatoid arthritis, so the possibility of using radiolabelled CD 4 -specific antibodies to localise diseased joints was studied. Prospectively six patients with rheumatoid arthritis were investigated in all. Five of them received 200-300 μg of a 555 MBq technetium 99m CD 4 -specific antibody (MAX.16H5) and were examined with three phase bone scans. Max.16H5 (IgG1) was labelled according to the mercaptoethanol (Schwarz) method. Lumphocytes of one patient were isolated on a Ficoll-Hypaque gradient and labelled with the antibody in vitro. Scans were performed 1.5 h, 4 and 24 h post injection in anterior and posterior views. In all patients, diseased joints could be clearly imaged at as early as 1.5 h. The localisation of the diseased joints correlated (P 0.05). According to these data we conclude that 99m Tc-labelled CD 4 -specific antibodies specifically image actively diseased joints in rheumatoid arthritis. (orig.)

  7. Occurrence of anti-Brucella abortus and anti-Leptospira spp. antibodies in buffaloes from Paraíba state, Northeastern Brazil

    Directory of Open Access Journals (Sweden)

    Arthur Willian de Lima Brasil


    Full Text Available The objective of this study was to determine the frequency of animals that tested positive for brucellosis and leptospirosis and the risk factors for these diseases in the State of Paraíba. A total of 136 buffaloes from 14 herds were examined. For brucellosis, we used the buffered acidified plate antigen (BAPA test as screening method and 2-mercaptoethanol as confirmatory test. For leptospirosis, we conducted a microscopic agglutination test (MAT, with a cut-off point of 1:100. Of the animals examined, two [1.5%; 95% CI = 0.4%–5.2%] were positive for brucellosis, and 38 (27.9%; 95% CI = 21.1%–36.0% were positive for leptospirosis. The brucellosis-positive animals were from two (14.3% herds, while nine (64.3% herds had leptospirosis-seropositive animals. The more frequent Leptospira spp. serotypes were Bratislava, Pomona, and Canicola. We concluded that leptospirosis was widespread in buffaloes in the state of Paraíba and suggested that breeding alongside horses and pigs might be na important factor in the spread of leptospirosis-positive animals. The presence of brucellosispositive animals indicated the possibility of negative-impacting measures on disease control in bovines, and it is therefore recommended that greater attention be given to these animals for brucellosis control.

  8. Biochemical properties and primary structure of elastase inhibitor AFUEI from Aspergillus fumigatus. (United States)

    Okumura, Yoshiyuki; Matsui, Takeshi; Ogawa, Kenji; Uchiya, Kei-ichi; Nikai, Toshiaki


    An elastase inhibitor from Aspergillus fumigatus (AFUEI) was isolated, and its biochemical properties and primary structure examined. The inhibitor was purified by column chromatography using DE52 cellulose and Sephadex G-75, and was found to be homogeneous as indicated by a single band following discontinuous PAGE and SDS-PAGE. A molecular mass of 7525.1 Da was observed by matrix-assisted desorption/ionization time-of-flight mass spectroscopy. The elastolytic activity of elastases from A. fumigatus, Aspergillus flavus and human leukocytes was inhibited by AFUEI. However, the elastolytic activity of porcine pancreas elastase, Pseudomonas aeruginosa elastase and elastase from snake venom was not affected by AFUEI. No inhibitory effect of DTT or 2-mercaptoethanol on the elastase inhibitory activity of AFUEI was observed. The amino acid sequence of AFUEI peptides derived from digests utilizing clostripain was determined by Edman sequencing. AFUEI was composed of 68 aa and had a calculated molecular mass of 7526.2 Da. The search for amino acid homology with other proteins demonstrated that aa 1-68 of AFUEI are 100 % identical to aa 20-87 of the hypothetical protein AFUA 3G14940 of A. fumigatus.

  9. High-performance pure and Fe{sup 3+}-ion doped ZnS quantum dots as green nanophotocatalysts for the removal of malachite green under UV-light irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Rajabi, Hamid Reza, E-mail: [Chemistry Department, Yasouj University, Yasouj 75918-74831 (Iran, Islamic Republic of); Khani, Omid [Electroceramics Research Center, Malek-Ashtar University of Technology, Shahin Shahr (Iran, Islamic Republic of); Shamsipur, Mojtaba [Department of Chemistry, Razi University, Kermanshah (Iran, Islamic Republic of); Vatanpour, Vahid [Faculty of Chemistry, Kharazmi (Tarbiat Moallem) University, Tehran (Iran, Islamic Republic of)


    Highlights: ► Synthesis of ultra-small ZnS and ZnS:Fe QDs in aqueous media. ► Characterization of QDs by TEM, XRD, FAAS and UV–vis absorption techniques. ► The new report on the photocatalytic behavior of QDs on removal of MG. ► Effective degrade of dye by novel modified QDs up to 98%. -- Abstract: The heterogeneous photocatalysis using UV-radiation and quantum dots (QDs) is an interesting method for the treatment of water polluted with the organic substances. In this study, ZnS QDs, as a pure and doped with Fe{sup 3+}, were prepared for photodecolorization of malachite green (MG) as a model dye. The synthesis of QDs was carried out using a chemical precipitation method in aqueous solution, in the presence of 2-mercaptoethanol as a capping agent. The XRD patterns indicated that the doped nanoparticles are crystalline, with cubic zinc blend structure. The effects of dopant content, pH, nanophotocatalyst amount, irradiation time, and initial dye concentration on the removal efficiency of MG were studied. Results showed that the QDs presented high MG decolorization efficiency, and doping with Fe{sup 3+} promoted the dye removal. The maximum removal of dyes was obtained at 80 mg/L of photocatalyst as an optimum value for the dosage of photocatalyst in pH of 8.0.

  10. Ex Situ Formation of Metal Selenide Quantum Dots Using Bacterially Derived Selenide Precursors

    Energy Technology Data Exchange (ETDEWEB)

    Fellowes, Jonathan W.; Pattrick, Richard; Lloyd, Jon; Charnock, John M.; Coker, Victoria S.; Mosselmans, JFW; Weng, Tsu-Chien; Pearce, Carolyn I.


    Luminescent quantum dots were synthesized using bacterially derived selenide (SeII-) as the precursor. Biogenic SeII- was produced by the reduction of Se-IV by Veillonella atypica and compared directly against borohydride-reduced Se-IV for the production of glutathione-stabilized CdSe and beta-mercaptoethanol-stabilized ZnSe nanoparticles by aqueous synthesis. Biological SeII- formed smaller, narrower size distributed QDs under the same conditions. The growth kinetics of biologically sourced CdSe phases were slower. The proteins isolated from filter sterilized biogenic SeII- included a methylmalonyl-CoA decarboxylase previously characterized in the closely related Veillonella parvula. XAS analysis of the glutathione-capped CdSe at the S K-edge suggested that sulfur from the glutathione was structurally incorporated within the CdSe. A novel synchrotron based XAS technique was also developed to follow the nucleation of biological and inorganic selenide phases, and showed that biogenic SeII- is more stable and more resistant to beam-induced oxidative damage than its inorganic counterpart. The bacterial production of quantum dot precursors offers an alternative, 'green' synthesis technique that negates the requirement of expensive, toxic chemicals and suggests a possible link to the exploitation of selenium contaminated waste streams.

  11. Immobilization of maltase from Saccharomyces cerevisiae on thiosulfonate supports

    Directory of Open Access Journals (Sweden)

    Mihailović Mladen


    Full Text Available In this study, two commercial supports (Eupergit® C and Purolite® A109 were chemically modified in order to introduce thiosulfonate groups, which could subsequently exclusively react with cysteine residues on enzyme surface. Thereafter, the immobilization of maltase from Saccharomyces cerevisiae onto obtained thiosulfonate-activated supports was performed, resulting in high expressed enzymatic activities (around 50%, while on the other hand, immobilization on unmodified supports yielded expressed activities less than 5%. Moreover, protein loadings up to 12.3 mg g-1 and immobilized activities up to 3580 IU g-1 were achieved by employment of theses thiosulfonate supports. Desorption experiments, performed on samples taken during immobilization, proved that immobilization on thiosulfonate supports encompass first step of fast adsorption on support and second slower step of the covalent bond formation between thiosulfonate groups and thiol groups of cysteine. More importantly, although enzyme coupling occurs via covalent bond formation, performed immobilization proved to be reversible, since it was shown that 95% of immobilized activity can be detached from support after treatment with thiol reagent (β-mercaptoethanol, thus support can be reused after enzyme inactivation. [Projekat Ministarstva nauke Republike Srbije, br. III 46010

  12. Identification of a Passiflora alata Curtis dimeric peptide showing identity with 2S albumins. (United States)

    Ribeiro, Suzana M; Almeida, Renato G; Pereira, Camila A A; Moreira, João S; Pinto, Michelle F S; Oliveira, Antonio C; Vasconcelos, Ilka M; Oliveira, José T A; Santos, Marcelo O; Dias, Simoni C; Franco, Octávio L


    Antifungal proteins and peptides, essential compounds for plant defense, have been isolated from several tissues of various plants. These proteins could be used as a natural alternative to control phytopathogenic fungi. In this report a heterodimeric antifungal protein named Pa-AFP1, showing higher identity with the 2S albumin family, was purified by using 70-100% ammonium sulfate saturation and further purification steps such as anionic exchange Q-Sepharose chromatography associated with HPLC reversed-phase C4 chromatography. Analysis by Tricine-SDS-PAGE revealed two peptidic molecular masses of approximately 4500 Da and 7000 Da, in the presence of β-mercaptoethanol, while by removing the reducing agent a single protein with molecular mass of about 11,500 Da was obtained. Moreover, dimer mass was confirmed by MALDI-TOF analyses (11,569.76 Da). The antifungal protein, named Pa-AFP1, efficiently inhibited the growth of filamentous fungi Colletotrichum gloeosporioides, and was added to a short list of 2S albumins with antimicrobial properties. Otherwise, this same peptide showed no activity toward bacteria and yeasts. In summary, this compound could be used in the future to develop biotechnological products for the control of phytopathogenic fungi. Copyright © 2010 Elsevier Inc. All rights reserved.

  13. Antimicrobial and antifungal activities of Lactobacillus curvatus strain isolated from homemade Azerbaijani cheese. (United States)

    Ahmadova, Aynur; Todorov, Svetoslav Dimitrov; Hadji-Sfaxi, Imen; Choiset, Yvan; Rabesona, Hanitra; Messaoudi, Soumaya; Kuliyev, Akif; Franco, Bernadette Dora Gombossy de Melo; Chobert, Jean-Marc; Haertlé, Thomas


    The aims of this study were to characterize inhibitory activity spectra, some probiotic properties and safety of Lactobacillus curvatus A61 for its future application in production of fermented foods. The studied strain was isolated from traditional homemade cheese manufactured in Azerbaijan. The cell-free supernatant of culture of Lb. curvatus A61 inhibited the growth of tested LAB, as well as of Listeria monocytogenes and Bacillus cereus strains. The strain presented antifungal activity and inhibited the growth of Cladosporium and Fusarium ssp. during co-cultivation on agar media. PCR amplification with specific primers revealed the presence of curvacin A encoding gene in Lb. curvatus A61. Bacteriocin produced by the studied strain was heat stable and active in a broad pH range, and in the presence of Triton X-20, Triton X-80, Triton X-100, β-mercaptoethanol, Na-EDTA, SDS and NaCl. The mode of action of bacteriocin against selected indicator strains was found to be bacteriostatic. Lb. curvatus A61 was resistant to physiological concentrations of bile salts and showed high auto-aggregation ability, as well as co-aggregation ability with pathogenic L. monocytogenes strains. It was sensitive to chloramphenicol, penicillin, tetracycline, ciprofloxacin and vancomycin, but resistant to ampicillin and gentamicin. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. Effect of controlled redox potential and dissolved oxygen on the in vitro refolding of an E. coli alkaline phosphatase and chicken lysozyme. (United States)

    Meneses-Acosta, Angélica; Vizcaíno-Meza, Luis Rodolfo; Ayala-Castro, Hector G; Contreras, Martha A; Ortega-López, Jaime; Ramírez, Octavio T


    The development of efficient purification strategies of recombinant active protein derived from inclusion bodies requires the knowledge of the effect of environmental variables, such as redox potential (RP) and dissolved oxygen tension (DOT), in order to control the protein folding process. However, that information is scarce and only few in vitro studies of the impact of such variables have been reported under constant controlled conditions. In this work, the effect of controlled RP and DOT on the refolding of E. coli alkaline phosphatase (AP) and chicken lysozyme (CL) enzymes were studied. Disulphide bonds of both enzymes were reduced in an instrumented vessel using 2-mercaptoethanol and nitrogen. In the latter case, guanidine hydrochloride was also used to denature the protein. Such conditions caused protein conformational changes, as determined by the intrinsic fluorescence spectra that correlated with a decrease on the activity in both cases. Reduced enzymes were then oxidized, under different constant and predetermined RP or DOT, by manipulating the gas composition in the vessel. Folding kinetics were followed as the recovery of enzyme activity. Results showed that the percentage of recovery and rate of increase of enzymatic activity directly depended on the RP and DOT. A higher folding efficiency was found under controlled DOT compared to controlled RP conditions. These results are useful for establishing protein folding strategies to improve the recovery of active protein from inclusion bodies. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Highly efficient generation of definitive endoderm lineage from human induced pluripotent stem cells. (United States)

    Sekine, K; Takebe, T; Suzuki, Y; Kamiya, A; Nakauchi, H; Taniguchi, H


    Although hepatocytes can be an option for liver transplantation, the shortage of donor organs continues to worsen. Since the development of induced pluripotent stem (iPS) cell technology, it is eagerly anticipated to produce functional elements from pluripotent stem cells. These functional cells differentiated from iPS cells could be used for transplantation, drug screening, and in vitro toxicology. Human iPS cells are maintained on Mitomycin C-treated mouse embryonic fibroblast layers in DMEM-Ham F12-based medium supplemented with Knockout Serum Replacement, nonessential amino acids, 2-mercaptoethanol, and Glutamax. Differentiation of human iPS cells into a definitive endodermal lineage was induced with PRMI 1640 medium supplemented with B27 and 100 ng/mL human activin A. Two B27 supplements were examined with and without insulin. Furthermore, the PI3 kinase inhibitor LY294002 was used to examine the effect of inhibiting insulin signaling. We established efficient induction of definitive endodermal differentiation from iPS cells. Quantitative analysis revealed efficient (93.03 ± 2.74%) differentiation of human iPS cells into definitive endoderm cells using B27 minus insulin. This protocol may contribute as a fundamental technique to promote human iPS studies to develop cellular sources for transplantation. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. Isolation and characterization of a cold-active, alkaline, detergent stable α-amylase from a novel bacterium Bacillus subtilis N8. (United States)

    Arabacı, Nihan; Arıkan, Burhan


    A cold-active alkaline amylase producer Bacillus subtilis N8 was isolated from soil samples. Amylase synthesis optimally occurred at 15°C and pH 10.0 on agar plates containing starch. The molecular weight of the enzyme was found to be 205 kDa by performing SDS-PAGE. While the enzyme exhibited the highest activity at 25°C and pH 8.0, it was highly stable in alkaline media (pH 8.0-12.0) and retained 96% of its original activity at low temperatures (10-40°C) for 24 h. While the amylase activity increased in the presence of β-mercaptoethanol (103%); Ba 2+ , Ca 2+ , Na + , Zn 2+ , Mn 2+ , H 2 O 2 and TritonX-100 slightly inhibited the activity. The enzyme showed resistance to some denaturants: such as SDS, EDTA, and urea (52, 65 and 42%, respectively). N8 α -amylase displayed the maximum remaining activity of 56% with 3% NaCl. The major final products of starch were glucose, maltose, and maltose-derived oligosaccharides. This novel cold-active α -amylase has the potential to be used in the industries of detergent and food, bioremediation process and production of prebiotics.

  17. Generation of Insulin-Producing Cells from Human Bone Marrow-Derived Mesenchymal Stem Cells: Comparison of Three Differentiation Protocols

    Directory of Open Access Journals (Sweden)

    Mahmoud M. Gabr


    Full Text Available Introduction. Many protocols were utilized for directed differentiation of mesenchymal stem cells (MSCs to form insulin-producing cells (IPCs. We compared the relative efficiency of three differentiation protocols. Methods. Human bone marrow-derived MSCs (HBM-MSCs were obtained from three insulin-dependent type 2 diabetic patients. Differentiation into IPCs was carried out by three protocols: conophylline-based (one-step protocol, trichostatin-A-based (two-step protocol, and β-mercaptoethanol-based (three-step protocol. At the end of differentiation, cells were evaluated by immunolabeling for insulin production, expression of pancreatic endocrine genes, and release of insulin and c-peptide in response to increasing glucose concentrations. Results. By immunolabeling, the proportion of generated IPCs was modest (≃3% in all the three protocols. All relevant pancreatic endocrine genes, insulin, glucagon, and somatostatin, were expressed. There was a stepwise increase in insulin and c-peptide release in response to glucose challenge, but the released amounts were low when compared with those of pancreatic islets. Conclusion. The yield of functional IPCs following directed differentiation of HBM-MSCs was modest and was comparable among the three tested protocols. Protocols for directed differentiation of MSCs need further optimization in order to be clinically meaningful. To this end, addition of an extracellular matrix and/or a suitable template should be attempted.

  18. Radical-Scavenging Activity of Thiols, Thiobarbituric Acid Derivatives and Phenolic Antioxidants Determined Using the Induction Period Method for Radical Polymerization of Methyl Methacrylate

    Directory of Open Access Journals (Sweden)

    Seiichiro Fujisawa


    Full Text Available The radical-scavenging activities of two thiols, eight (thiobarbituric acid derivatives and six chain-breaking phenolic antioxidants were investigated using the induction period method for polymerization of methyl methacrylate (MMA initiated by thermal decomposition of 2,2’-azobisisobutyronitrile (AIBN and monitored by differential scanning calorimetry (DSC. The induction period (IP for the thiols 2-mercaptoethanol (ME and 2-mercapto-1-methylimidazole (MMI was about half that for phenolic antioxidants. Except for the potent inhibitor 5,5-dimethyl-2-thiobarbituric acid (3, the IP for thiobarbituric acid derivatives was about one tenth of that for phenolic antioxidants. The IP for 1,3,5-trimethyl-2-thiobarbituric acid (1 and 5-allyl-1, 3-dimethyl-2-thiobarbituric acid (7 was less than that of the control, possibly due to inhibition by a small amount of atmospheric oxygen in the DSC container. The ratio of the chain inhibition to that of chain propagation (CI/CP for the thiols and thiobarbituric acid compounds except for 1, 3 and 7 was about 10 times greater or greater than that for phenolic compounds. A kinetic chain length (KCL about 10% greater than that of the control was observed for 1, suggesting that 1 had chain transfer reactivity in the polymerization of MMA. The average molecular weight of polymers formed from thiobarbituric acid derivatives is discussed.

  19. Optimization of DNA Extraction for RAPD and ISSR Analysis of Arbutus unedo L. Leaves

    Directory of Open Access Journals (Sweden)

    Paula Baptista


    Full Text Available Genetic analysis of plants relies on high yields of pure DNA. For the strawberry tree (Arbutus unedo this represents a great challenge since leaves can accumulate large amounts of polysaccharides, polyphenols and secondary metabolites, which co-purify with DNA. For this specie, standard protocols do not produce efficient yields of high-quality amplifiable DNA. Here, we present for the first time an improved leaf-tissue protocol, based on the standard cetyl trimethyl ammonium bromide protocol, which yields large amounts of high-quality amplifiable DNA. Key steps in the optimized protocol are the addition of antioxidant compounds—namely polyvinyl pyrrolidone (PVP, 1,4-dithiothreitol (DTT and 2-mercaptoethanol, in the extraction buffer; the increasing of CTAB (3%, w/v and sodium chloride (2M concentration; and an extraction with organic solvents (phenol and chloroform with the incubation of samples on ice. Increasing the temperature for cell lyses to 70 °C also improved both DNA quality and yield. The yield of DNA extracted was 200.0 ± 78.0 µg/µL and the purity, evaluated by the ratio A260/A280, was 1.80 ± 0.021, indicative of minimal levels of contaminating metabolites. The quality of the DNA isolated was confirmed by random amplification polymorphism DNA and by inter-simple sequence repeat amplification, proving that the DNA can be amplified via PCR.

  20. Purification and characterization of glutaminase-free L-asparaginase from Pectobacterium carotovorum MTCC 1428. (United States)

    Kumar, Sanjay; Venkata Dasu, V; Pakshirajan, K


    An intracellular glutaminase-free L-asparaginase from Pectobacterium carotovorum MTCC 1428 was isolated to apparent homogeneity. The homotetramer enzyme has a molecular mass of 144.4 kDa (MALDI-TOF MS) and an isoelectric point of approximately 8.4. The enzyme is very specific for its natural substrate, L-asparagine. The activity of L-asparaginase is activated by mono cations and various effectors including Na+, K+, L-cystine, L-histidine, glutathione and 2-mercaptoethanol whereas it is moderately inhibited by various divalent cations and thiol group blocking reagents. Kinetic parameters, Km, Vmax and kcat of purified L-asparaginase from P. carotovorum MTCC 1428 were found to be 0.657 mM, 4.45 U μg(-1) and 2.751×10(3) s(-1), respectively. Optimum pH of purified L-asparaginase for the hydrolysis of L-asparagine was in the range of 8.0-10.0, and its optimum temperature was found to be 40 °C. The purified L-asparaginase has no partial glutaminase activity, which can reduce the possibility of side effects during the course of anti-cancer therapy. Copyright © 2010 Elsevier Ltd. All rights reserved.

  1. Preparation of reusable bioreactors using reversible immobilization of enzyme on monolithic porous polymer support with attached gold nanoparticles. (United States)

    Lv, Yongqin; Lin, Zhixing; Tan, Tianwei; Svec, Frantisek


    Porcine lipase has been reversibly immobilized on a monolithic polymer support containing thiol functionalities prepared within confines of a fused silica capillary and functionalized with gold nanoparticles. Use of gold nanoparticles enabled rejuvenation of the activity of the deactivated reactor simply by stripping the inactive enzyme from the nanoparticles using 2-mercaptoethanol and subsequent immobilization of fresh lipase. This flow through enzymatic reactor was then used to catalyze the hydrolysis of glyceryl tributyrate (tributyrin). The highest activity was found within a temperature range of 37-40°C. The reaction kinetics is characterized by Michaelis-Menten constant, Km  = 10.9 mmol/L, and maximum reaction rate, Vmax  = 5.0 mmol/L min. The maximum reaction rate for the immobilized enzyme is 1,000 times faster compared to lipase in solution. The fast reaction rate enabled to achieve 86.7% conversion of tributyrin in mere 2.5 min and an almost complete conversion in 10 min. The reactor lost only less than 10% of its activity even after continuous pumping through it a solution of substrate equaling 1,760 reactor volumes. Finally, potential application of this enzymatic reactor was demonstrated with the transesterification of triacylglycerides from kitchen oil to fatty acid methyl esters thus demonstrating the ability of the reactor to produce biodiesel. © 2013 Wiley Periodicals, Inc.

  2. Control of bovine brucellosis from 1998 to 2009 in the State of Mato Grosso do Sul, Brazil

    Directory of Open Access Journals (Sweden)

    Jamil Manoel Leal Filho


    Full Text Available This study used disease prevalence as an indicator to assess the effectiveness of the bovine brucellosis vaccination program implemented by the state of Mato Grosso do Sul. The state was divided into three regions: Pantanal, Planalto Sul, and Planalto Norte. For each region, a predetermined number of properties was sampled; in each, blood samples were collected from randomly selected cows aged at least 24 months. Sera from animals were subjected to a protocol that called for serial testing, with screening and confirmation using the buffered acidified antigen and 2-mercaptoethanol tests, respectively. In each property, a questionnaire was completed in order to identify risk factors associated with the disease. The overall prevalence rates of infected herds and infected animals in the state were 30.6% [27.4; 34.0] and 7.0% [5.6; 8.7], respectively. The regional prevalence rates of infected herds and infected animals were 39.1% and 8.9%, respectively, in Pantanal, 25.3% and 6.1% in Planalto Sul, and 32.1% and 6.4% in Planalto Norte. Bovine brucellosis in the state is associated with the purchase of breeding animals, herd size, and beef and mixed types of enterprise. Thus, Mato Grosso do Sul should reassess its vaccination program in order to improve its effectiveness. The state must also increase education and supervision to encourage producers to test breeding animals for brucellosis before introducing them to their properties.

  3. Specific localization of the annexin II heterotetramer in brain lipid raft fractions and its changes in spatial learning. (United States)

    Zhao, Wei-Qin; Waisman, David M; Grimaldi, Maurizio


    Annexin-II (AII) is a Ca(2+)-dependent phospholipid-binding protein that is present in both intracellular and extracellular compartments. In the present study AII immunoreactivity was found in a subpopulation of neurons in specific brain regions, including the cerebral cortex and the surface of hippocampal pyramidal neurons from adult rats. AII from synaptic membranes was detected by immunoblotting as multiple species containing the monomer (AII36) and heterotetramer (AIIt). AIIt was resistant to beta-mercaptoethanol and dithiothreitol in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but was completely reduced to monomers (36 kDa) by two-dimensional electrophoresis. AIIt resided exclusively in the detergent-resistant lipid rafts concentrated in neuronal dendrites, and its recruitment to those structures was enhanced by antibody cross-link. AII abundantly distributed on the outer leaflet of neuronal membranes and between spaces of neurons appeared to be neuronal adhesive. The formation of AIIt required synthesis of sphingolipids and cholesterol, and its stability depended on Ca2+. Increases in neuronal activities such as depolarization and learning were shown to promote formation of AIIt. Our results suggest that, via a dynamic association with dendritic lipid rafts, AII may play a role in synaptic signal transduction and remodeling. This probably involves focal adhesion and interactions with actin that are associated with brain development and memory consolidation.

  4. Rocket and Two Dimensional Immunoelectrophoresis in Diagnosis of Caprine Brucellosis. (United States)

    Mehrabani, Davood; Gholami, Zahra; Kohanteb, Jamshid; Sepehrimanesh, Masood; Hosseini, Seyed Mohammad Hossein


    Brucellosis is a major bacterial zoonosis of global importance with the causative organisms of Gram-negative facultative intracellular pathogens. The aims of this study were to standardize two immunoelectrophoretic techniques, rocket and cross immunoelectrophoresis, and compare their results with other conventional serodiagnostic tests. Sera from 15 sheep, without any history of brucellosis vaccination, infected with Brucella melitensis M16 subcutaneously, were employed in a comparison of culture, precipitating, and immunoelectrophoretic tests. A 125 days serologic follow-up was performed after the infection was started. As a reference, these tests also done in the five healthy sheep. The results obtained with the rocket immunoelectrophoresis test correlated very well with those of the cross immunoelectrophoresis, whereas results of other tests such as culture, Rose Bengal, standard tube agglutination and 2-mercaptoethanol seruagglutination tests were inferior. As agglutination test shows cross reaction and a prozone phenomenon, and in blood culture, the bacteria is not always detectable, so they are time consuming rocket and cross immunoelectrophoresis are recommended because their results can be obtained in a shorter time.

  5. Crystallization and preliminary crystallographic analysis of recombinant human galectin-1

    Energy Technology Data Exchange (ETDEWEB)

    Scott, Stacy A. [Institute for Glycomics, Gold Coast Campus, Griffith University, Queensland 4222 (Australia); Scott, Ken [School of Biological Sciences, University of Auckland, Auckland (New Zealand); Blanchard, Helen, E-mail: [Institute for Glycomics, Gold Coast Campus, Griffith University, Queensland 4222 (Australia)


    Human galectin-1 has been cloned, expressed in E. coli, purified and crystallized in the presence of both lactose (ligand) and β-mercaptoethanol under six different conditions. The X-ray diffraction data obtained have enabled the assignment of unit-cell parameters for two novel crystal forms of human galectin-1. Galectin-1 is considered to be a regulator protein as it is ubiquitously expressed throughout the adult body and is responsible for a broad range of cellular regulatory functions. Interest in galectin-1 from a drug-design perspective is founded on evidence of its overexpression by many cancers and its immunomodulatory properties. The development of galectin-1-specific inhibitors is a rational approach to the fight against cancer because although galectin-1 induces a plethora of effects, null mice appear normal. X-ray crystallographic structure determination will aid the structure-based design of galectin-1 inhibitors. Here, the crystallization and preliminary diffraction analysis of human galectin-1 crystals generated under six different conditions is reported. X-ray diffraction data enabled the assignment of unit-cell parameters for crystals grown under two conditions, one belongs to a tetragonal crystal system and the other was determined as monoclinic P2{sub 1}, representing two new crystal forms of human galectin-1.

  6. Screening for Stress Resistance Mutations in the Mouse

    Directory of Open Access Journals (Sweden)

    Wallace S Chick


    Full Text Available Longevity is correlated with stress resistance in many animal models. However, previous efforts through the boosting of the antioxidant defense system did not extend life span, suggesting that longevity related stress resistance is mediated by other uncharacterized pathways. We have developed a high-throughput platform for screening and rapid identification of novel genetic mutants in the mouse that are stress-resistant. Selection for resistance to stressors occurs in mutagenized mouse embryonic stem (ES cells, which are carefully treated so as to maintain pluripotency for mouse production. Initial characterization of these mutant ES cells revealed mutations in Pigl, Tiam1, and Rffl, among others. These genes are implicated in glycosylphosphatidylinositol biosynthesis, NADPH oxidase function, and inflammation. These mutants: (1 are resistant to two different oxidative stressors, paraquat and the omission of 2-mercaptoethanol, (2 have reduced levels of endogenous reactive oxygen species (ROS, (3 are capable of generating live mice, and (4 transmit the stress resistance phenotype to the mice. This strategy offers an efficient way to select for new mutants expressing a stress resistance phenotype, to rapidly identify the causative genes, and to develop mice for in vivo studies.

  7. Semiconservative and unscheduled DNA-synthesis of rat thymocytes under the influence of some radioprotecting and radiosensitizing agents

    Energy Technology Data Exchange (ETDEWEB)

    Tempel, K.; Wulffius-Kock, M.; Winkle, J.; Schmerold, I.


    The effects of aminoethylisothiuroniumbromide (AET), cysteamine (CY-A), cysteine (CY-E), glutathione (GLU), mercaptoethanol (MA), mercaptopropionylglycine (MPG), N-ethylmaleimide (NEM), metronidazole (MNA), nitroacetophenone (NAP), nitrofurazone (NFA), arabinofuranosylcytosine (araC), fluorouracil (FU), adriamycin (AM), ethidiumbromide (E), bleomycin (BM), and diethyldithiocarbamate (DDC) on the semiconservative and unscheduled incorporation of /sup 3/H-thymidine into the DNA were tested on rat thymocytes in vitro. DNA damage has been measured using the hydroxylapatite system. Unscheduled DNA synthesis was induced by UV-light and/or X-irradiation. The semiconservative DNA synthesis was inhibited by the above substances-with exception of MA and MPG. Aminothioles, NAP, NFA, and BM enhanced, araC, FU, AM, E, and DDC diminished unscheduled DNA synthesis. After alkaline unwinding, the duplex form of DNA decreased under the influence of CY-A, CY-E, GLU, MPG, NEM, NAP, NFA, araC, FU, AM, E, and BM. It is suggested that stimulation of unscheduled DNA synthesis combined with a transient decrease of semiconservative DNA synthesis will amplify the DNA repair capacity of thymocytes, whereas radiation damage may be intensified by araC, FU, AM,E, and DDC - at least partly, through inhibition of unscheduled DNA synthesis. With respect to the action of NAP, NFA, and BM, DNA repair may be concerned in a more indirect manner.

  8. Ex situ formation of metal selenide quantum dots using bacterially derived selenide precursors

    International Nuclear Information System (INIS)

    Fellowes, J W; Pattrick, R A D; Lloyd, J R; Charnock, J M; Coker, V S; Mosselmans, J F W; Weng, T-C; Pearce, C I


    Luminescent quantum dots were synthesized using bacterially derived selenide (Se II− ) as the precursor. Biogenic Se II− was produced by the reduction of Se IV by Veillonella atypica and compared directly against borohydride-reduced Se IV for the production of glutathione-stabilized CdSe and β-mercaptoethanol-stabilized ZnSe nanoparticles by aqueous synthesis. Biological Se II− formed smaller, narrower size distributed QDs under the same conditions. The growth kinetics of biologically sourced CdSe phases were slower. The proteins isolated from filter sterilized biogenic Se II− included a methylmalonyl-CoA decarboxylase previously characterized in the closely related Veillonella parvula. XAS analysis of the glutathione-capped CdSe at the S K-edge suggested that sulfur from the glutathione was structurally incorporated within the CdSe. A novel synchrotron based XAS technique was also developed to follow the nucleation of biological and inorganic selenide phases, and showed that biogenic Se II− is more stable and more resistant to beam-induced oxidative damage than its inorganic counterpart. The bacterial production of quantum dot precursors offers an alternative, ‘green’ synthesis technique that negates the requirement of expensive, toxic chemicals and suggests a possible link to the exploitation of selenium contaminated waste streams. (paper)

  9. Single-step purification and characterization of an extreme halophilic, ethanol tolerant and acidophilic xylanase from Aureobasidium pullulans NRRL Y-2311-1 with application potential in the food industry. (United States)

    Yegin, Sirma


    An extracellular xylanase from Aureobasidium pullulans NRRL Y-2311-1 produced on wheat bran was purified by a single-step chromatographic procedure. The enzyme had a molecular weight of 21.6kDa. The optimum pH and temperature for xylanase activity were 4.0 and 30-50°C, respectively. The enzyme was stable in the pH range of 3.0-8.0. The inactivation energy of the enzyme was calculated as 218kJmol -1 . The xylanase was ethanol tolerant and kept complete activity in the presence of 10% ethanol. Likewise, it retained almost complete activity at a concentration range of 0-20% NaCl. In general, the enzyme was resistant to several metal ions and reagents. Mg 2+ , Zn 2+ , Cu 2+ , K 1+ , EDTA and β-mercaptoethanol resulted in enhanced xylanase activity. The K m and V max values on beechwood xylan were determined to be 19.43mgml -1 and 848.4Uml -1 , respectively. The enzyme exhibits excellent characteristics and could, therefore, be a promising candidate for application in food and bio-industries. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Seroepidemiological Study of Brucellosis in High Risk Groups in Boyerahmad 1384

    Directory of Open Access Journals (Sweden)

    AM Khosravani


    Full Text Available ABSTRACT: Introduction & Objective: Brucellosis is a zoonotic disease that may have a major public health and economic impact in most countries. The disease appears as a Malt fever in humans and abortion in animals. This study was designed to determine the serologic titer of Brucella in high risk and non high risk people in Boyerahmad. Materials & Methods: A retrospective seroepidemiological study was performed on samples collected from 604 high risk and non high risk people using Rose Bengol test, tube standard test as a rapid test and 2 mercaptoethanol (2ME and comb's wright as a confirmatory test. The data collected were analyzed by X2 test via SPSS. Results: Seroprevalence of Brucellosis in high risk people appeared to be high in the Rose Bengal and tube standard test (TST 6.62 at titer ≥1/40 whereas for non high risk it was 0%. Confirmation test in high risk people was shown with 2ME in four people. Conclusion: Brucellosis is a major cause of disease in high risk people which can be due to direct or indirect contact with diary products of the related animals.

  11. The maize benzoxazinone DIMBOA reacts with glutathione and other thiols to form spirocyclic adducts. (United States)

    Dixon, David P; Sellars, Jonathan D; Kenwright, Alan M; Steel, Patrick G


    Maize, wheat and other grasses synthesise large quantities of benzoxazinones and their glucosides, which act as antifeedant and allelopathic agents. These activities are probably due to the electrophilic nature of the aglycones, however, the mechanism of their action is unclear. In biological systems, glutathione (GSH) is the major electrophile-reactive compound so the reaction of the major maize benzoxazinone DIMBOA with GSH was studied. GSH reacts with DIMBOA to form eight isomeric mono-conjugates and eight isomeric di-conjugates. Through NMR studies with the model thiol 2-mercaptoethanol, these were structurally elucidated as unusual spirocycles. Similar reactivity was observed with proteins, with cysteinyl thiols being modified by DIMBOA. The thioether bonds formed were stable and not easily reduced to the parent thiol. DIMBOA can therefore readily deplete GSH levels and irreversibly inactivate enzymes with active-site cysteine residues, with clear implications for potentially toxic effects when young grasses are ingested, whether by insect pests or humans. Copyright © 2012 Elsevier Ltd. All rights reserved.

  12. A freeze dried kit formulation for the preparation of 99m Tc labelled human polyclonal IgG for the detection of infection and inflammation

    International Nuclear Information System (INIS)

    Pedraza L, M.


    A freeze dried kit formulation for the preparation of 99m Tc-labelled human polyclonal IgG for the detection of infection and inflammation employing direct methods for protein labeling was developed. The method comprises reduction of intrinsic disulphide bridges within the antibody molecule by the use of the reductant 2-mercaptoethanol. Following a subsequent purification, the resulting reduced antibody is labeled via Sn 2+ reduction of pertechnetate in the presence of a weak competing ligand ethane-1-hydroxy-1,1-diphosphonate (EHDP). High labeling efficiencies (>90%) were obtained with high in vitro stability and without effect upon antibody immunoreactivity. Methods of analysis were also established permitting identification of radiochemical impurities which may be present in radiopharmaceutical solution. 99m Tc-polyclonal IgG prepared by the kit method was evaluated for scintigraphic localization of inflammatory lesions and abscesses in rabbits. Data demonstrated that the 99m Tc-polyclonal IgG after kit-reconstitution shows excellent stability and is an effective imaging agent of infection and/or inflammation. (Author)

  13. Effect of β-mercaptoetanol and cysteine on post-thawing quality and oxidative activity of ram sperm and on the viability of vitrified sheep embryos

    Directory of Open Access Journals (Sweden)

    J. Pradieé

    Full Text Available ABSTRACT The effects of β-mercaptoethanol (BME and cysteine on the viability and oxidative activity of ram sperm after thawing and on development in vitro and viability of vitrified sheep embryos were evaluated. Ejaculates from four rams were pooled and extended, composing six treatments: no antioxidants; 2mM BME; 5mM BME; 2mM BME and 5mM cysteine; 5mM BME and 5mM cysteine; and 5mM cysteine. Sperm motility, membrane and acrosome integrity, mitochondrial functionality, production of reactive oxygen species and total antioxidant capacity were similar across treatments (P>0.05. A medium with no antioxidant presented cleavage and blastocyst development rates (60.3% and 33.6%, respectively similar (P>0.05 to those of a medium with 50μM BME and 600μM cysteine (64.3% and 36.6%, respectively. Post-thawing viability of vitrified embryos was similar between media (P>0.05. Cysteine and BME had no influence on the post-thawing viability and oxidative activity of ram sperm and on the viability of vitrified sheep embryos.

  14. Inactivation of Escherichia coli glycerol kinase by 5'-[p-(fluorosulfonyl)benzoyl]adenosine: protection by the hydrolyzed reagent

    International Nuclear Information System (INIS)

    Pettigrew, D.W.


    Incubation of Escherichia coli glycerol kinase with 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSO 2 BzAdo) at pH 8.0 and 25 0 C results in the loss of enzyme activity, which is not restored by the addition of β-mercaptoethanol or dithiothreitol. The FSO 2 BzAdo concentration dependence of the inactivation kinetics is described by a mechanism that includes the equilibrium binding of the reagent to the enzyme prior to a first-order inactivation reaction in addition to effects of reagent hydrolysis. The hydrolysis of the reagent has two effects on the observed kinetics. The first effect is deviation from pseudo-first-order kinetic behavior due to depletion of the reagent. The second effect is the novel protection of the enzyme from inactivation due to binding of the sulfonate hydrolysis product. Determinations of the reaction stoichiometry with 3 H-labeled FSO 2 BzAdo show that the inactivation is associated with the covalent incorporation of 1.08 mol of reagent/mol of enzyme subunit. Ligand protection experiments show that ATP, AMP, dAMP, NADH, 5'-adenylyl imidodiphosphate, and the sulfonate hydrolysis product of FSO 2 BzAdo provide protection from inactivation. The protection obtained with ATMP is not dependent on Mg 2+ . The results are consistent with modification by FSO 2 BzAdo of a single adenine nucleotide binding site per enzyme subunit

  15. Non-covalent interactions of cadmium sulphide and gold nanoparticles with DNA (United States)

    Atay, Z.; Biver, T.; Corti, A.; Eltugral, N.; Lorenzini, E.; Masini, M.; Paolicchi, A.; Pucci, A.; Ruggeri, G.; Secco, F.; Venturini, M.


    Mercaptoethanol-capped CdS nanoparticles (CdSnp) and monohydroxy-(1-mercaptoundec-11-yl)tetraethylene-glycol-capped Au nanoparticles (Aunp) were synthesised, characterised and their interactions with DNA were investigated. Aunp are stable in different aqueous solvents, whereas CdSnp do precipitate in 0.1 M NaCl and form two different cluster types in 0.1 M NaNO3. As regards the CdSnp/DNA interaction, absorbance and fluorescence titrations, ethidium bromide displacement assays and gel electrophoresis experiments indicate that a non-covalent interaction between DNA and the CdSnp external surface does take place. The binding constant was evaluated to be equal to (2.2 ± 0.5) × 105 M-1. On the contrary, concerning Aunp, no direct interaction with DNA could be observed. Possible interaction with serum albumin was also checked, but no effects could be observed for either CdSnp or Aunp. Finally, short-time exposure of cultured cells to nanoparticles revealed the ability of CdSnp to enter the cells and allocate both in cytosol and nucleus, thus promoting cell proliferation at low concentration ( p resulted in a significant inhibition of cell growth, accompanied by apoptotic cell death. Aunp neither enter the cells, nor do affect cell proliferation. In conclusion, our data indicate that CdSnp can strongly interact with living cells and nucleic acid while no effects or interactions were observed for Aunp.

  16. Binding of ligands to the catalytic zinc ion in horse liver alcohol dehydrogenase. (United States)

    Syvertsen, C; McKinley-McKee, J S


    The affinity of nitrogen and sulfur ligands for the catalytic zinc ion in horse liver alcohol dehydrogenase has been investigated by their influence on the affinity labeling reaction with iodoacetate. All the nitrogen compounds including ammonia, a primary and a secondary amine, and heterocycles containing a pyridine-type nitrogen with the exception of 2,2-dipyridyl were found to activate the affinity labeling reaction. Activation results from inner-sphere ligand coordination to the catalytic zinc ion. Closely related pyridine compounds gave a regular increase in affinity for the enzyme with increasing basicity, as expected for coordination to a metal ion. The sulfur compounds penicillamine and mercaptoethanol also activated the affinity labeling reaction, but dimercaptopropanol bound very tightly as a bidentate inhibited the reaction. The anions hydrosulfide, diethyldithiocarbamate, and cyanide coordinated to the catalytic zinc ion, whereas azide, thiocyanate, tetrazole, and iodide complexed the anion-binding site. The anionic metal ligands increased the rate of inactivation of the enzyme with iodoacetamide by binding to the catalytic zinc ion, while the binding of iodoacetate to the anion-binding site was prevented.

  17. Highly cytotoxic bioconjugated gold(I) complexes with cysteine-containing dipeptides. (United States)

    Gutiérrez, Alejandro; Marzo, Isabel; Cativiela, Carlos; Laguna, Antonio; Gimeno, M Concepción


    Several gold(I) complexes with cysteine-containing dipeptides have been prepared starting from cystine by coupling different amino acids and using several orthogonal protections. The first step is the reaction of cystine, where the sulfur centre is protected as disulfide, with Boc2 O in order to protect the amino group, followed by coupling of an amino acid ester; finally the disulfide bridge is broken with mercaptoethanol to afford the dipeptide derivative. Further reaction with [AuCl(PPh3 )] gives the gold-dipeptide-phosphine species. Starting from these formally gold(I) thiolate-dipeptide phosphine complexes with the general formula [Au(SR)(PR3 )] different structural modifications, such as change in the type of the amino protecting group, the type of phosphine, the number of gold(I) atoms per molecule, or the use of a non-proteinogenic conformationally restricted amino acid ester, were introduced in order to evaluate their influence in the biological activity of the final complexes. The cytotoxic activity, in vitro, of these complexes was evaluated against different tumour human cell lines (A549, MiaPaca2 and Jurkat). The complexes show an outstanding cytotoxic activity with IC50 values in the very low micromolar range. Structure-activity relationship studies from the complexes open the possibility of designing more potent and promising gold(I) anticancer agents. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. An improved method for the isolation of total RNA from Avicennia germinans leaves. (United States)

    Gonzalez-Mendoza, Daniel; Moreno, Adriana Quiroz; Zapata-Perez, Omar


    Isolation of high-quality RNA of Avicennia germinans L. tissue is difficult due to high levels of phenols and other substances that interfere when using conventional procedures for the isolation. These substances not only decrease the yield but also the quality of RNA is almost poor. We present here a simple RNA protocol and fast methodology that effectively removes these contaminating substances without affecting the yield. The protocol developed is based on the SDS/phenol method with modifications including beta-mercaptoethanol to prevent oxidation of phenolic complexes, and phenol/chloroform extraction is introduced to remove proteins, genomic DNA, and secondary metabolites, and co-precipitated polysaccharides. Both A260/A230 and A260/A280 absorbance ratios of isolated RNA were around 2 and the yield was about 0.3 mg g(-1) fresh weight. Good-quality total RNA from leaves of Avicennia germinans could be easily isolated within 2 h by this protocol which avoided the limitation of plant materials and could provide total RNA for all kinds of further molecular studies.

  19. Seroprevalence of Brucellosis in Human Immunodeficiency Virus Infected Patients in Hamadan, Iran (United States)

    Keramat, Fariba; Majzobi, Mohammad Mehdi; Poorolajal, Jalal; Ghane, Zohreh Zarei; Adabi, Maryam


    Objectives Brucellosis is a systemic disease with a wide spectrum of clinical manifestations. This study aimed to determine the seroprevalence of brucellosis in human immunodeficiency virus (HIV) infected patients in Hamadan Province in the west of Iran. Methods A total of 157 HIV-infected patients were screened through standard serological tests, including Wright’s test, Coombs’ Wright test, and 2-mercaptoethanol Brucella agglutination test (2ME test), blood cultures in Castaneda media, and CD4 counting. Data were analyzed using Stata version 11. Results Wright and Coombs’ Wright tests were carried out, and only 5 (3.2%) patients had positive serological results. However, all patients had negative 2ME results, and blood cultures were negative for Brucella spp. Moreover, patients with positive serology and a mean CD4 count of 355.8 ± 203.11 cells/μL had no clinical manifestations of brucellosis, and, and the other patients had a mean CD4 count of 335.55 ± 261.71 cells/μL. Conclusion Results of this study showed that HIV infection is not a predisposing factor of acquiring brucellosis. PMID:28904852

  20. Evaluation of an indirect elisa for the diagnosis of bovine brucellosis in Patagonia, Argentina

    International Nuclear Information System (INIS)

    Uzal, F.A.; Carrasco, E.A.; Robles, C.A.; Echaide, S.


    Control and eradication of bovine brucellosis is usually based on the serological detection of antibodies. In Argentina, the Rose Bengal test (RB) and the Buffered Plate antigen test (BPA) are the two screening test officially recognized, while the 2-mercaptoethanol test (2ME) and the Tube Agglutination test (SAT) are the confirmatory assays currently in use. In order to improve the serological diagnosis of bovine brucellosis in Patagonia, Argentina, an indirect ELISA kit produced by the Joint FAO/IAEA Division was evaluated. Sera from negative non-vaccinated, negative but vaccinated and positive animals were tested by all the above techniques. The specificity of the I-ELISA (99.6% and 99.7%) was similar to that of the BPA, RB, 2ME and Complement Fixation test (CF) when used to test sera from non-vaccinated, negative and vaccinated, negative animals, respectively. The sensitivity of the I-ELISA (98%) was higher than the BPA test (96%) and the CF test (95,2%). The I-ELISA kit evaluated in this study was thought to be a valuable tool for the diagnosis of bovine brucellosis in Patagonia region where little epidemiological information is available about this disease and where large numbers of sera should be tested to obtain such information. (author)

  1. Seroepidemiology of human brucellosis in nomads in a rural area of Iran

    Directory of Open Access Journals (Sweden)

    Ali Sharafi Chegeni


    Full Text Available Objective: To survey the seroprevalence of brucellosis among nomads in Shoulabad, Lorestan province, Iran. Methods: In this descriptive study, a total of 5 847 person took part by randomized cluster sampling and 312 of them with clinical feature of brucellosis were selected. The diagnosis of brucellosis was made with a standard tube agglutination test (STAT, Coombs test, and 2-mercaptoethanol (2ME test, and the results with titers equal 1:80 or more were considered positive. Results: The seroprevalnce of anti-Brucella titer was 29.5% for STAT, 29.9% for Coombs test and 21.1% for 2ME test. Overall, 92, 93 and 66 patients had titers of 1:80 or higher in STAT, Coombs test and 2ME test, respectively. Seroprevalence was more among males (54.3% than females (45.7%. There is statistical difference between seroprevalence of infection and age (P<0.05. Based on age group, 10-19 years age group was more affected (37%. Conclusions: This results confirmed the necessity of conducting comprehensive and scheduled program of seroprevalence survey, particularly in nomad area, which aims at reducing the incidence of brucellosis as well as to guide planning programs by decision makers to improve community health.

  2. Development of a serum-free medium for in vitro expansion of human cytotoxic T lymphocytes using a statistical design

    Directory of Open Access Journals (Sweden)

    Lee Gyun


    Full Text Available Abstract Background Serum-containing medium (SCM, which has a number of poorly defined components with varying concentrations, hampers standardization of lymphocyte cultures. In order to develop a serum-free medium (SFM for the expansion of human lymphocytes from peripheral blood mononuclear cells (PBMCs, a statistical optimization approach based on a fractional factorial method and a response surface method was adopted. A basal medium was prepared by supplementing RPMI1640 medium with insulin, albumin, ferric citrate, ethanolamine, fatty acids, glutamine, sodium pyruvate, 2-mercaptoethanol, 1-thioglycerol, nonessential amino acids, and vitamins. We identified additional positive determinants and their optimal concentrations for cell growth through a statistical analysis. Results From a statistical analysis using the fractional factorial method, cholesterol and polyamine supplement were identified as positive determinants for cell growth. Their optimal concentrations were determined by the response surface method. The maximum viable cell concentration in the developed SFM was enhanced by more than 1.5-fold when compared to that in RPMI1640 supplemented with 10% fetal bovine serum (FBS. Furthermore, a cytotoxicity assay and an enzyme-linked immunospot assay revealed that the effector function of cytotoxic T lymphocytes generated from PBMCs grown in SFM, by stimulation of peptide-presenting dendritic cells, was retained or even better than that in SCM. Conclusions The use of a developed SFM with cholesterol and polyamine supplement for human lymphocyte culture resulted in better growth without loss of cellular function when compared to SCM.

  3. Development of a serum-free medium for in vitro expansion of human cytotoxic T lymphocytes using a statistical design. (United States)

    Jeon, Min Kyoung; Lim, Jong-Baeck; Lee, Gyun Min


    Serum-containing medium (SCM), which has a number of poorly defined components with varying concentrations, hampers standardization of lymphocyte cultures. In order to develop a serum-free medium (SFM) for the expansion of human lymphocytes from peripheral blood mononuclear cells (PBMCs), a statistical optimization approach based on a fractional factorial method and a response surface method was adopted. A basal medium was prepared by supplementing RPMI1640 medium with insulin, albumin, ferric citrate, ethanolamine, fatty acids, glutamine, sodium pyruvate, 2-mercaptoethanol, 1-thioglycerol, nonessential amino acids, and vitamins. We identified additional positive determinants and their optimal concentrations for cell growth through a statistical analysis. From a statistical analysis using the fractional factorial method, cholesterol and polyamine supplement were identified as positive determinants for cell growth. Their optimal concentrations were determined by the response surface method. The maximum viable cell concentration in the developed SFM was enhanced by more than 1.5-fold when compared to that in RPMI1640 supplemented with 10% fetal bovine serum (FBS). Furthermore, a cytotoxicity assay and an enzyme-linked immunospot assay revealed that the effector function of cytotoxic T lymphocytes generated from PBMCs grown in SFM, by stimulation of peptide-presenting dendritic cells, was retained or even better than that in SCM. The use of a developed SFM with cholesterol and polyamine supplement for human lymphocyte culture resulted in better growth without loss of cellular function when compared to SCM.

  4. {sup 99m} Tc labelled monoclonal antibodies: preclinical and clinical evaluation of anti granulocyte monoclonal antibody

    Energy Technology Data Exchange (ETDEWEB)

    Janoki, Gyozo A. [National FJC Research Institute for Radiobiology and Radiohygiene, Budapest (Hungary)


    Full text. The {sup 99} {sup m} Tc-anti-NCA-95 Mo Ab could be used to image bone marrow or inflammatory processes. In our experiment M Ab 47 was reduced with 2-mercaptoethanol and thereafter purified on Sephadex G-50 column. Protein fractions were polled. Samples containing 1 mg of reduce M Ab 47, stannous-pyrophosphate and filling materials were frozen and lyophilized. The number of free S H group by Ellman`s reagent were determined. Structural integrity and labelling efficiency were determined by non-reduced PAGE and size exclusion HPLC Serum stability and cysteine challenge assay were performed also. The in vitro immunoreactivity of {sup 99m} Tc-M ab 47 was evaluation in cell binding assay when human granulocytes and H T-29 adenocarcinoma (CEA+) cells were used. The number of endogenous sulfhydryl groups generated were 6.3 S H group/antibody molecule. Stannous-salt added prior freeze-drying prevented the rearrangement of Sh groups. The amount of 2- mercaptoethanol (M E) survived the gel filtration was 0.4%/mg M Ab 47 as determined by {sup 14} C-M E. The results of HPLC experiments showed, that after purification of reduced M Ab 47 only one component seen on HPLC which has retention time identical with starting Mo Ab. The ratio HPLC analysis of {sup 99m} Tc-M Ab 47 shows two radioactive peak. Non reduced PAGE experiments showed three major component with signification tailing activity.From the results of TLC and ITLC experiments can be seen that the amount of free pertechnetate + radiocolloid are low less than 10%. The results of cysteine challenge assay showed that significant amount of activity dissociated from protein molecule as determined by the followed paper chromatography. Serum stability was 90% at 1 h and 85% at 4 h. In vitro immunoreactivity retained ranged between 80-85% (granulocytes) and 75-90% (CEA+cells). Biodistribution of {sup 99m} Tc-M Ab 47 in normal mice showed high activity in blood pool and well perfused organs. Experiments using tumor

  5. Soy-based Polymers for Surface Modification and Interactions with Lignocellulosic Materials (United States)

    Salas Araujo, Carlos Luis

    -conglycinin was reduced (by 25 and 57 % on silica and cellulose, respectively). Similarly, the addition of 10 mM of 2-mercaptoethanol (a denaturing agent) reduced the mass adsorbed for both proteins. The amounts of 11S and 7S adsorbed on lignin and self-assembled 1-dodecanethiol monolayers were higher when the protein was in the native state if compared to that after chemical denaturation (by using urea and 2-mercaptoethanol). Urea-denatured proteins adsorbed more extensively onto the hydrophobic SAM monolayes. The reduction in water contact angle after protein adsorption (≈40° and 35° for native 11S and 7S, respectively) suggests strong nonspecific interactions between the protein and the substrates, favoring conformational changes at the interface that contribute to exposure and rearrangement of hydrophobic and hydrophilic amino acid residues. The adsorption on polypropylene thin films and nonwovens of different grades of soy proteins in their native as well as thermally-denatured states, including purified glycinin and beta-conglycinin as well as commercial soy flour and isolate was investigated at 25 °C in PBS buffer (pH 7.4). It was found that application of a primer layer of a cationic surfactant, dioctadecyldimethylammonium bromide (DODA) dramatically enhanced protein adsorption, which resulted in fully wettable systems. Fluorescence imaging experiments with tagged proteins confirmed the contribution of a fully-covering layer facilitated by the cationic surfactant pre-treatment. Furthermore, complementary wicking tests indicated that the nonwoven fabrics absorbed a significant amount of water (≈25 times their weight) when the fibers carried pre-adsorbed proteins.

  6. Food allergen analysis for processed food using a novel extraction method to eliminate harmful reagents for both ELISA and lateral-flow tests. (United States)

    Ito, Kaori; Yamamoto, Takayuki; Oyama, Yuriko; Tsuruma, Rieko; Saito, Eriko; Saito, Yoshikazu; Ozu, Takeshi; Honjoh, Tsutomu; Adachi, Reiko; Sakai, Shinobu; Akiyama, Hiroshi; Shoji, Masahiro


    Enzyme-linked immunosorbent assay (ELISA) is commonly used to determine food allergens in food products. However, a significant number of ELISAs give an erroneous result, especially when applied to highly processed food. Accordingly, an improved ELISA, which utilizes an extraction solution comprising the surfactant sodium lauryl sulfate (SDS) and reductant 2-mercaptoethanol (2-ME), has been specially developed to analyze food allergens in highly processed food by enhancing analyte protein extraction. Recently, however, the use of 2-ME has become undesirable. In the present study, a new extraction solution containing a human- and eco-friendly reductant, which is convenient to use at the food manufacturing site, has been established. Among three chemicals with different reducing properties, sodium sulfite, tris(3-hydroxypropyl)phosphine, and mercaptoethylamine sodium sulfite was selected as a 2-ME substitute. The protein extraction ability of SDS/0.1 M sodium sulfite solution was comparable to that of SDS/2-ME solution. Next, the ELISA performance for egg, milk, wheat, peanut, and buckwheat was evaluated by using model-processed foods and commercially available food products. The data showed that the SDS/0.1 M sulfite ELISA significantly correlated with the SDS/2-ME ELISA for all food allergens examined (p food allergens in processed food, showing consistency with the SDS/0.1 M sulfite ELISA results. Accordingly, a harmonized analysis system for processed food comprising a screening LF test and a quantitative ELISA with identical extraction solution has been established. The ELISA based on the SDS/0.1 M sulfite extraction solution has now been authorized as the revised official method for food allergen analysis in Japan.

  7. Synthesis of Waterborne Polyurethane by the Telechelic α,ω-Di(hydroxypoly(n-butyl acrylate

    Directory of Open Access Journals (Sweden)

    Xin Chen


    Full Text Available A key for the preparation of polyacrylate-based polyurethane is the synthesis of hydroxyl-terminated polyacrylate. To our knowledge, exactly one hydroxyl group of every polyacrylate chain has not been reported. The hydroxyl-terminated poly(butyl acrylate (PBA has been successfully synthesized by degenerative iodine transfer polymerization (DITP of the n-butyl acrylate (n-BA using 4,4′-azobis(4-cyano-1-pentanol (ACPO and diiodoxylene (DIX as initiator and chain transfer agent, respectively, and subsequently substituted reaction of the iodine-terminated PBA with β-mercaptoethanol in alkaline condition. The latter reaction was highly efficient, and the terminal iodine at the end of polymer chains were almost quantitatively transformed to a hydroxyl group. 2,2′-Azobis(isobutyronitrile (AIBN and ACPO were used as initiators in the DITPs of n-BA. The results demonstrated that they had a significant influence on the terminal groups of the formed polymer chains. The structure, molecular weight, and molecular weight distribution of the hydroxyl-terminated PBA have been studied by 1H, 13C NMR, and GPC results. The components of hydroxyl-terminated PBA were determined by MALDI-TOF MS spectra, and their formation is discussed. The broad molecular weight distribution of the PBA and the difference in the polymerization behaviors from typical living radical polymerization are explained based on the results of 1H NMR and MALDI-TOF MS spectra. The hydroxyl-terminated PBA has been successfully used in the preparation of PBA-based polyurethane dispersions (PUDs. The aqueous PUDs were stable, and based on the DSC results it can be said that the miscibility of hard segments with PBA chains was improved.

  8. Accumulation of β-Conglycinin in Soybean Cotyledon through the Formation of Disulfide Bonds between α′- and α-Subunits1[W][OA (United States)

    Wadahama, Hiroyuki; Iwasaki, Kensuke; Matsusaki, Motonori; Nishizawa, Keito; Ishimoto, Masao; Arisaka, Fumio; Takagi, Kyoko; Urade, Reiko


    β-Conglycinin, one of the major soybean (Glycine max) seed storage proteins, is folded and assembled into trimers in the endoplasmic reticulum and accumulated into protein storage vacuoles. Prior experiments have used soybean β-conglycinin extracted using a reducing buffer containing a sulfhydryl reductant such as 2-mercaptoethanol, which reduces both intermolecular and intramolecular disulfide bonds within the proteins. In this study, soybean proteins were extracted from the cotyledons of immature seeds or dry beans under nonreducing conditions to prevent the oxidation of thiol groups and the reduction or exchange of disulfide bonds. We found that approximately half of the α′- and α-subunits of β-conglycinin were disulfide linked, together or with P34, prior to amino-terminal propeptide processing. Sedimentation velocity experiments, size-exclusion chromatography, and two-dimensional polyacrylamide gel electrophoresis (PAGE) analysis, with blue native PAGE followed by sodium dodecyl sulfate-PAGE, indicated that the β-conglycinin complexes containing the disulfide-linked α′/α-subunits were complexes of more than 720 kD. The α′- and α-subunits, when disulfide linked with P34, were mostly present in approximately 480-kD complexes (hexamers) at low ionic strength. Our results suggest that disulfide bonds are formed between α′/α-subunits residing in different β-conglycinin hexamers, but the binding of P34 to α′- and α-subunits reduces the linkage between β-conglycinin hexamers. Finally, a subset of glycinin was shown to exist as noncovalently associated complexes larger than hexamers when β-conglycinin was expressed under nonreducing conditions. PMID:22218927

  9. Subunit heterogeneity in the lima bean lectin. (United States)

    Roberts, D D; Etzler, M E; Goldstein, I J


    Three forms of lectin (components I, II, and III) from lima beans (Phaseolus lunatus) have been purified on an affinity support containing the synthetic type A blood group trisaccharide alpha-D-GalNAc-(1 leads to 3)-[alpha-L-Fuc-(1 leads to 2)]-beta-D-Gal-(1 leads to). Conversion of components I and II to component III has been achieved by reduction in 10(-2) M dithiothreitol. Isoelectric focusing of lima bean lectin in the presence of 8 M urea and beta-mercaptoethanol revealed charge heterogeneity of the lectin subunits. Three major subunit classes of apparent pI 7.05, 6.65, and 6.45, designated alpha, beta, and alpha', respectively, were identified; they occur in a relative abundance of 2:5:3. Green lima beans harvested before maturity lacked the alpha' subunit (pI 6.45) which appears to accumulate during seed maturation. The three subunits are glycoproteins of identical size and immunochemical reactivity. Identical NH2-terminal sequences were found for the three subunits. Amino acid analysis and tryptic peptide mapping indicated that the observed charge heterogeneity is probably due to differences in the primary structure of the subunits. Studies of subunit composition of charge isolectins provided evidence of nonrandom subunit assembly. A model is proposed involving pairing of a pI 6.65 subunit with either a pI 7.06 or 6.45 subunit to form dimeric units. Possible roles for subunit heterogeneity and ordered subunit assembly in determining the metal and sugar binding properties of lima bean lectin are discussed.

  10. Bovine brucellosis in Argentina and bordering countries: update. (United States)

    Aznar, M N; Samartino, L E; Humblet, M-F; Saegerman, C


    Bovine brucellosis is a zoonotic disease spread worldwide. The infection in cattle is predominantly caused by Brucella abortus and is usually detected in pregnant females through abortions. The disease is endemic in Argentina; however, infection in humans is underestimated and often not reported. The prevalence of bovine brucellosis in countries bordering Argentina is quite variable: 0.04% in Uruguay, 10.20% in the north and 0.06% in the south of Brazil, 0.2% in Chile, 3.15% in Paraguay and 2.27% in Bolivia. In 1999, the Argentine National Control and Eradication Program was implemented. Its strategies include identification of vaccinated animals, compulsory vaccination with B. abortus S19 of 100% of 3- to 8-month-old females, negative serological tests before animal movements and categorization of farms in terms of their brucellosis status. The epidemiological surveillance in milk is performed through the milk ring test and the indirect ELISA. The result of a national brucellosis survey performed in 2004 indicates that 12.4% (95% CI: 10.89-14.0) of Argentine beef farms are seropositive to Brucella and that the apparent prevalence in cattle is 2.10% (95% CI: 1.90-2.40). The official serological diagnostic tests are as follows: buffered plate antigen test, as screening, serum agglutination test, 2-mercaptoethanol and fluorescence polarization assay, competitive ELISA, as confirmatory tests, and complement fixation test, as definitive test. Santa Fe and a district in Córdoba have 'Outstanding Plans'. Tierra del Fuego is a 'Zone free from bovine brucellosis'. One question arising when studying the Argentine situation is why the disease remains endemic if good regulations exist to control and eradicate it. In future, some different aspects might be evaluated to understand it, and further studies should be performed to prioritize, select and refine control strategies. © 2012 Blackwell Verlag GmbH.

  11. A study of the cytoplasmic expression of a form of human prolactin and of its solubilization and renaturation from bacterial inclusion bodies; Estudo da expressao citoplasmatica bacteriana de uma forma de prolactina humana e de sua solubilizacao e renaturacao a partir de corpos de inclusao

    Energy Technology Data Exchange (ETDEWEB)

    Affonso, Regina


    Different vector elements, that can determine a high expression level of a form of human prolactin (taghPrl) in bacterial cytoplasm, were studied. Expression conditions were first optimized for a reference vector, which was used to transform different strains of E. coli: HB2151, RRI and RB791. The highest expression level (113 {+-}16 {mu}g/mL.A{sub 600}) was obtained in HB2151, after activation with only 0.1 mM IPTG. At this point the influence of the transcription terminator (g32 from bacteriophage T4), of the translation enhancer (g10 from bacteriophage T7), of the promoter ({lambda}P{sub L} or tac) and of the antibiotic resistance gene (amp{sup r} or kan{sup r}) were studied. The first three elements did not show any significant influence, at least in our systems. On the contrary, the analysis of the influence of amp{sup r} and kan{sup r} genes showed, unexpectedly, that the presence of the last one provides an approximately 5-fold higher expression for taghPrl in E. coli cytoplasm. Finally, an appropriate extraction, solubilization, renaturation and purification process, able to provide a monomeric form of taghPrl, was studied. A method utilizing urea and mercaptoethanol as solubilizing agents and a dialysis as a renaturation procedure, provided with some modifications, one of the highest yields ever reported in the literature: 35.4 {+-} 4.5% of total recovery. Moreover, the biological activity of the taghPrl obtained, when tested in the Nb2 cell proliferation assay, was of the same order of that shown by the International Standard of human prolactin of pituitary origin. These data show that the cytoplasmic expression system here described, which can provide an expression efficiency 50-100 - fold higher than the periplasmic expression, can represent a valid alternative for the production of this and of other hormones of pharmaceutical interest and grade. (author)

  12. Two skin cell lines from wild-type and albino Japanese flounder (Paralichthys olivaceus): establishment, characterization, virus susceptibility, efficient transfection, and application to albinism study. (United States)

    Wang, Ruoqing; Zhang, Nianwei; Wang, Renkai; Wang, Shengpeng; Wang, Na


    In order to provide an applicable cell platform to study fish pathology and skin pigmentation, two cell lines derived from skin tissues of wild-type and albino Japanese flounder were established and named JFSK_wt and JFSK_alb, respectively. These two cell lines were cultured for 45 passages within approximately 300 days. JFSK_wt and JFSK_alb cells were maintained in Dulbecco's Modified Eagle's Medium and Ham's F-12 Nutrient Mixture (DMEM/F12) supplemented with antibiotics, fetal bovine serum (FBS), 2-mercaptoethanol (2-Me), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and basic fibroblast growth factor (bFGF). The optimal growth temperature for JFSK_wt and JFSK_alb cells was 24 °C, and microscopically, the two cell lines were composed of fibroblast-like cells. Chromosomal analysis revealed that JFSK_wt and JFSK_alb cells had an identical diploid karyotype with 2n = 48t. Results of viral inoculation assays revealed that both cell lines shared similar patterns of viral susceptibility to nervous necrosis virus (NNV). High transfection efficiency was observed in JFSK_wt and JFSK_alb cells transfected with a pEGFP-N3 reporter plasmid and Cy3-siRNA. The detection of dermal marker Dermo-1 showed that these two cells were both derived from the dermis. Finally, three genes involved in the melanogenesis pathway, including adenylate cyclase type 5 (adcy5), microphthalmia-associated transcription factor (mitf), and endothelin B receptor (ednrb), were downregulated in JFSK_alb versus JFSK_wt cells. Thus, the two cell lines, sampled from skin tissue of wild-type and albino Japanese flounder will be not only helpful for fish pathogen research but also beneficial for albinism-related gene function studies.

  13. Purification and sequencing of radish seed calmodulin antagonists phosphorylated by calcium-dependent protein kinase. (United States)

    Polya, G M; Chandra, S; Condron, R


    A family of radish (Raphanus sativus) calmodulin antagonists (RCAs) was purified from seeds by extraction, centrifugation, batch-wise elution from carboxymethyl-cellulose, and high performance liquid chromatography (HPLC) on an SP5PW cation-exchange column. This RCA fraction was further resolved into three calmodulin antagonist polypeptides (RCA1, RCA2, and RCA3) by denaturation in the presence of guanidinium HCl and mercaptoethanol and subsequent reverse-phase HPLC on a C8 column eluted with an acetonitrile gradient in the presence of 0.1% trifluoroacetic acid. The RCA preparation, RCA1, RCA2, RCA3, and other radish seed proteins are phosphorylated by wheat embryo Ca(2+)-dependent protein kinase (CDPK). The RCA preparation contains other CDPK substrates in addition to RCA1, RCA2, and RCA3. The RCA preparation, RCA1, RCA2, and RCA3 inhibit chicken gizzard calmodulin-dependent myosin light chain kinase assayed with a myosin-light chain-based synthetic peptide substrate (fifty percent inhibitory concentrations of RCA2 and RCA3 are about 7 and 2 microM, respectively). N-terminal sequencing by sequential Edman degradation of RCA1, RCA2, and RCA3 revealed sequences having a high homology with the small subunit of the storage protein napin from Brassica napus and with related proteins. The deduced amino acid sequences of RCA1, RCA2, RCA3, and RCA3' (a subform of RCA3) have agreement with average molecular masses from electrospray mass spectrometry of 4537, 4543, 4532, and 4560 kD, respectively. The only sites for serine phosphorylation are near or at the C termini and hence adjacent to the sites of proteolytic precursor cleavage.

  14. Characterization of biotechnologically relevant extracellular lipase produced by Aspergillus terreus NCFT 4269.10

    Directory of Open Access Journals (Sweden)

    Bijay Kumar Sethi


    Full Text Available Abstract Enzyme production by Aspergillus terreus NCFT 4269.10 was studied under liquid static surface and solid-state fermentation using mustard oil cake as a substrate. The maximum lipase biosynthesis was observed after incubation at 30 °C for 96 h. Among the domestic oils tested, the maximum lipase biosynthesis was achieved using palm oil. The crude lipase was purified 2.56-fold to electrophoretic homogeneity, with a yield of 8.44%, and the protein had a molecular weight of 46.3 kDa as determined by SDS-PAGE. Enzyme characterization confirmed that the purified lipase was most active at pH 6.0, temperature of 50 °C, and substrate concentration of 1.5%. The enzyme was thermostable at 60 °C for 1 h, and the optimum enzyme–substrate reaction time was 30 min. Sodium dodecyl sulfate and commercial detergents did not significantly affect lipase activity during 30-min incubation at 30 °C. Among the metal ions tested, the maximum lipase activity was attained in the presence of Zn2+, followed by Mg2+ and Fe2+. Lipase activity was not significantly affected in the presence of ethylenediaminetetraacetic acid, sodium lauryl sulfate and Triton X-100. Phenylmethylsulfonyl fluoride (1 mM and the reducing, β-mercaptoethanol significantly inhibited lipase activity. The remarkable stability in the presence of detergents, additives, inhibitors and metal ions makes this lipase unique and a potential candidate for significant biotechnological exploitation.

  15. Purification and Characterization of Extracellular enzyme from Aspergillus fumigatus and Its Application on a pennisetum sp for enhanced glucose production

    Directory of Open Access Journals (Sweden)

    Sonali Mohapatra


    Full Text Available Aspergillus species are saprophytic fungi widely distributed in nature and are associated with a number of human diseases. The present study was investigated for production of extracellular cellulase from Aspergillus fumigatus which could be potentially used for degradation of cellulose in lignocellulosic biomass for bioethanol production. In the present work, A. fumigatus were grown in fungal basal medium and preserved at 30 °C for 72 h. The cellulase enzyme was filtered (using Whatman filter paper, precipitated (using ammonium sulphate, dialysed and then purified on a Sepharose 6B ion exchange column. The cellulase enzyme showed a purification of 0.4 fold and the molecular weight was determined as 100 kDa by SDS-PAGE. The optimum pH, temperature, incubation time of the enzyme was determined to be pH 7.0, 35 °C and 24 h respectively. The presence of metal ion Mn2+, followed by Ca2+ and Co2+ was found to increase the cellulase activity. Notably, the cellulase activity was not significantly affected in the presence of additives like EDTA, and Triton X-100 and β-mercaptoethanol. Response surface methodology was used to design optimisation experiments for saccharification of lignocellulosic biomass (hybrid napier grass and the response i.e. glucose yield was considered as the product. The glucose yield was considerably increased from 101.4 mg/g to 856.5 mg/g in the optimised conditions of 35°C, pH 5.2 with substrate concentration (ultrasono assisted alkali pretreated biomass of 3.5 g, with enzyme concentration of 3 ml was incubated for 24 h. Further, the statistical analysis using ANNOVA demonstrated a p- value of less than 0.005 and the R2 value of 90.18.

  16. Characterization of purified α-amylase produced by Aspergillus terreus NCFT 4269.10 using pearl millet as substrate

    Directory of Open Access Journals (Sweden)

    Bijay Kumar Sethi


    Full Text Available α-amylase was produced by Aspergillus terreus NCFT 4269.10 using both liquid static surface (LSSF and solid-state fermentation using pearl millet residues as substrate. The maximum production of α-amylase was noticed at 30°C incubated for 96h. The crude α-amylase was purified to electrophoretic homogeneity and characterized. Characterization of amylase confirmed that the purified α-amylase was found to be most stable at pH 5.0, 60°C temperature, and a substrate concentration of 1.25%. The enzyme was active for 40 min at 70°C with an optimum enzyme–substrate reaction time of 60 min. Amylase was compatible with all detergents tested having highest activity with Surf excel followed by Henko and Ariel. SDS and Tween 20 reduced the activity. Among the metal ions tested, the maximum α-amylase activity was attained in the presence of Ca2+, followed by Mg2+ and Mn2+. The activity of α-amylase was not considerably affected in the presence of ethylenediaminetetraacetic acid and Triton X-100. Amylase activity was accelerated in the presence of sodium lauryl sulfate and phenylmethylsulfonyl fluoride did not significantly (or slightly affect the activity and stability. Tween 20, urea (5%, and the reducing agent, β-mercaptoethanol significantly inhibited the activity of α-amylase. Owing to its noteworthy stability in the presence of detergents, additives, inhibitors, and metal ions, this α-amylase could be an impending enzyme for significant industrial exploitations.

  17. Production and some properties of crude alkaline proteases of indigenous Central Amazonian rhizobia strains

    Directory of Open Access Journals (Sweden)

    Arlem Nascimento de Oliveira


    Full Text Available Two rhizobia strains isolated from soils of the Central Amazonian floodplain produced appreciable quantities of crude alkaline protease extracts with inexpensive carbon and nitrogen sources. These protease crude extracts were optimally active at pH 9.0-11.0. The optimum temperatures were 35 ºC for Rhizobium sp. strain R-986 and 55 ºC for Bradyrhizobium sp. strain R-993. Protease activities in the crude extracts were enhanced in the presence of 5 mM metal ions, such as Na+, Ca2+, Mg2+ and Mn2+. Rhizobia proteases were strongly inhibited by PMSF, a serine-protease inhibitor. The enzymes were active in the presence of surfactants (SDS and Triton X-100 and stable in oxidizing (H2O2 and reducing agents (β-mercaptoethanol, and organic solvents (acetone, hexane, methanol, 1-propanol and toluene.Duas estirpes de rizóbia isoladas de solos de várzea da Amazônia Central produziram grandes quantidades de proteases alcalinas extracelulares, usando fontes baratas de carbono e nitrogênio. Os extratos brutos de proteases foram ativos em pH 9,0-11,0. As temperaturas ótimas foram de 35 ºC para a enzima do Rhizobium R-986 e de 55 ºC para a do Bradyrhizobium R-993. As atividades proteolíticas aumentaram na presença de 5 mM dos íons Na+, Ca2+ , Mg2+ e Mn2+ . As proteases secretadas pelos rizóbios foram fortemente inibidas por PMSF, um inibidor de serina protease. As enzimas foram ativas na presença de surfactantes (SDS e Triton X-100, e estáveis na presença de agentes oxidantes (H2O2 e redutores (β-mercaptoetanol e solventes orgânicos (acetona, hexano, metanol, 1-propanol e tolueno.


    Directory of Open Access Journals (Sweden)

    Muhammad Irfan


    Full Text Available High levels of extracellular xylanase activity (994.50 IU/ml produced by Geobacillus thermodenitrificans C5 originated gene was detected when it was expressed in E. coli BL21 host. Thermostable xylanase (GthC5Xyl was purified to homogeneity and showed a molecular mass of approximately 44 kDa according to SDS-PAGE. The specific activity of the purified GthC5Xyl was up to 1243.125IU/mg with a 9.89-fold purification. The activity of GthC5Xyl was stimulated by CoCl2, MnSO4, CuSO4, MnCl2 but was inhibited by FeSO4, Hg, CaSO4. GthC5Xyl showed resistant to SDS, Tween 20, Triton X-100, β- Mercaptoethanol, PMSF, DTT, NEM and DEPC, SDS, and EDTA. A greater affinity for oat spelt xylan was exhibited by GthC5Xyl with maximum enzymatic activity at 60°C and 6.0 pH. The activity portrayed by GthC5Xyl was found to be acellulytic with stability at high temperature (70°C-80°C and low pH (4.0 to 8.0. Xylobiose and xylopentose were the end products of proficient oat spelt xylanase hydrolysis by GthC5Xyl. High SDS resistance and broader stability of GthC5Xyl proves it to be worthy of impending application in numerous industrial processes especially textile, detergents and animal feed industry.

  19. Production, Characterization and Antioxidant Potential of Protease from Streptomyces sp. MAB18 Using Poultry Wastes

    Directory of Open Access Journals (Sweden)

    Panchanathan Manivasagan


    Full Text Available Poultry waste is an abundant renewable source for the recovery of several value-added metabolites with potential industrial applications. This study describes the production of protease on poultry waste, with the subsequent use of the same poultry waste for the extraction of antioxidants. An extracellular protease-producing strain was isolated from Cuddalore coast, India, and identified as Streptomyces sp. MAB18. Its protease was purified 17.13-fold with 21.62% yield with a specific activity of 2398.36 U/mg and the molecular weight was estimated as 43 kDa. The enzyme was optimally active at pH 8–10 and temperature 50–60°C and it was most stable up to pH 12 and 6–12% of NaCl concentration. The enzyme activity was reduced when treated with Hg2+, Pb2+, and SDS and stimulated by Fe2+, Mg2+, Triton X-100, DMSO (dimethyl sulfoxide, sodium sulphite, and β-mercaptoethanol. Furthermore, the antioxidant activities of protease were evaluated using in vitro antioxidant assays, such as DPPH radical-scavenging activity, O2 scavenging activity, NO scavenging activity, Fe2+ chelating activity, and reducing power. The enzyme showed important antioxidant potential with an IC50 value of 78±0.28 mg/mL. Results of the present study indicate that the poultry waste-derived protease may be useful as supplementary protein and antioxidant in the animal feed formulations.

  20. Two efficient methods for isolation of high-quality genomic DNA from entomopathogenic fungi. (United States)

    Serna-Domínguez, María G; Andrade-Michel, Gilda Y; Arredondo-Bernal, Hugo C; Gallou, Adrien


    Conventional and commercial methods for isolation of nucleic acids are available for fungal samples including entomopathogenic fungi (EPF). However, there is not a unique optimal method for all organisms. The cell wall structure and the wide range of secondary metabolites of EPF can broadly interfere with the efficiency of the DNA extraction protocol. This study compares three commercial protocols: DNeasy® Plant Mini Kit (Qiagen), Wizard® Genomic DNA Purification Kit (Promega), and Axygen™ Multisource Genomic DNA Miniprep Kit (Axygen) and three conventional methods based on different buffers: SDS, CTAB/PVPP, and CTAB/β-mercaptoethanol versus three cell lysis procedures: liquid nitrogen homogenization and two bead-beating materials (i.e., tungsten-carbide and stainless-steel) for four representative species of EPF (i.e., Beauveria bassiana, Hirsutella citriformis, Isaria javanica, and Metarhizium anisopliae). Liquid nitrogen homogenization combined with DNeasy® Plant Mini Kit (i.e., QN) or SDS buffer (i.e., SN) significantly improved the yield with a good purity (~1.8) and high integrity (>20,000 bp) of genomic DNA in contrast with other methods, also, these results were better when compared with the two bead-beating materials. The purified DNA was evaluated by PCR-based techniques: amplification of translation elongation factor 1-α (TEF) and two highly sensitive molecular markers (i.e., ISSR and AFLP) with reliable and reproducible results. Despite a variation in yield, purity, and integrity of extracted DNA across the four species of EPF with the different DNA extraction methods, the SN and QN protocols maintained a high-quality of DNA which is required for downstream molecular applications. Copyright © 2018 Elsevier B.V. All rights reserved.

  1. A fusion protein consisting of the exopeptidases PepN and PepX-production, characterization, and application. (United States)

    Stressler, Timo; Pfahler, Nina; Merz, Michael; Hubschneider, Larissa; Lutz-Wahl, Sabine; Claaßen, Wolfgang; Fischer, Lutz


    Nowadays, general and specific aminopeptidases are of great interest, especially for protein hydrolysis in the food industry. As shown previously, it is confirmed that the general aminopeptidase N (PepN; EC and the proline-specific peptidase PepX (EC from Lactobacillus helveticus ATCC 12046 show a synergistic effect during protein hydrolysis which results in high degrees of hydrolysis and reduced bitterness. To combine both activities, the enzymes were linked and a fusion protein called PepN-L1-PepX (FUS-PepN-PepX) was created. After production and purification, the fusion protein was characterized. Some of its biochemical characteristics were altered in favor for an application compared to the single enzymes. As an example, the optimum temperature for the PepN activity increased from 30 °C for the single enzyme to 35 °C for FUS-PepN. In addition, the temperature stability of PepX was higher for FUS-PepX than for the single enzyme (50 % compared to 40 % residual activity at 50 °C after 14 days, respectively). In addition, the disulfide bridge-reducing reagent β-mercaptoethanol did not longer inactivate the FUS-PepN activity. Furthermore, the K M values decreased for both enzyme activities in the fusion protein. Finally, it was found that the synergistic hydrolysis performance in a casein hydrolysis was not reduced for the fusion protein. The increase of the relative degree of hydrolysis of a prehydrolyzed casein solution was the same as it was for the single enzymes. As a benefit, the resulting hydrolysate showed a strong antioxidative capacity (ABTS-IC50 value: 5.81 μg mL(-1)).

  2. Purification and Characterization of Thermostable and Detergent-Stable α-Amylase from Anoxybacillus sp. AH1

    Directory of Open Access Journals (Sweden)

    Fatma Matpan Bekler


    Full Text Available A thermostable and detergent-stable α-amylase from a newly isolated Anoxybacillus sp. AH1 was purifi ed and characterized. Maximum enzyme production (1874.8 U/mL was obtained at 24 h of incubation. The amylase was purified by using Sephadex G-75 gel filtration, after which an 18-fold increase in specific activity and a yield of 9 % were achieved. The molecular mass of the purified enzyme was estimated at 85 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE. The optimum pH and temperature values of the enzyme were 7.0 and 60 °C, respectively. The enzyme was highly stable in the presence of 30 % glycerol, retaining 85 % of its original activity at 60 °C within 120 min. Km and vmax values were 0.102 μmol and 0.929 μmol/min, espectively, using Lineweaver-Burk plot. The enzyme activity was increased by various detergents, but it was significantly inhibited in the presence of urea. Mg2+ and Ca2+ also significantly activated α-amylase, while Zn2+, Cu2+ and metal ion chelators ethylenediaminetetraacetic acid (EDTA and 1,10-phenanthroline (phen greatly inhibited the enzyme activity. α-Amylase activity was enhanced by β-mercaptoethanol (β-ME and dithiothreitol (DTT to a great extent, but inhibited by p-chloromercuribenzoic acid (PCMB. Iodoacetamide (IAA and N-ethylmaleimide (NEM had a slight, whereas phenylmethylsulfonyl fluoride (PMSF had a strong inhibitory effect on the amylase activity.

  3. Purification and Characterization of Agarase from Marine Bacteria Acinetobacter sp. PS12B and Its Use for Preparing Bioactive Hydrolysate from Agarophyte Red Seaweed Gracilaria verrucosa. (United States)

    Leema Roseline, T; Sachindra, N M


    Acinetobacter strain PS12B was isolated from marine sediment and was found to be a good candidate to degrade agar and produce agarase enzyme. The extracellular agarase enzyme from strain PS12B was purified by ammonium sulfate precipitation followed by DEAE-cellulose ion-exchange chromatography. The specific activity of the crude enzyme which was 1.52 U increased to 45.76 U, after two-stage purification, with an enzyme yield of 9.76%. Purified enzyme had a molecular mass of 24 kDa. The optimum pH and temperature for activity of purified agarase were found to be 8.0 and 40 °C, respectively. The K m and V max values for agarase were 4.69 mg/ml and 0.5 μmol/min, respectively. Treatment with EDTA reduced the agarase activity by 58% at 5 mM concentration. The enzyme activity was stimulated by the presence of Fe 2+ , Mn 2+ , and Ca2 + ions while reducing reagents (β-mercaptoethanol and dithiothreitol, DTT) enhanced its activity by 30-40%. The purified agarase exhibited tolerance to both detergents and organic solvents. Major hydrolysis products of agar were DP4 and also a mixture of longer oligosaccharides DP6 and DP7. The enzyme hydrolysed seaweed (Gracilaria verrucosa) exhibited strong antioxidant activity in vitro. Successful hydrolysis of seaweed indicates the potential use of the enzyme to produce seaweed hydrolysate having health benefits as well as the industrial application like the production of biofuels.

  4. Effects of Cysteamine on Sheep Embryo Cleavage Rates

    Directory of Open Access Journals (Sweden)

    Sinem Ö. ENGİNLER


    Full Text Available Oxidative stress during in vitro culture leads to defects in development of gametes and embryos. Several antioxidants such as cysteamine, L-ascorbic acid, beta mercaptoethanol, cysteine, glutathione, proteins, vitamins have been used to supplement culture media to counter the oxidative stress. This study was conducted to detect the effect of adding cysteamine to the maturation medium to subsequent cleavage rates of sheep embryos. Totally 604 ovaries were obtained by ten replica and 2060 oocytes were collected. The cumulus oocyte complexes were recovered by the slicing method. A total of 1818 selected oocytes were divided into two groups and used for maturation (88.25%. The first group was created as supplemented with cysteamine (Group A and second group (Group B, control without cysteamine in TCM-199. The two groups were incubated for 24 h at 38.8 °C in an atmosphere of 5% CO2 in humidified air for in vitro maturation (IVM. After IVM, oocytes were fertilized with 50 x 107 / mL fresh ram semen in BSOF medium for 18 h. After fertilization, maturation groups were divided into two subgroups with different culture media: Group AI-SOF (Synthetic Oviduct Fluid medium, Group AII-CR1aa (Charles Rosencrans medium, Group BI-SOF and Group BII-CR1aa were achieved. Cleavage rates were evaluated at day 2. post insemination. The rates of cleavage were detected as 59.54% (184/309, 55.44% (173/312, 65.34% (215/329, 59.34% (200/337 respectively, with showing no statistically significant difference between the groups at the level of P>0.05. In conclusion, supplementing cysteamine to maturation media in TCM-199 did not affect the cleavage rates of sheep embryos in SOF and CR1aa culture media.

  5. Formation of the Fe-S cluster of ferredoxin in lysed spinach chloroplasts

    International Nuclear Information System (INIS)

    Takahashi, Yasuhiro; Mitsui, Akira; Matsubara, Hiroshi


    In vitro formation of the 35 S-labeled Fe-S cluster of ferredoxin (Fd) has been achieved by incubating apo-Fd and [ 35 S]cysteine with osmotically lysed chloroplasts of spinach (Spinacia oleracea). Correct integration of the 35 S-labeled Fe-S cluster into Fd was verified on the basis of the following: (a) Under nondenaturing conditions, 35 S-labeled holo-Fd showed the same electrophoretic mobility as authentic holo-Fd; (b) 35 S-labeled holo-Fd showed an ability to bind Fd-NADP + reductase; (c) the 35 S-labeled moiety was removed from the Fd polypeptide by TCA treatment but not by 2-mercaptoethanol treatment; (d) externally added pea II apo-Fd was converted to 35 S-labeled holo-Fd. This reconstitution was dependent on both ATP and light, and formation of the 35 S-labeled Fe-S cluster was observed upon addition of ATP or when an ATP generation-system was constructed in the light. In contrast, ATP-consuming systems abolished the Fe-S cluster formation. A non-hydrolyzable ATP analog was unable to serve as an ATP substitute, indicating the requirement of ATP hydrolysis for cluster formation. GTP was able to substitute for ATP, but CTP and UTP were less effective. Fe-S cluster formation in lysed chloroplasts was stimulated by light even in the presence of added ATP. Light stimulation was inhibited by DCMU or methyl viologen but not by NH 4 + . NADPH was able to substitute for light, indicating that light energy is required for the production of reducing compounds such as NADPH in addition to the generation of ATP

  6. Radiolabeling of anti-CD20 with Re-188 for treatment of Non-Hodgkin's lymphoma: radiochemical control

    International Nuclear Information System (INIS)

    Dias, C.R.; Osso Junior, J.A.


    Radioimmunotherapy (RIT) uses target-specific monoclonal antibodies or fragments labeled with a radioactive isotope to combine humoral and radiolytic functions and has the advantage of targeting not only the cell to which the antibody is bound but also the surrounding tumor cells and microenvironment. The most successful clinical studies of RIT in patients with Non-Hodgkin's Lymphoma (NHL) have targeted CD20+ Bcell tumors. Antibody therapy directed against the CD20 antigen on the surface of B-cells is considered one of the first successful target-specific therapies in oncology. The radionuclide rhenium-188 ( 188 Re) is currently produced from the father nuclide 188 W through a transportable generator system. Because of its easy availability and suitable nuclear properties (E βMAX = 2.1 MeV, t1/2 = 16.9 h, E γ = 155 keV), this radionuclide is considered an attractive candidate for application as therapeutic agent and could be conveniently utilized for imaging and dosimetric purposes. The objective of this work is the optimization of direct radiolabeling method of anti-CD20 with 188 Re using a liquid formulation. Anti-CD20 was reduced by incubation with 2-mercaptoethanol at room temperature. The number of resulting free sulphydryl groups was assayed with Ellman's reagent. Optimization of radiolabeling was achieved by varying parameters: antibody mass, reducing agent mass, tartrate mass, stability and reaction time, 188 Re volume and activity. Radiochemical purity of 188 Re-anti-CD20 was evaluated using instant thin layer chromatography-silica gel (ITLC-SG). Quality control methods for evaluation of radiochemical purity showed good labeling yield of the antibody but further studies will be carried out in order to improve the labeling yields and consequently the specific activity of the product. (author)

  7. Purification of camel liver catalase by zinc chelate affinity chromatography and pH gradient elution: An enzyme with interesting properties. (United States)

    Chafik, Abdelbasset; Essamadi, Abdelkhalid; Çelik, Safinur Yildirim; Mavi, Ahmet


    Climate change and increasing temperatures are global concerns. Camel (Camelus dromedarius) lives most of its life under high environmental stress in the desert and represent ideal model for studying desert adaptation among mammals. Catalase plays a key role in protecting cells against oxidative stress. For the first time, catalase from camel liver was purified to homogeneity by zinc chelate affinity chromatography using pH gradient elution, a better separation was obtained. A purification fold of 201.81 with 1.17% yield and a high specific activity of 1132539.37U/mg were obtained. The native enzyme had a molecular weight of 268kDa and was composed of four subunits of equal size (65kDa). The enzyme showed optimal activity at a temperature of 45°C and pH 7.2. Thiol reagents, β-Mercaptoethanol and D,L-Dithiothreitol, inhibited the enzyme activity. The enzyme was inhibited by Al 3+ , Cd 2+ and Mg 2+ , whereas Ca 2+ , Co 2+ and Ni 2+ stimulated the catalase activity. Reduced glutathione has no effect on catalase activity. The K m and V max of the enzyme for hydrogen peroxide were 37.31mM and 6185157U/mg, respectively. Sodium azide inhibited the enzyme noncompetitively with K i value of 14.43μM, the IC 50 was found to be 16.71μM. The properties of camel catalase were different comparing to those of mammalian species. Relatively higher molecular weight, higher optimum temperature, protection of reduced glutathione from hydrogen peroxide oxidation and higher affinity for hydrogen peroxide and sodium azide, these could be explained by the fact that camel is able to live in the intense environmental stress in the desert. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. In vitro and in vivo inhibitory effects of some fungicides on catalase produced and purified from white-rot fungus Phanerochaete chrysosporium. (United States)

    Kavakçıoğlu, Berna; Tarhan, Leman


    In this study, in vitro and in vivo effects of some commonly used fungicides, antibiotics, and various chemicals on isolated and purified catalase from Phanerochaete chrysosporium were investigated. The catalase was purified 129.10-fold by using 60% ammonium sulfate and 60% ethanol precipitations, DEAE-cellulose anion exchange and Sephacryl-S-200 gel filtration chromatographies from P. chrysosporium growth in carbon- and nitrogen-limited medium for 12 days. The molecular weight of native purified catalase from P. chrysosporium was found to be 290 ± 10 kDa, and sodium dodecyl sulfate (SDS)-PAGE results indicated that enzyme consisted of four apparently identical subunits, with a molecular weight of 72.5 ± 2.5 kDa. Kinetic characterization studies showed that optimum pH and temperature, Km and Vmax values of the purified catalase which were stable in basic region and at comparatively high temperatures were 7.5, 30°C, 289.86 mM, and 250,000 U/mg, respectively. The activity of purified catalase from P. chrysosporium was significantly inhibited by dithiothreitol (DTT), 2-mercaptoethanol, iodoacetamide, EDTA, and sodium dodecyl sulfate (SDS). It was found that while antibiotics had no inhibitory effects, 45 ppm benomyl, 144 ppm captan, and 47.5 ppm chlorothalonil caused 14.52, 10.82, and 38.86% inhibition of purified catalase, respectively. The inhibition types of these three fungicides were found to be non-competitive inhibition with the Ki values of 1.158, 0.638, and 0.145 mM and IC50 values of 0.573, 0.158, 0.010 mM, respectively. The results of in vivo experiments also showed that benomyl, captan and chlorothalonil caused 15.25, 1.96, and 36.70% activity decreases after 24-h treatments compared to that of the control.

  9. Resolution of NADH:ubiquinone oxidoreductase from bovine heart mitochondria into two subcomplexes, one of which contains the redox centers of the enzyme. (United States)

    Finel, M; Skehel, J M; Albracht, S P; Fearnley, I M; Walker, J E


    NADH:ubiquinone oxidoreductase (complex I) was purified from bovine heart mitochondria by solubilization with n-dodecyl beta-D-maltoside (lauryl maltoside), ammonium sulfate fractionation, and chromatography on Mono Q in the presence of the detergent. Its subunit composition was very similar to complex I purified by conventional means. Complex I was dissociated in the presence of N,N-dimethyldodecylamine N-oxide and beta-mercaptoethanol, and two subcomplexes, I alpha and I beta, were isolated by chromatography. Subcomplex I alpha catalyzes electron transfer from NADH to ubiquinone-1. It is composed of about 22 different and mostly hydrophilic subunits and contains 2.0 nmol of FMN/mg of protein. Among its subunits is the 51-kDa subunit, which binds FMN and NADH and probably contains a [4Fe-4S] cluster also. Three other potential Fe-S proteins, the 75- and 24-kDa subunits and a 23-kDa subunit (N-terminal sequence TYKY), are also present. All of the Fe-S clusters detectable by EPR in complex I, including cluster 2, are found in subcomplex I alpha. The line shapes of the EPR spectra of the Fe-S clusters are slightly broadened relative to spectra measured on complex I purified by conventional means, and the quinone reductase activity is insensitive to rotenone. Similar changes were found in samples of the intact chromatographically purified complex I, or in complex I prepared by the conventional method and then subjected to chromatography in the presence of lauryl maltoside. Subcomplex I beta contains about 15 different subunits. The sequences of many of them contain hydrophobic segments that could be membrane spanning, including at least two mitochondrial gene products, ND4 and ND5. The role of subcomplex I beta in the intact complex remains to be elucidated.

  10. Characterization of endo-β-mannanase from Enterobacter ludwigii MY271 and application in pulp industry. (United States)

    Yang, Miao; Cai, Jun; Wang, Changgao; Du, Xin; Lin, Jianguo


    β-Mannanases are the second most important enzymes for the hydrolysis of hemicelluloses. An endo-β-mannanase from Enterobacter ludwigii MY271 was purified at 11.7 ± 0.2-fold to homogeneity with a final recovery of 15.2 ± 0.2 %. Using purified β-mannanase protein and SDS-PAGE, the molecular mass was found to be 43.16 kDa. The optimal pH and temperature of the enzyme was found to be 7.0 and 55 °C, respectively. The β-mannanase activity was stable over a broad pH range of pH 2.0-10.0. In addition, the purified enzyme was highly activated by several metal ions and chemical reagents, such as Mg 2+ , L-cysteine, glutathione (GSH) and β-mercaptoethanol. Whereas the enzyme was strongly inhibited by Hg 2+ , Cu 2+ , N-bromosuccinimide (NBS), 1-ethyl-3-(3-dimethyl-amino-propyl)-carbodiimide (EDC), phenylmethanesulfonyl fluoride (PMSF), and sodium dodecyl sulfate (SDS). The β-mannanase was highly active towards glucomannan, and showed endo-activity by producing a mixture of oligosaccharides. Moreover, the enzyme displayed a classical endo-type mode on mannooligosaccharides. The β-mannanase coupled with xylanase significantly improved the brightness of kraft pulp, whereas it has no remarkable effect on the tensile strength of the pulp. Our functional studies of the purified β-mannanase indicate that the enzyme is beneficial to industrial applications, in particular, biotechnological processes, such as food, feed and pulp industry.

  11. The subunit structure of the extracellular hemoglobin of Biomphalaria glabrata

    International Nuclear Information System (INIS)

    Arndt, Marcio H.L.; Naves, Cristiani F.; Xavier, Luciana P.; Santoro, Marcelo M.


    Full text. The hemoglobin of Biomphalaria glabrata was purified to homogeneity by a two step purification protocol using a gel filtration column (Superose 6 HR/Pharmacia ) followed by an anion exchange chromatography (MONO-Q Sepharose/Pharmacia). The dissociation products were analysed by a 5 - 15 % Polyacrylamide gel electrophoresis containing Sodium Dodecyl Sulfate (SDS-PAGE) giving a band of 270 K Daltons and a band of 180 K Daltons after reduction with β-mercaptoethanol. The same profile was obtained in a 3.5 % Agarose gel electrophoresis containing SDS (SDS-AGE) showing additional bands of higher molecular weight. These bands were proposed to be monomers, dimers and trimers and, after reduction in a Bidimensional SDS-AGE, the proposed monomers and dimers were decomposed in two and four bands that were interpreted as 1 - 4 chains. The hemoglobin was digested by four different proteases ( Thrombin, Trypsin, Chymotrypsin and Subtilisin ) showing several equivalent fragments with molecular weights multiples of its minimum molecular weight ( 17.7 K Daltons). The circular dichroism spectrum of the protein showed a characteristic high α-helix content. We proposed that this hemoglobin is a pentamer of approx. 360 K Daltons subunits each formed by two 180 K Daltons chains linked in pairs by disulfide bridges and each of these chains comprises ten Heme binding domains. These data were compared to other Planorbidae extracellular hemoglobins. Up to now, the quaternary structure of this hemoglobin (shape and disposition of the subunits) is unknown. It is intended to elucidate its structure by Small Angle X-Ray Scattering in Brazilian National Laboratory of Synchrotron Light (LNLS). (author)

  12. Isolation and Culture of Pig Spermatogonial Stem Cells and Their in Vitro Differentiation into Neuron-Like Cells and Adipocytes

    Directory of Open Access Journals (Sweden)

    Xiaoyan Wang


    Full Text Available Spermatogonial stem cells (SSCs renew themselves throughout the life of an organism and also differentiate into sperm in the adult. They are multipopent and therefore, can be induced to differentiate into many cells types in vitro. SSCs from pigs, considered an ideal animal model, are used in studies of male infertility, regenerative medicine, and preparation of transgenic animals. Here, we report on a culture system for porcine SSCs and the differentiation of these cells into neuron-like cells and adipocytes. SSCs and Sertoli cells were isolated from neonatal piglet testis by differential adhesion and SSCs were cultured on a feeder layer of Sertoli cells. Third-generation SSCs were induced to differentiate into neuron-like cells by addition of retinoic acid, β-mercaptoethanol, and 3-isobutyl-1-methylxanthine (IBMX to the induction media and into adipocytes by the addition of hexadecadrol, insulin, and IBMX to the induction media. The differentiated cells were characterized by biochemical staining, qRT-PCR, and immunocytochemistry. The cells were positive for SSC markers, including alkaline phosphatase and SSC-specific genes, consistent with the cells being undifferentiated. The isolated SSCs survived on the Sertoli cells for 15 generations. Karyotyping confirmed that the chromosomal number of the SSCs were normal for pig (2n = 38, n = 19. Pig SSCs were successfully induced into neuron-like cells eight days after induction and into adipocytes 22 days after induction as determined by biochemical and immunocytochemical staining. qPCR results also support this conclusion. The nervous tissue markers genes, Nestin and β-tubulin, were expressed in the neuron-like cells and the adipocyte marker genes, PPARγ and C/EBPα, were expressed in the adipocytes.

  13. Efficacy of strain RB51 vaccine in heifers against experimental brucellosis. (United States)

    Poester, Fernando P; Gonçalves, Vítor S P; Paixão, Tatiane A; Santos, Renato L; Olsen, Steven C; Schurig, Gerhardt G; Lage, Andrey P


    With the goal of providing an additional tool for controlling bovine brucellosis in Brazil and evaluating the full calf dose in adult cattle, the efficacy of the rough Brucella abortus strain RB51 vaccine was tested in heifers. Thirty-three females of approximately 24 months of age were divided in two groups: one group (n=20) received the RB51 vaccine and the other group (n=13) were used as non-vaccinated control. Animals in the vaccinated group were split in two sub-groups. One sub-group (n=12) was vaccinated subcutaneously with 1.5x10(10) colony forming units (CFU) of RB51 at Day 0 of the experiment and the other sub-group (n=8) was vaccinated subcutaneously with 1.6x10(10) CFU of RB51 at 60 days of gestation (Day 260 of the experiment). All cattle were challenged between 6 and 7 months of pregnancy with 3x10(8) CFU of the virulent strain 2308 of B. abortus by the conjunctival route. Vaccination with RB51 vaccine did not result in the production of any antibodies against the O-side chain of lipopolysaccharide (LPS), as measured by conventional serological tests (rose bengal plate agglutination test (RBPAT), standard tube agglutination test (STAT), and 2-mercaptoethanol test (2ME)). A total of 25% cumulative incidence of abortions was found in the vaccinated group, whereas in the control group the cumulative incidence was 62%. B. abortus RB51 was not isolated from any sample, and no abortions were produced by RB51 vaccination of females at 60 days of pregnancy. The results indicate that vaccination with RB51 prevented 59.4% of abortions, 58.6% of cow infections, and 61.0% of fetal infections. The relative risk (RR) revealed that non-vaccinated animals have 2.462 (95% CI 1.029-5.889) times higher risk of aborting than RB51-vaccinated animals.

  14. Transition metal ions and selenite modulate the methylation of arsenite by the recombinant human arsenic (+3 oxidation state) methyltransferase (hAS3MT). (United States)

    Song, Xiaoli; Geng, Zhirong; Li, Chengying; Hu, Xin; Wang, Zhilin


    This report demonstrates that transition metal ions and selenite affect the arsenite methylation by the recombinant human arsenic (+3 oxidation state) methyltransferase (hAS3MT) in vitro. Co(2+), Mn(2+), and Zn(2+) inhibited the arsenite methylation by hAS3MT in a concentration-dependent manner and the kinetics indicated Co(2+) and Mn(2+) to be mixed (competitive and non-competitive) inhibitors while Zn(2+) to be a competitive inhibitor. However, only a high concentration of Fe(2+) could restrain the methylation. UV-visible, CD and fluorescence spectroscopy were used to study the interactions between the metal ions above and hAS3MT. Further studies showed that neither superoxide anion nor hydrogen peroxide was involved in the transition metal ion or selenite inhibition of hAS3MT activity. The inhibition of arsenite methylating activity of hAS3MT by selenite was reversed by 2mM DTT (dithiothreitol) but neither by cysteine nor by beta-mercaptoethanol. Whereas, besides DTT, cysteine can also prevent the inhibition of hAS3MT activity by Co(2+), Mn(2+), and Zn(2+). Free Cys residues were involved in the interactions of transition metal ions or selenite with hAS3MT. It is proposed that the inhibitory effect of the ions (Co(2+), Mn(2+), and Zn(2+)) or selenite on hAS3MT activity might be via the interactions of them with free Cys residues in hAS3MT to form inactive protein adducts.

  15. Preparations of meiotic pachytene chromosomes and extended DNA fibers from cotton suitable for fluorescence in situ hybridization.

    Directory of Open Access Journals (Sweden)

    Renhai Peng

    Full Text Available Fluorescence in situ hybridization (FISH has become one of the most important techniques applied in plant molecular cytogenetics. However, the application of this technique in cotton has lagged behind because of difficulties in chromosome preparation. The focus of this article was FISH performed not only on cotton pachytene chromosomes, but also on cotton extended DNA fibers. The cotton pollen mother cells (PMCs instead of buds or anthers were directly digested in enzyme to completely breakdown the cell wall. Before the routine acetic acid treatment, PMCs were incubated in acetic acid and enzyme mixture to remove the cytoplasm and clear the background. The method of ice-cold Carnoy's solution spreading chromosome was adopted instead of nitrogen removed method to avoid chromosomes losing and fully stretch chromosome. With the above-improved steps, the high-quality well-differentiated pachytene chromosomes with clear background were obtained. FISH results demonstrated that a mature protocol of cotton pachytene chromosomes preparation was presented. Intact and no debris cotton nuclei were obtained by chopping from etiolation cotyledons instead of the conventional liquid nitrogen grinding method. After incubating the nuclei with nucleus lysis buffer on slide, the parallel and clear background DNA fibers were acquired along the slide. This method overcomes the twist, accumulation and fracture of DNA fibers compared with other methods. The entire process of DNA fibers preparation requires only 30 min, in contrast, it takes 3 h with routine nitrogen grinding method. The poisonous mercaptoethanol in nucleus lysis buffer is replaced by nonpoisonous dithiothreitol. PVP40 in nucleus isolation buffer is used to prevent oxidation. The probability of success in isolating nuclei for DNA fiber preparation is almost 100% tested with this method in cotton. So a rapid, safe, and efficient method for the preparation of cotton extended DNA fibers suitable for FISH

  16. Use of Residual Biomass from the Textile Industry as Carbon Source for Production of a Low-Molecular-Weight Xylanase from Aspergillus oryzae

    Directory of Open Access Journals (Sweden)

    Gilvan Caetano Duarte


    Full Text Available Pretreated dirty cotton residue (PDCR from the textile industry was used as an alternative carbon source for the submerged cultivation of Aspergillus oryzae and the production of xylanases. The filtered culture supernatant was fractionated by ultrafiltration followed by three chromatographic steps, which resulted in the isolation of a homogeneous low-molecular-weight xylanase (Xyl-O1 with a mass of 21.5 kDa as determined by sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE co-polymerized with 0.1% oat spelt xylan. Enzyme catalysis was the most efficient at 50 °C and pH 6.0. The Km values (mg·mL−1 for the soluble fraction of oat spelt and birchwood xylans were 10.05 and 3.34, respectively. Xyl-O1 was more stable in the presence of 5,5-dithio-bis-(2-nitrobenzoic acid (DTNB, 1,4-dithiothreitol (DTT, l-cysteine or β-mercaptoethanol, which increased the rate of catalysis by 40%, 14%, 40% or 37%, respectively. The enzyme stability was improved at pH 7.0 in the presence of 20 mM l-cysteine, with the retention of nearly 100% of the activity after 6 h at 50 °C. Xyl-O1 catalyzed the cleavage of internal β-1,4 linkages of the soluble substrates containing d-xylose residues, with a maximum efficiency of 33% for the hydrolysis of birchwood xylan after 12 h of incubation. Identification of the hydrolysis products by high-performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD indicated the predominance of the hydrolysis products X2-X6 during the first 12 h of incubation and the accumulation of higher xylooligomers after the elution of the last xylooligomer standard, xylohexaose.

  17. X-ray-induced mutation of Bacillus sp. MR10 for manno-oligosaccharides production from copra meal. (United States)

    Chaikaew, Siriporn; Kanpiengjai, Apinun; Intatep, Jenjira; Unban, Kridsada; Wongputtisin, Pairote; Takata, Goro; Khanongnuch, Chartchai


    The present study demonstrates the effectiveness of X-ray radiation in strain improvement for defective lipase production by Bacillus sp. MR10 for further application in the fermentative production of manno-oligosaccharides (MOS) from agricultural by-product, defatted copra meal (DCM). The mutants obtained were screened based on their defective lipase activity together with their β-mannanase production performance. Among 10 selected mutants, the strain M7 was the highest promising mutant regarding the smallest lipase activity (0.05 U/ml) and the retained β-mannanase activity similar to the parental strain (22 U/ml) were detected. The mutant M7 effectively hydrolyzed DCM to MOS with low-degree of polymerization (DP) oligomers including mannotriose (M3), mannotetraose (M4), and mannopentose (M5) as the main products. Although the pattern of DCM hydrolysis products of mutant M7 was distinctly different from wild type, the biochemical and catalytic properties of purified β-mannanase of mutant were similar to those of wild type. Both purified β-mannanases with apparent molecular mass of 38 kDa displayed optimal activity at pH 5-7 and 45-55°C. Co 2+ and Hg 2+ nearly completely inhibited activities of both enzymes, whereas Ba 2+ , Fe 3+ , and 2-mercaptoethanol obviously activated enzyme activities. Both enzymes showed high specificity for locust bean gum, konjac mannan, DCM, and guar gum. Thus, the mutant M7 has a potential for commercial production of high-quality MOS from low-cost DCM for further application in the feed industry.

  18. Expression of the chitinase family glycoprotein YKL-40 in undifferentiated, differentiated and trans-differentiated mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Daniel J Hoover

    Full Text Available The glycoprotein YKL-40 (CHI3L1 is a secreted chitinase family protein that induces angiogenesis, cell survival, and cell proliferation, and plays roles in tissue remodeling and immune regulation. It is expressed primarily in cells of mesenchymal origin, is overexpressed in numerous aggressive carcinomas and sarcomas, but is rarely expressed in normal ectodermal tissues. Bone marrow-derived mesenchymal stem cells (MSCs can be induced to differentiate into various mesenchymal tissues and trans-differentiate into some non-mesenchymal cell types. Since YKL-40 has been used as a mesenchymal marker, we followed YKL-40 expression as undifferentiated MSCs were induced to differentiate into bone, cartilage, and neural phenotypes. Undifferentiated MSCs contain significant levels of YKL-40 mRNA but do not synthesize detectable levels of YKL-40 protein. MSCs induced to differentiate into chondrocytes and osteocytes soon began to express and secrete YKL-40 protein, as do ex vivo cultured chondrocytes and primary osteocytes. In contrast, MSCs induced to trans-differentiate into neurons did not synthesize YKL-40 protein, consistent with the general absence of YKL-40 protein in normal CNS parenchyma. However, these trans-differentiated neurons retained significant levels of YKL-40 mRNA, suggesting the mechanisms which prevented YKL-40 translation in undifferentiated MSCs remained in place, and that these trans-differentiated neurons differ in at least this way from neurons derived from neuronal stem cells. Utilization of a differentiation protocol containing β-mercaptoethanol resulted in cells that expressed significant amounts of intracellular YKL-40 protein that was not secreted, which is not seen in normal cells. Thus the synthesis of YKL-40 protein is a marker for MSC differentiation into mature mesenchymal phenotypes, and the presence of untranslated YKL-40 mRNA in non-mesenchymal cells derived from MSCs reflects differences between differentiated and

  19. A new monoclonal antibody radiopharmaceutical for radioimmunoscintigraphy of breast cancer: direct labeling of antibody and its quality control

    Directory of Open Access Journals (Sweden)

    Mojtaba Salouti


    Full Text Available Radioimmunoscintigraphy (RIS has found widespread clinical application in tumor diagnosis. The antibody (Ab PR81 is a new murine anti-MUC1 monoclonal antibody (MAb against human breast carcinoma. In this study a very simple, rapid and efficient method for labeling of this MAb with 99mTc, particularly suitable for development of a ‘kit’is described. The reduction of Ab was performed with 2-mercaptoethanol (2-ME at a molar ratio of 2000:1 (2-ME:MAb and the reduced Ab was labeled with 99mTc via methylene diphosphonate (MDP as a transchelator. The labeling efficiency which was determined by instant thin layer chromatography (ITLC was 94.2%±2.3. Radiocolloides measured by cellulose nitrate electrophoresis were 2.5%±1.7. In vitro stability of the labeled product in human serum which was measured by gel filtration chromatography (FPLC was 70%±5.7 over 24 hr. The integrity of labeled MAb was checked by means of SDS-PAGE and no significant fragmentation was observed. The results of the cell-binding studies showed that both labeled and unlabeled PR81 were able to compete for binding to MCF 7 cells. Biodistribution studies were performed in normal BALB/c mice at 4 and 24 hrs post-injection and no important accumulation was observed in vital organs. These results show that the new radiopharmaceutical may be considered as a promising candidate for imaging of breast cancer.

  20. A novel Ca2+-activated protease from germinating Vigna radiata seeds and its role in storage protein mobilization. (United States)

    Khan, Sadaf; Verma, Giti; Sharma, Samir


    Calcium (Ca(2+))-dependent/activated proteases make decisive cleavages in proteins, affecting their further degradation/activation. Few such Ca(2+)-dependent proteases have been reported from plants, and none during germination-related events. Seeds are woken up from their quiescent state upon imbibition of water. The subsequent process of germination is strongly influenced by hormones (mainly gibberellins) and light, with both resulting in change in intracellular Ca(2+). We have investigated the effect of Ca(2+) on protease activity in extracts prepared from dry Vigna radiata (L.) Wilczec seeds and cotyledons 4, 24, 48 and 72h post-imbibition. Ca(2+)-activated protease activity is present at a very low level in dry seeds, rises with imbibition and peaks 24h post-imbibition. Subsequently, the activity rapidly declines, even as total protease activity continues to rise. Calcium activation of proteolysis was reversed by ethylene diamine tetraacetic acid (EDTA), ethylene glycol-bis (2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), 1,10, phenanthroline, chlorpromazine and by beta-mercaptoethanol in a concentration-dependent manner. Protease activity was also inhibited by para chloro mercuribenzoate (pCMB) and l-trans-epoxysuccinyl-leucylamido(4-guanidino) butane (E 64), while phenyl methyl sulfonyl fluoride (PMSF) and pepstatin did not effect Ca(2+) activation. The protease could be separated from the calmodulin fraction by size-exclusion chromatography, while retaining its ability for Ca(2+) activation, excluding the possibility of activation through calmodulin-based pathways. The presence of a Ca(2+)-activated protease in the cotyledons suggests its role in a predetermined program of germination involving elevation of cytosolic Ca(2+) levels during germination. This protease could be an important enzyme interfacing cytoplasmic signaling events and initiation of storage protein mobilization during seed germination. Copyright (c) 2010 Elsevier GmbH. All rights

  1. Electrophoretic and chemical studies on the X-ray damage of malate synthase

    International Nuclear Information System (INIS)

    Durchschlag, H.; Zipper, P.


    1) Both X-irradiation and treatment with H 2 O 2 caused a decrease of total and an increase of available sulfhydryl groups of the enzyme and led to a loss of enzymic activity. The presence of dithiothreitol turned out to be able to protect the enzyme against X-ray or H 2 O 2 induced inactivation. Moreover, addition of dithiothreitol after X-irradiation or H 2 O 2 treatment allowed a considerable repair of enzymic activity. 2) Polyacrylamide gel disc electrophoreses of X-irradiated enzyme solutions, performed in the presence of sodium dodecyl sulfate, showed the occurrence of covalently cross-linked subunits (preferably dimers and trimers) and of various definite fragments. Electrophoreses in the absence of the denaturant indicated the occurrence of enzyme aggregation. The effects were more pronounced with increasing X-ray doses. The electrophoreses also clearly reflected a radioprotection by dithiothreitol against cross-linking, but not against fragmentation. Addition of excess of 2-mercaptoethanol or of dithiothreitol to the X-irradiated enzyme clearly demonstrated that part of the covalent cross-links were disulfide bridges; the aggregates themselves, however, were held together primarily by non-covalent bonds. Blocking of exposed enzyme sulfhydryls by means of Ellman's reagent prevented both covalent cross-linking and enzyme aggregation. 3) Similar electrophoretic patterns as found for the X-irradiated enzyme were obtained for the unirradiated enzyme after treatment with H 2 O 2 . The similarity of the electropherograms, as well as the reversible diminution of enzymic activity and the loss of sulfhydryls in the presence of H 2 O 2 , suggest an involvement of H 2 O 2 in the radiation damage of the enzyme. It seems plausible that oxidation reactions are responsible for the effects caused by X-irradiation or H 2 O 2 treatment. (orig./AJ)

  2. An unusual presentation of brucellosis, involving multiple organ systems, with low agglutinating titers: a case report

    Directory of Open Access Journals (Sweden)

    Khorvash Farzin


    Full Text Available Abstract Background Brucellosis is a multi-system disease that may present with a broad spectrum of clinical manifestations. While hepatic involvement in brucellosis is not rare, it may rarely involve the kidney or display with cardiac manifestations. Central nervous system involvement in brucellosis sometimes can cause demyelinating syndromes. Here we present a case of brucella hepatitis, myocarditis, acute disseminated encephalomyelitis, and renal failure. Case presentation A 26-year-old man presented with fever, ataxia, and dysarthria. He was a shepherd and gave a history of low grade fever, chilly sensation, cold sweating, loss of appetite, arthralgia and 10 Kg weight loss during the previous 3 months. He had a body temperature of 39°C at the time of admission. On laboratory tests he had elevated level of liver enzymes, blood urea nitrogen, Creatinine, Creatine phosphokinase (MB, and moderate proteinuria. He also had abnormal echocardiography and brain MRI. Enzyme-linked immunosorbent assay for IgG and IgM was negative. Standard tube agglutination test (STAT and 2-mercaptoethanol (2-ME titers were 1:80 and 1:40 respectively. Finally he was diagnosed with brucellosis by positive blood culture and the polymerase chain reaction for Brucella mellitensis. Conclusion In endemic areas clinicians should consider brucellosis in any unusual presentation involving multiple organ systems, even if serology is inconclusive. In endemic areas low STAT and 2-ME titers should be considered as an indication of brucellosis and in these cases additional testing is recommended to rule out brucellosis.

  3. Evaluation and standardization of different purification procedures for fish bile and liver metallothionein quantification by spectrophotometry and SDS-PAGE analyses. (United States)

    Tenório-Daussat, Carolina Lyrio; Resende, Marcia Carolina Martinho; Ziolli, Roberta L; Hauser-Davis, Rachel Ann; Schaumloffel, Dirk; Saint'Pierre, Tatiana D


    Fish bile metallothioneins (MT) have been recently reported as biomarkers for environmental metal contamination; however, no studies regarding standardizations for their purification are available. Therefore, different procedures (varying centrifugation times and heat-treatment temperatures) and reducing agents (DTT, β-mercaptoethanol and TCEP) were applied to purify MT isolated from fish (Oreochromis niloticus) bile and liver. Liver was also analyzed, since these two organs are intrinsically connected and show the same trend regarding MT expression. Spectrophotometrical analyses were used to quantify the resulting MT samples, and SDS-PAGE gels were used to qualitatively assess the different procedure results. Each procedure was then statistically evaluated and a multivariate statistical analysis was then applied. A response surface methodology was also applied for bile samples, in order to further evaluate the responses for this matrix. Heat treatment effectively removes most undesired proteins from the samples, however results indicate that temperatures above 70 °C are not efficient since they also remove MTs from both bile and liver samples. Our results also indicate that the centrifugation times described in the literature can be decreased in order to analyze more samples in the same timeframe, of importance in environmental monitoring contexts where samples are usually numerous. In an environmental context, biliary MT was lower than liver MT, as expected, since liver accumulates MT with slower detoxification rates than bile, which is released from the gallbladder during feeding, and then diluted by water. Therefore, bile MT seems to be more adequate in environmental monitoring scopes regarding recent exposure to xenobiotics that may affect the proteomic and metalloproteomic expression of this biological matrix. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. Colorimetric assay for lead ions based on the leaching of gold nanoparticles. (United States)

    Chen, Yi-You; Chang, Huan-Tsung; Shiang, Yen-Chun; Hung, Yu-Lun; Chiang, Cheng-Kang; Huang, Chih-Ching


    A colorimetric, label-free, and nonaggregation-based gold nanoparticles (Au NPs) probe has been developed for the detection of Pb(2+) in aqueous solution, based on the fact that Pb(2+) ions accelerate the leaching rate of Au NPs by thiosulfate (S(2)O(3)(2-)) and 2-mercaptoethanol (2-ME). Au NPs reacted with S(2)O(3)(2-) ions in solution to form Au(S(2)O(3))(2)(3-) complexes on the Au NP surfaces, leading to slight decreases in their surface plasmon resonance (SPR) absorption. Surface-assisted laser desorption/ionization time-of-flight ionization mass spectrometry (SALDI-TOF MS) data reveals the formation of Pb-Au alloys on the surfaces of the Au NPs in the presence of Pb(2+) ions and 2-ME. The formation of Pb-Au alloys accelerated the Au NPs rapidly dissolved into solution, leading to dramatic decreases in the SPR absorption. The 2-ME/S(2)O(3)(2-)-Au NP probe is highly sensitive (LOD = 0.5 nM) and selective (by at least 1000-fold over other metal ions) toward Pb(2+) ions, with a linear detection range (2.5 nM-10 muM) over nearly 4 orders of magnitude. The cost-effective probe allows rapid and simple determination of the concentrations of Pb(2+) ions in environmental samples (Montana soil and river), with results showing its great practicality for the detection of lead in real samples.

  5. Visual detection of copper(II) ions in blood samples by controlling the leaching of protein-capped gold nanoparticles. (United States)

    Lee, Yen-Fei; Deng, Ting-Wei; Chiu, Wei-Jane; Wei, Tsao-Yen; Roy, Prathik; Huang, Chih-Ching


    We have developed a simple, low-cost, paper-based probe for the selective colorimetric detection of copper ions (Cu(2+)) in aqueous solutions. The bovine serum albumin (BSA)-modified 13.3-nm Au nanoparticle (BSA-Au NP) probe was designed to detect Cu(2+) ions using lead ions (Pb(2+)) and 2-mercaptoethanol (2-ME) as leaching agents in a glycine-NaOH (pH 12.0) solution. In addition, a nitrocellulose membrane (NCM) was used to trap the BSA-Au NPs, leading to the preparation of a nanocomposite film consisting of a BSA-Au NP-decorated membrane (BSA-Au NPs/NCM). The BSA-Au NPs probe operates on the principle that Cu deposition on the surface of the BSA-Au NPs inhibits their leaching ability, which is accelerated by Pb(2+) ions in the presence of 2-ME. Under optimal solution conditions (5 mM glycine-NaOH (pH 12.0), Pb(2+) (50 μM), and 2-ME (1.0 M)), the Pb(2+)/2-ME-BSA-Au NPs/NCM enabled the detection of Cu(2+) at nanomolar concentrations in aqueous solutions by the naked eye with high selectivity (at least 100-fold over other metal ions). In addition, this cost-effective probe allowed for the rapid and simple determination of Cu(2+) ions in not only natural water samples but also in a complex biological sample (in this case, blood sample).

  6. Role of a non-ionic surfactant in direct electron transfer-type bioelectrocatalysis by fructose dehydrogenase

    International Nuclear Information System (INIS)

    Kawai, Shota; Yakushi, Toshiharu; Matsushita, Kazunobu; Kitazumi, Yuki; Shirai, Osamu; Kano, Kenji


    Highlights: • Addition of Triton ® X-100 (1%) completely quenches the FDH-catalyzed current at hydrophobic electrode, but causes only small competitive effect at hydrophilic electrode. • Quartz crystal microbalance measurements support the adsorption of FDH and Triton ® X-100 on both of the electrodes. • The surfactant forms a monolayer on the hydrophobic electrode and FDH adsorbs on the surfactant monolayer. • The surfactant forms a bilayer on the hydrophilic electrode and FDH is embedded in the bilayer to communicate with the electrode. - ABSTRACT: A heterotrimeric membrane-bound fructose dehydrogenase (FDH) from Gluconobacter japonicus NBRC3260 contains FAD in subunit I and three heme C moieties in subunit II as the redox centers, and is one of the direct electron transfer (DET)-type redox enzymes. FDH-catalyzed current density of fructose oxidation at hydrophilic mercaptoethanol (MEtOH)-modified Au electrode is much larger than that at hydrophobic mercaptoethane (MEtn)-modified Au electrode. Addition of a non-ionic surfactant Triton ® X-100 (1%) completely quenches the catalytic current at the MEtn-modified Au electrode, while only small competitive effect is observed at the MEtOH-modified Au electrode. Quartz crystal microbalance measurements support the adsorption of FDH and Triton ® X-100 on both of the modified electrodes. We propose a model to explain the phenomenon as follows. The surfactant forms a monolayer on the hydrophobic MEtn-modified electrode with strong hydrophobic interaction, and FDH adsorbs on the surface of the surfactant monolayer. The monolayer inhibits the electron transfer from FDH to the electrode. On the other hand, the surfactant forms a bilayer on the hydrophilic MEtOH-modified electrode. The interaction between the surfactant bilayer and the hydrophilic electrode is relatively weak so that FDH replaces the surfactant and is embedded in the bilayer to communicate electrochemically with the hydrophilic electrode

  7. Preliminary study of an immunochromatography test for serological diagnosis of canine brucellosis. (United States)

    Wanke, M M; Cairó, F; Rossano, M; Laiño, M; Baldi, P C; Monachesi, N E; Comercio, E A; Vivot, M M


    The most widely used screening test for the diagnosis of brucellosis in the dog is the rapid slide agglutination test in the presence of 2-mercaptoethanol (2ME-RSAT). The diagnosis is partially confirmed by the agar gel immunodiffusion test (AGID) and definitively confirmed by bacteriological isolation. Some chronic cases not detected by these tests may be detected by ELISA tests. The use of 2ME-RSAT in routine clinical practice requires a microscope and an experienced operator. An immunochromatographic diagnostic test for canine brucellosis (FASTest(®) Brucella c., Megacor, Hörbranz, Austria) has been recently released. In this study, we compared the diagnostic performance of the FASTest with those of 2ME-RSAT, AGID and ELISAs. Sera from 17 healthy dogs used as negative controls yielded negative results by FASTest, indicating a 100% specificity in this sample. Among 27 sera of dogs with acute or subacute brucellosis confirmed by B. canis isolation, all of which were positive by RSAT and ELISAs, the FASTest was positive in 24 cases and AGID in 23. In acute and subacute cases, the sensitivity of FASTest was 89%. Sera from six dogs with bacteriologically confirmed chronic brucellosis, which were positive by ELISAs but negative by 2ME-RSAT, were also tested; 1 was positive by FASTest and 4 were positive by AGID. These preliminary results indicate a good specificity of the FASTest (100% in this sample) but an unacceptable sensitivity as a screening test. In cases with chronic brucellosis, the sensitivity of the FASTest was lower than that of ELISAs but this assay could make a good intermediate test to be run after a positive RSAT and before running an AGID. © 2012 Blackwell Verlag GmbH.

  8. Generation of Hepatocyte-like Cells from Human Induced Pluripotent Stem (iPS) Cells By Co-culturing Embryoid Body Cells with Liver Non-parenchymal Cell Line TWNT-1

    International Nuclear Information System (INIS)

    Javed, M. S.; Yaqoob, N.; Iwamuro, M.; Kobayashi, N.; Fujiwara, T.


    Objective: To generate a homogeneous population of patient-specific hepatocyte-like cells (HLCs) from human iPS cells those show the morphologic and phenotypic properties of primary human hepatocytes. Study Design: An experimental study. Place and Duration of Study: Department of Surgery, Okayama University, Graduate School of Medicine, Japan, from April to December 2011. Methodology: Human iPS cells were generated and maintained on ES qualified matrigel coated plates supplemented with mTeSR medium or alternatively on mitotically inactivated MEF feeder layer in DMEM/F12 medium containing 20% KOSR, 4ng/ml bFGF-2, 1 x 10-4 M 2-mercaptoethanol, 1 mmol/L NEAA, 2mM L-glutamine and 1% penicillin-streptomycin. iPS cells were differentiated to HLCs by sequential culture using a four step differentiation protocol: (I) Generation of embryoid bodies (EBs) in suspension culture; (II) Induction of definitive endoderm (DE) from 2 days old EBs by growth in human activin-A (100 ng/ml) and basic fibroblasts growth factor (bFGF2) (100 ng/ml) on matrigel coated plates; (III) Induction of hepatic progenitors by co-culture with non-parenchymal human hepatic stellate cell line (TWNT-1); and (IV) Maturation by culture in dexamethasone. Characterization was performed by RT-PCR and functional assays. Results: The generated HLCs showed microscopically morphological phenotype of human hepatocytes, expressed liver specific genes (ASGPR, Albumin, AFP, Sox17, Fox A2), secreted human liver-specific proteins such as albumin, synthesized urea and metabolized ammonia. Conclusion: Functional HLCs were generated from human iPS cells, which could be used for autologus hepatocyte transplantation for liver failure and as in vitro model for determining the metabolic and toxicological properties of drug compounds. (author)

  9. Isolation and characterization of a novel cold-adapted esterase, MtEst45, from Microbulbifer thermotolerans DAU221

    Directory of Open Access Journals (Sweden)

    Yong-Suk eLee


    Full Text Available A novel esterase, MtEst45, was isolated from a fosmid genomic library of Microbulbifer thermotolerans DAU221. The encoding gene is predicted to have a mass of 45,564 Da and encodes 495 amino acids, excluding a 21 amino acid signal peptide. MtEst45 showed a low amino acid identity (approximately 23–24% compared with other lipolytic enzymes belonging to Family III, a closely related bacterial lipolytic enzyme family. MtEst45 also showed a conserved GXSXG motif, G131IS133YG135, which was reported as active site of known lipolytic enzymes, and the putative catalytic triad composed of D237 and H265. Because these mutants of MtEst45, which was S133A, D237N, and H265L, had no activity, these catalytic triad essential for the enzyme catalysis. MtEst45 was overexpressed in Escherichia coli BL21 (DE3 and purified via His-tag affinity chromatography. The optimal pH and temperature of MtEst45 were estimated to be 8.17 and 46.27°C by response surface methodology, respectively. Additionally, MtEst45 was also active between 1–15°C. The optimal hydrolysis substrate for MtEst45 among p-nitrophenyl esters (C2–C18 was p-nitrophenyl butyrate, and the Km and Vmax values were 0.0998 mM and 550 μmol/min/mg of protein, respectively. MtEst45 was strongly inhibited by Hg2+, Zn2+, and Cu2+ ions; by phenylmethanesulfonyl fluoride; and by β-mercaptoethanol. Ca2+ did not affect the enzyme’s activity. These biochemical properties, sequence identity, and phylogenetic analysis suggest that MtEst45 represents a novel and valuable bacterial lipolytic enzyme family and is useful for biotechnological applications.

  10. Aromatic Polyketide Synthases (Purification, Characterization, and Antibody Development to Benzalacetone Synthase from Raspberry Fruits). (United States)

    Borejsza-Wysocki, W.; Hrazdina, G.


    p-Hydroxyphenylbutan-2-one, the characteristic aroma compound of raspberries (Rubus idaeus L.), is synthesized from p-coumaryl-coenzyme A and malonyl-coenzyme A in a two-step reaction sequence that is catalyzed by benzalacetone synthase and benzalacetone reductase (W. Borejsza-Wysocki and G. Hrazdina [1994] Phytochemistry 35: 623-628). Benzalacetone synthase condenses one malonate with p-coumarate to form the pathway intermediate p-hydroxyphenylbut-3-ene-2-one (p-hydroxybenzalacetone) in a reaction that is similar to those catalyzed by chalcone and stilbene synthases. We have obtained an enzyme preparation from ripe raspberries that was preferentially enriched in benzalacetone synthase (approximately 170-fold) over chalcone synthase (approximately 14-fold) activity. This preparation was used to characterize benzalacetone synthase and to develop polyclonal antibodies in rabbits. Benzalacetone synthase showed similarity in its molecular properties to chalcone synthase but differed distinctly in its substrate specificity, response to 2-mercaptoethanol and ethylene glycol, and induction in cell-suspension cultures. The product of the enzyme, p-hydroxybenzalacetone, inhibited mycelial growth of the raspberry pathogen Phytophthora fragariae var rubi at 250 [mu]M. We do not know whether the dual activity in the benzalacetone synthase preparation is the result of a bifunctional enzyme or is caused by contamination with chalcone synthase that was also present. The rapid induction of the enzyme in cell-suspension cultures upon addition of yeast extract and the toxicity of its product, p-hydroxybenzalacetone, to phytopathogenic fungi also suggest that the pathway may be part of a plant defense response.

  11. Proteins isolated from regenerating sciatic nerves of rats form aggregates following posttranslational amino acid modification

    International Nuclear Information System (INIS)

    Ingoglia, N.A.; Chakroborty, G.; Yu, M.; Luo, D.; Sturman, J.A.


    Soluble proteins of regenerating sciatic nerves of rats can be posttranslationally, covalently modified by a variety of radioactive amino acids. The present study shows that once modified by a mixture of 15 amino acids, many of those proteins form aggregates that are unable to pass through a 0.45-micron filter and pellet following 20,000g centrifugation (suggesting a size of greater than 2 x 10(6) Da). Aggregation of proteins also occurs following modification by Arg or Lys alone, but does not occur following protein modification in nonregenerating nerves or in brain. Aggregates are not disrupted by treatment with 100 mM beta mercaptoethanol or by exposure to 1.0 M NaCl, but aggregates are solubilized by treatment with urea and by boiling in 1.5% SDS. Amino acid analysis of proteins modified by a mixture of [3H]amino acids shows a similar proportion of posttranslationally incorporated Ser, Pro, Val, Ala, Leu, Phe, Lys, and Arg in the soluble and pelletable fractions. Two-dimensional PAGE profiles of soluble and pelletable modified proteins show that the modified proteins in both fractions are in similar pI and molecular weight ranges, except that the soluble modified proteins include a high-molecular-weight component that is absent in the pelleted modified proteins. Kinetic studies show that while half-maximal levels of protein modification occur within 30 seconds of incubation, the appearance of the pelletable modified protein fraction is delayed significantly. These results indicate that amino acid modification of soluble proteins in regenerating sciatic nerves of rats results in physical changes in those proteins so that they form high-molecular-weight aggregates

  12. Localization of the 5-phospho-alpha-D-ribosyl-1-pyrophosphate binding site of human hypoxanthine-guanine phosphoribosyltransferase. (United States)

    Keough, D T; Emmerson, B T; de Jersey, J


    Human erythrocyte hypoxanthine-guanine phosphoribosyltransferase (HPRT) is inactivated by iodoacetate in the absence, but not in the presence, of the substrate, 5-phospho-alpha-D-ribosyl-1-pyrophosphate (PRib-PP). Treatment of HPRT with [14C]iodoacetate followed by tryptic digestion, peptide separation and sequencing has shown that Cys-22 reacts with iodoacetate only in the absence of PRib-PP; this strongly suggests that Cys-22 is in or near the PRib-PP binding site. In contrast, Cys-105 reacts with [14C]iodoacetate both in the presence and absence of PRib-PP. Carboxymethylation of Cys-22 resulted in an increase in the Km for PRib-PP, but no change in Vmax. Storage of HPRT also resulted in an increase in the Km for PRib-PP and a decrease in its susceptibility to inactivation by iodoacetate. Dialysis of stored enzyme against 1 mM dithiothreitol resulted in a marked decrease in Km for PRib-PP. The stoichiometry of the reaction of [14C]iodoacetate with Cys-22 in HPRT leading to inactivation (approx. 1 residue modified per tetramer) showed that, in this preparation of HPRT purified from erythrocytes, only about 25% of the Cys-22 side chains were present as free and accessible thiols. Titration of thiol groups [with 5,5'-dithiobis(2-nitrobenzoic acid)] and the effect of dithiothreitol on Km for PRib-PP indicate that oxidation of thiol groups occurs on storage of HPRT, even in the presence of 1 mM beta-mercaptoethanol.

  13. Microsomal prostaglandin E synthase type 2 (mPGES2) is a glutathione-dependent heme protein, and dithiothreitol dissociates the bound heme to produce active prostaglandin E2 synthase in vitro. (United States)

    Takusagawa, Fusao


    An x-ray study indicated that microsomal prostaglandin E synthase type 2 (mPGES2) is a heme-bound protein and catalyzes prostaglandin (PG) H2 degradation, but not PGE2 formation (Yamada, T., and Takusagawa, F. (2007) Biochemistry 46, 8414-8424). In response to the x-ray study, Watanabe et al. claimed that mPGES2 is a heme-free protein and that both the heme-free and heme-bound proteins have PGE2 synthesis activity in the presence of dithiothreitol (Watanabe, K., Ito, S., and Yamamoto, S. (2008) Biochem. Biophys. Res. Commun. 367, 782-786). To resolve the contradictory results, the heme-binding scheme of mPGES2 was further characterized in vivo and in vitro by absorption and fluorescence spectroscopies. A substantial amount of heme-bound mPGES2 was detected in cell extracts. The heme content in mPGES2 was increased along with an increase in Fe(3+) in the culture medium. Heme-free mPGES2 was converted to the heme-bound form by mixing it with pig liver extract, indicating that mPGES2 is capable of forming a complex with heme in mammalian cells. Heme binds to mPGES2 only in the presence of glutathione. The newly determined heme dissociation constant (2.9 nM) supports strongly that mPGES2 is a heme-bound protein in vivo. The bound heme was not dissociated by oxidation by H2O2 or reduction by glutathione or 2-mercaptoethanol. However, reduction by dithiothreitol (an artificial reducing compound) induced the bound heme to dissociate from mPGES2 and released heme-free mPGES2, which exhibited PGE2 synthesis activity in vitro. Imidazole bound to mPGES2 by stacking on the bound heme and inhibited heme oxidation by H2O2 and reduction by dithiothreitol.

  14. Combining cattle and wound-derived synthetic attractants, POC and Bezzilure B, for sampling Chrysomya bezziana in Indonesia. (United States)

    Sulston, E C J; Wardhana, A H; Hall, M J R; Logan, J G; Gezan, S A; Cameron, M M


    The myiasis fly Chrysomya bezziana Villeneuve (Diptera: Calliphoridae) is an obligate parasite of body orifices and unnatural openings or wounds of vertebrates. It is a major pest of livestock and responsible for economic losses throughout the Old World. Two chemical lures were tested in the present study to assess their effectiveness at trapping Ch. bezziana. Bezzilure B contains a synthetic lure of 2-mercaptoethanol originally identified from cattle wound volatiles. POC is a synthetic lure comprising 4-methyl phenol, 3-propyl phenol and racemic 1-octen-3-ol, originally identified from volatiles of ox odour and now used to control tsetse flies (Diptera: Glossina). Bezzilure B and POC lures were tested alone, and in combination, in a laboratory free flight room bioassay and in a field trial. Sticky traps were used to capture males and females of Ch. bezziana and to determine the efficacy of the lures. In the laboratory, Bezzilure B caught significantly more male and female flies than Bezzilure B+POC (p<0.001). Similarly, in the field, treatment significantly affected the total number of Ch. bezziana caught (p<0.001) but, contrastingly, Bezzilure B+POC caught significantly more flies than Bezzilure B (p<0.05). The differing results between the laboratory and field may simply reflect the different assays and could suggest different roles of semiochemicals at different operational distances. Although ineffective on its own, POC has a synergistic effect with Bezzilure B to increase fly catches in the field: by proportion, Bezzilure B+POC was 2.17 times as efficient as Bezzilure B for Ch. bezziana, and 1.16-2.68 times as efficient for other flies depending on the species collected. Copyright © 2014 International Atomic Energy Agency 2014. Published by Elsevier B.V. All rights reserved.

  15. Study on the preparation and stability of 188Re biomolecules via EHDP

    International Nuclear Information System (INIS)

    Ferro-Flores, G.; Garcia-Salinas, L.; Paredes-Gutierrez, L.; Hashimoto, K.; Melendez-Alafort, L.; Murphy, C.A.


    A direct labelling technique via ethane-1-hydroxy-1,1-diphosphonic acid (EHDP) as a weak competing ligand was developed for the preparation of several biomolecules: 188 Re-monoclonal antibody ior cea1 against carcinoembryonic antigen ( 188 Re-MoAb), biotinylated 188 Re-MoAb ( 188 Re-MoAb-biotin), 188 Re-polyclonal IgG ( 188 Re-IgG), 188 Re-peptide (somatostatine analogue peptide b-(2-naphtyl)-D-Ala-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-amide), 188 Re-MoAb fragments ( 188 Re-F(ab') 2 ) and biotinylated 188 Re-F(ab') 2 ( 188 Re-F(ab') 2 -biotin). The reaction conditions such as pH, temperature, weak ligand concentration and stannous chloride concentration were optimized during the radiolabelling of each biomolecule. Before the labelling procedure, disulphide bridge groups of the biomolecules were reduced with 2-mercaptoethanol (2-ME). To obtain 188 Re labelled antibodies and peptides in high radiochemical yields (>90%) via EHDP, it was necessary to use acidic conditions and a high concentration of stannous chloride to allow the redox reaction Re +7 →Re +5 :Re +4 . The labelling of MoAb and F(ab') 2 with 188 Re via EHDP was also evaluated employing a pretargeted technique by avidin-biotin strategy in normal mice, demonstrating that the 188 Re-labelled biotinylated antibodies are stable complexes in vivo. The 188 Re-peptide complex prepared by this method, was stable for 24 h and no radiolytic degradation was observed. (author)

  16. Purification and characterization of a halophilic α-amylase with increased activity in the presence of organic solvents from the moderately halophilic Nesterenkonia sp. strain F. (United States)

    Shafiei, Mohammad; Ziaee, Abed-Ali; Amoozegar, Mohammad Ali


    An extracellular halophilic α-amylase was purified from Nesterenkonia sp. strain F using 80 % ethanol precipitation and Q-Sepharose anion exchange chromatography. The enzyme showed a single band with an apparent molecular weight of 110 kDa by SDS-PAGE. The amylase exhibited maximal activity at pH 7-7.5, being relatively stable at pH 6.5-7.5. Optimal temperature for the amylase activity and stability was 45 °C. The purified enzyme was highly active in the broad range of NaCl concentrations (0-4 M) with optimal activity at 0.25 M NaCl. The amylase was highly stable in the presence of 3-4 M NaCl. Amylase activity was not influenced by Ca²⁺, Rb⁺, Li⁺, Cs⁺, Mg²⁺ and Hg²⁺, whereas Fe³⁺, Cu²⁺, Zn²⁺ and Al³⁺) strongly inhibited the enzyme activity. The α-amylase was inhibited by EDTA, but was not inhibited by PMSF and β-mercaptoethanol. K(m) value of the amylase for soluble starch was 6.6 mg/ml. Amylolytic activity of the enzyme was enhanced not only by 20 % of water-immiscible organic solvents but also by acetone, ethanol and chloroform. Higher concentration (50 %) of the water-miscible organic solvents had no significant effect on the amylase activity. To the best of our knowledge, this is the first report on increased activity of a microbial α-amylase in the presence of organic solvents.

  17. Selective and irreversible inhibitors of aphid acetylcholinesterases: steps toward human-safe insecticides.

    Directory of Open Access Journals (Sweden)

    Yuan-Ping Pang

    Full Text Available Aphids, among the most destructive insects to world agriculture, are mainly controlled by organophosphate insecticides that disable the catalytic serine residue of acetylcholinesterase (AChE. Because these agents also affect vertebrate AChEs, they are toxic to non-target species including humans and birds. We previously reported that a cysteine residue (Cys, found at the AChE active site in aphids and other insects but not mammals, might serve as a target for insect-selective pesticides. However, aphids have two different AChEs (termed AP and AO, and only AP-AChE carries the unique Cys. The absence of the active-site Cys in AO-AChE might raise concerns about the utility of targeting that residue. Herein we report the development of a methanethiosulfonate-containing small molecule that, at 6.0 microM, irreversibly inhibits 99% of all AChE activity extracted from the greenbug aphid (Schizaphis graminum without any measurable inhibition of the human AChE. Reactivation studies using beta-mercaptoethanol confirm that the irreversible inhibition resulted from the conjugation of the inhibitor to the unique Cys. These results suggest that AO-AChE does not contribute significantly to the overall AChE activity in aphids, thus offering new insight into the relative functional importance of the two insect AChEs. More importantly, by demonstrating that the Cys-targeting inhibitor can abolish AChE activity in aphids, we can conclude that the unique Cys may be a viable target for species-selective agents to control aphids without causing human toxicity and resistance problems.

  18. Optimization of Thermostable Alpha-Amylase Production Via Mix Agricultural-Residues and Bacillus amyloliquefaciens

    Directory of Open Access Journals (Sweden)

    Shalini RAI


    Full Text Available This study reports utilization of mixture of wheat and barley bran (1:1 for the production of thermostable alpha-amylase enzyme through a spore former, heat tolerant strain of Bacillus amyloliquefaciens in solid state fermentation. Maximum yield of alpha-amylase (252.77 U mL-1 was obtained in following optimized conditions, inoculums size 2 mL (2 × 106 CFU/mL, moisture 80%, pH 7±0.02, NaCl (3%, temperature 38±1°C, incubation for 72 h, maltose (1% and tryptone (1%. After SSF crude enzyme was purified via ammonium sulfate precipitation, ion exchange and column chromatography by DEAE Cellulose. Purified protein showed a molecular weight of 42 kDa by SDS-PAGE electrophoresis. After purification, purified enzyme was characterized against several enzymes inhibitors such as temperature, NaCl, pH, metal and surfactants. Pure enzyme was highly active over broad temperature (50-70°C, NaCl concentration (0.5-4 M, and pH (6-10 ranges, indicating it’s a thermoactive and alkali-stable nature. Moreover, CaCl2, MnCl2, =-mercaptoethanol were found to stimulate the amylase activity, whereas FeCl3, sodium dodecyl sulfate (SDS, CuCl3 and ethylenediaminetetraacetic acid (EDTA strongly inhibited the enzyme. Moreover, enzyme specificity and thermal stability conformed by degradation of different soluble starch up to 55°C. Therefore, the present study proved that the extracellular alpha-amylase extracted through wheat flour residues by organism B. amyloliquefaciens MCCB0075, both have considerable potential for industrial application owing to its properties.

  19. Biochemical and functional characterization of Bothropoidin: the first haemorrhagic metalloproteinase from Bothrops pauloensis snake venom. (United States)

    Gomes, Mário Sérgio R; Naves de Souza, Dayane L; Guimarães, Denise O; Lopes, Daiana S; Mamede, Carla C N; Gimenes, Sarah Natalie C; Achê, David C; Rodrigues, Renata S; Yoneyama, Kelly A G; Borges, Márcia H; de Oliveira, Fábio; Rodrigues, Veridiana M


    We present the biochemical and functional characterization of Bothropoidin, the first haemorrhagic metalloproteinase isolated from Bothrops pauloensis snake venom. This protein was purified after three chromatographic steps on cation exchange CM-Sepharose fast flow, size-exclusion column Sephacryl S-300 and anion exchange Capto Q. Bothropoidin was homogeneous by SDS-PAGE under reducing and non-reducing conditions, and comprised a single chain of 49,558 Da according to MALDI TOF analysis. The protein presented an isoelectric point of 3.76, and the sequence of six fragments obtained by MS (MALDI TOF\\TOF) showed a significant score when compared with other PIII Snake venom metalloproteinases (SVMPs). Bothropoidin showed proteolytic activity on azocasein, Aα-chain of fibrinogen, fibrin, collagen and fibronectin. The enzyme was stable at pH 6-9 and at lower temperatures when assayed on azocasein. Moreover, its activity was inhibited by EDTA, 1.10-phenanthroline and β-mercaptoethanol. Bothropoidin induced haemorrhage [minimum haemorrhagic dose (MHD) = 0.75 µg], inhibited platelet aggregation induced by collagen and ADP, and interfered with viability and cell adhesion when incubated with endothelial cells in a dose and time-dependent manner. Our results showed that Bothropoidin is a haemorrhagic metalloproteinase that can play an important role in the toxicity of B. pauloensis envenomation and might be used as a tool for studying the effects of SVMPs on haemostatic disorders and tumour metastasis. © The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  20. Biochemical, biological and molecular characterization of an L-Amino acid oxidase (LAAO) purified from Bothrops pictus Peruvian snake venom. (United States)

    Lazo, Fanny; Vivas-Ruiz, Dan E; Sandoval, Gustavo A; Rodríguez, Edith F; Kozlova, Edgar E G; Costal-Oliveira, F; Chávez-Olórtegui, Carlos; Severino, Ruperto; Yarlequé, Armando; Sanchez, Eladio F


    An L-amino acid oxidase from Peruvian Bothrops pictus (Bpic-LAAO) snake venom was purified using a combination of size-exclusion and ion-exchange chromatography. Bpic-LAAO is a homodimeric glycosylated flavoprotein with molecular mass of ∼65 kDa under reducing conditions and ∼132 kDa in its native form as analyzed by SDS-PAGE and gel filtration chromatography, respectively. N-terminal amino acid sequencing showed highly conserved residues in a glutamine-rich motif related to binding substrate. The enzyme exhibited optimal activity towards L-Leu at pH 8.5, and like other reported SV-LAAOs, it is stable until 55 °C. Kinetic studies showed that the cations Ca 2+ , Mg 2+ and Mn 2+ did not alter Bpic-LAAO activity; however, Zn 2+ is an inhibitor. Some reagents such as β-mercaptoethanol, glutathione and iodoacetate had inhibitory effect on Bpic-LAAO activity, but PMSF, EDTA and glutamic acid did not affect its activity. Regarding the biological activities of Bpic-LAAO, this enzyme induced edema in mice (MED = 7.8 μg), and inhibited human platelet aggregation induced by ADP in a dose-dependent manner and showed antibacterial activity on Gram (+) and Gram (-) bacteria. Bpic-LAAO cDNA of 1494 bp codified a mature protein with 487 amino acid residues comprising a signal peptide of 11 amino acids. Finally, the phylogenetic tree obtained with other sequences of LAAOs, evidenced its similarity to other homologous enzymes, showing two well-established monophyletic groups in Viperidae and Elapidae families. Bpic-LAAO is evolutively close related to LAAOs from B. jararacussu, B. moojeni and B. atrox, and together with the LAAO from B. pauloensis, form a well-defined cluster of the Bothrops genus. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Purification and properties of a novel quizalofop-p-ethyl-hydrolyzing esterase involved in quizalofop-p-ethyl degradation by Pseudomonas sp. J-2. (United States)

    Zhang, Hui; Li, Mengya; Li, Jie; Wang, Guangli; Liu, Yuan


    Quizalofop-p-ethyl (QPE) is a post-emergence herbicide that effectively controls grass weeds and is often detected in the environment. However, the biochemical and molecular mechanisms of QPE degradation in the environment remains unclear. In this study, a highly effective QPE-degrading bacterial strain J-2 was isolated from acclimated activated sludge and identified as a Pseudomonas sp., containing the QPE breakdown metabolite quizalofop acid (QA) identified by Liquid Chromatography-Ion Trap-Mass Spectrometry (LC-IT-MS n ) analysis. A novel QPE hydrolase esterase-encoding gene qpeH was cloned from strain J-2 and functionally expressed in Escherichia coli BL21 (DE3). The specific activity of recombinant QpeH was 198.9 ± 2.7 U mg -1 for QPE with K m and K cat values of 41.3 ± 3.6 μM and 127.3 ± 4.5 s -1 . The optimal pH and temperature for the recombinant QpeH were 8.0 and 30 °C, respectively and the enzyme was activated by Ca 2+ , Cd 2+ , Li + , Fe 3+ and Co 2+ and inhibited by Ni 2+ , Fe 2+ , Ag + , DEPC, SDS, Tween 80, Triton X, β-mercaptoethanol, PMSF, and pCMB. In addition, the catalytic efficiency of QpeH toward different AOPP herbicides in descending order was as follows: fenoxaprop-P-ethyl > quizalofop-P-tefuryl > QPE > haloxyfop-P-methyl > cyhalofopbutyl > clodinafop-propargyl. On the basis of the phylogenetic analysis and multiple sequence alignment, the identified enzyme QpeH, was clustered with esterase family V, suggesting a new member of this family because of its low similarity of amino acid sequence with esterases reported previously.

  2. Solubilization and identification of hen eggshell membrane proteins during different times of chicken embryo development using the proteomic approach. (United States)

    Kaweewong, Kritsda; Garnjanagoonchorn, Wunwiboon; Jirapakkul, Wannee; Roytrakul, Sittiruk


    A fertilized chicken egg is a unit of life. During hatching, transport of nutrients, including calcium, have been reported from the egg components to the developing embryo. Calcium is mobilized from the eggshell with the involvement of Ca(2+)-binding proteins. In addition, other unknown proteins may also play some important roles during embryo developing process. Therefore identification and prediction of biological functions of eggshell membrane (ESM) proteins during chick embryo development was conducted by proteome analysis. Comparison of different lysis solutions indicated that the highest ability to extract ESM proteins could be obtained with 1 % sodium dodecyl sulfate in 5 mM Tris-HCl buffer pH 8.8 containing 0.1 % 2-mercaptoethanol. In this study fertilized Cornish chicken eggs were incubated at 37 °C in humidified incubators for up to 21 days. At selected times (days 1, 9, 15 and 21), samples were taken and the ESMs were carefully separated by hand, washed with distilled water, and air-dried at room temperature. The ESM proteins were then solubilized and analyzed by proteome analysis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis combined with high performance liquid chromatography and mass spectrometry revealed 62 proteins in the ESM; only keratin is known ESM protein, 8 of which are egg white proteins and related while 53 others have not previously been reported. Some differences in the types of proteins and their molecular functions were noted in ESM at different incubation times. One protein which was present only at days 15 and 21 of egg incubation was identified as a calcium binding protein i.e. EGF like repeats and discoidin I like domain 3 (EDIL3 homologous protein).

  3. Humoral and Cellular Response of Pheasants Vaccinated against Newcastle Disease and Haemorrhagic Enteritis

    Directory of Open Access Journals (Sweden)

    S. Graczyk


    Full Text Available The purpose of the experiment was to define whether and to what extent can prophylactic vaccinations against Newcastle disease (ND and haemorrhagic enteritis (HE affect the humoral and cellular response in pheasants. The evaluation of humoral response was performed on a basis of agglutinin titre after administered antigen and the cellular immunity index was the delayed type hypersensitivity (DTH reaction. The pheasants were prophylactically vaccinated against Newcastle Disease (ND on the 1st, 28th and 56th day of life. Moreover, on the 49th day of life, part of the birds was given in the drinking water a vaccine containing the HEV (Haemorrhagic Enteritis Virus. Fourteen days after the HEV vaccination, the birds were intravenously given 0.5 ml of the 10% SRBC (sheep red blood cells suspension. Simultaneously with the SRBC administration the delayed hypersensitivity test was performed by intradermal administration of phytohaemagglutinin (PHA. It was shown that in pheasants vaccinated with NDV and additionally with HEV, the specific agglutinin anti-SRBC titre was significantly (p < 0.05 lower than in birds vaccinated against ND only. It also appeared that, the antibodies resistant to 2-mercaptoethanol were 43% of the total pool of specific anti-SRBC antibodies in the NDV vaccinated birds, whereas in birds vaccinated also with HEV they were 75%. No significant differences were found in the DTH test. Only in the HEV vaccinated pheasants the tendency to increase the wing index value was noted. The results confirm the observations concerning immunosuppressive effects of simultaneous vaccinations. They also indicate that overloading the pheasants with many antigens (ND and HEV vaccination may weaken the humoral response to administered SRBC.

  4. Biomimetic trapping cocktail to screen reactive metabolites: use of an amino acid and DNA motif mixture as light/heavy isotope pairs differing in mass shift. (United States)

    Hosaka, Shuto; Honda, Takuto; Lee, Seon Hwa; Oe, Tomoyuki


    Candidate drugs that can be metabolically transformed into reactive electrophilic products, such as epoxides, quinones, and nitroso compounds, are of special concern because subsequent covalent binding to bio-macromolecules can cause adverse drug reactions, such as allergic reactions, hepatotoxicity, and genotoxicity. Several strategies have been reported for screening reactive metabolites, such as a covalent binding assay with radioisotope-labeled drugs and a trapping method followed by LC-MS/MS analyses. Of these, a trapping method using glutathione is the most common, especially at the early stage of drug development. However, the cysteine of glutathione is not the only nucleophilic site in vivo; lysine, histidine, arginine, and DNA bases are also nucleophilic. Indeed, the glutathione trapping method tends to overlook several types of reactive metabolites, such as aldehydes, acylglucuronides, and nitroso compounds. Here, we introduce an alternate way for screening reactive metabolites as follows: A mixture of the light and heavy isotopes of simplified amino acid motifs and a DNA motif is used as a biomimetic trapping cocktail. This mixture consists of [ 2 H 0 ]/[ 2 H 3 ]-1-methylguanidine (arginine motif, Δ 3 Da), [ 2 H 0 ]/[ 2 H 4 ]-2-mercaptoethanol (cysteine motif, Δ 4 Da), [ 2 H 0 ]/[ 2 H 5 ]-4-methylimidazole (histidine motif, Δ 5 Da), [ 2 H 0 ]/[ 2 H 9 ]-n-butylamine (lysine motif, Δ 9 Da), and [ 13 C 0 , 15 N 0 ]/[ 13 C 1 , 15 N 2 ]-2'-deoxyguanosine (DNA motif, Δ 3 Da). Mass tag triggered data-dependent acquisition is used to find the characteristic doublet peaks, followed by specific identification of the light isotope peak using MS/MS. Forty-two model drugs were examined using an in vitro microsome experiment to validate the strategy. Graphical abstract Biomimetic trapping cocktail to screen reactive metabolites.

  5. Inactivation of Escherichia coli glycerol kinase by 5'-(p-(fluorosulfonyl)benzoyl))adenosine: protection by the hydrolyzed reagent

    Energy Technology Data Exchange (ETDEWEB)

    Pettigrew, D.W.


    Incubation of Escherichia coli glycerol kinase with 5'-(p-(fluorosulfonyl)benzoyl)adenosine (FSO/sub 2/BzAdo) at pH 8.0 and 25/sup 0/C results in the loss of enzyme activity, which is not restored by the addition of ..beta..-mercaptoethanol or dithiothreitol. The FSO/sub 2/BzAdo concentration dependence of the inactivation kinetics is described by a mechanism that includes the equilibrium binding of the reagent to the enzyme prior to a first-order inactivation reaction in addition to effects of reagent hydrolysis. The hydrolysis of the reagent has two effects on the observed kinetics. The first effect is deviation from pseudo-first-order kinetic behavior due to depletion of the reagent. The second effect is the novel protection of the enzyme from inactivation due to binding of the sulfonate hydrolysis product. Determinations of the reaction stoichiometry with /sup 3/H-labeled FSO/sub 2/BzAdo show that the inactivation is associated with the covalent incorporation of 1.08 mol of reagent/mol of enzyme subunit. Ligand protection experiments show that ATP, AMP, dAMP, NADH, 5'-adenylyl imidodiphosphate, and the sulfonate hydrolysis product of FSO/sub 2/BzAdo provide protection from inactivation. The protection obtained with ATMP is not dependent on Mg/sup 2 +/. The results are consistent with modification by FSO/sub 2/BzAdo of a single adenine nucleotide binding site per enzyme subunit.

  6. Optimization of Thermostable Alpha-Amylase Production Via Mix Agricultural-Residues and Bacillus amyloliquefaciens

    Directory of Open Access Journals (Sweden)

    Shalini RAI


    Full Text Available This study reports utilization of mixture of wheat and barley bran (1:1 for the production of thermostable alpha-amylase enzyme through a spore former, heat tolerant strain of Bacillus amyloliquefaciens in solid state fermentation. Maximum yield of alpha-amylase (252.77 U mL-1 was obtained in following optimized conditions, inoculums size 2 mL (2 × 106 CFU/mL, moisture 80%, pH 7±0.02, NaCl (3%, temperature 38±1°C, incubation for 72 h, maltose (1% and tryptone (1%. After SSF crude enzyme was purified via ammonium sulfate precipitation, ion exchange and column chromatography by DEAE Cellulose. Purified protein showed a molecular weight of 42 kDa by SDS-PAGE electrophoresis. After purification, purified enzyme was characterized against several enzymes inhibitors such as temperature, NaCl, pH, metal and surfactants. Pure enzyme was highly active over broad temperature (50-70°C, NaCl concentration (0.5-4 M, and pH (6-10 ranges, indicating it’s a thermoactive and alkali-stable nature. Moreover, CaCl2, MnCl2, =-mercaptoethanol were found to stimulate the amylase activity, whereas FeCl3, sodium dodecyl sulfate (SDS, CuCl3 and ethylenediaminetetraacetic acid (EDTA strongly inhibited the enzyme. Moreover, enzyme specificity and thermal stability conformed by degradation of different soluble starch up to 55°C. Therefore, the present study proved that the extracellular alpha-amylase extracted through wheat flour residues by organism B. amyloliquefaciens MCCB0075, both have considerable potential for industrial application owing to its properties.

  7. Purification and characterization of an extracellular, thermo-alkali-stable, metal tolerant laccase from Bacillus tequilensis SN4.

    Directory of Open Access Journals (Sweden)

    Sonica Sondhi

    Full Text Available A novel extracellular thermo-alkali-stable laccase from Bacillus tequilensis SN4 (SN4LAC was purified to homogeneity. The laccase was a monomeric protein of molecular weight 32 KDa. UV-visible spectrum and peptide mass fingerprinting results showed that SN4LAC is a multicopper oxidase. Laccase was active in broad range of phenolic and non-phenolic substrates. Catalytic efficiency (kcat/Km showed that 2, 6-dimethoxyphenol was most efficiently oxidized by the enzyme. The enzyme was inhibited by conventional inhibitors of laccase like sodium azide, cysteine, dithiothreitol and β-mercaptoethanol. SN4LAC was found to be highly thermostable, having temperature optimum at 85°C and could retain more than 80% activity at 70°C for 24 h. The optimum pH of activity for 2, 6-dimethoxyphenol, 2, 2'-azino bis[3-ethylbenzthiazoline-6-sulfonate], syringaldazine and guaiacol was 8.0, 5.5, 6.5 and 8.0 respectively. Enzyme was alkali-stable as it retained more than 75% activity at pH 9.0 for 24 h. Activity of the enzyme was significantly enhanced by Cu2+, Co2+, SDS and CTAB, while it was stable in the presence of halides, most of the other metal ions and surfactants. The extracellular nature and stability of SN4LAC in extreme conditions such as high temperature, pH, heavy metals, halides and detergents makes it a highly suitable candidate for biotechnological and industrial applications.

  8. Functional and evolutionary characterization of Ohr proteins in eukaryotes reveals many active homologs among pathogenic fungi

    Directory of Open Access Journals (Sweden)

    D.A. Meireles


    Full Text Available Ohr and OsmC proteins comprise two subfamilies within a large group of proteins that display Cys-based, thiol dependent peroxidase activity. These proteins were previously thought to be restricted to prokaryotes, but we show here, using iterated sequence searches, that Ohr/OsmC homologs are also present in 217 species of eukaryotes with a massive presence in Fungi (186 species. Many of these eukaryotic Ohr proteins possess an N-terminal extension that is predicted to target them to mitochondria. We obtained recombinant proteins for four eukaryotic members of the Ohr/OsmC family and three of them displayed lipoyl peroxidase activity. Further functional and biochemical characterization of the Ohr homologs from the ascomycete fungus Mycosphaerella fijiensis Mf_1 (MfOhr, the causative agent of Black Sigatoka disease in banana plants, was pursued. Similarly to what has been observed for the bacterial proteins, we found that: (i the peroxidase activity of MfOhr was supported by DTT or dihydrolipoamide (dithiols, but not by β-mercaptoethanol or GSH (monothiols, even in large excess; (ii MfOhr displayed preference for organic hydroperoxides (CuOOH and tBOOH over hydrogen peroxide; (iii MfOhr presented extraordinary reactivity towards linoleic acid hydroperoxides (k=3.18 (±2.13×108 M−1 s−1. Both Cys87 and Cys154 were essential to the peroxidase activity, since single mutants for each Cys residue presented no activity and no formation of intramolecular disulfide bond upon treatment with hydroperoxides. The pKa value of the Cysp residue was determined as 5.7±0.1 by a monobromobimane alkylation method. Therefore, eukaryotic Ohr peroxidases share several biochemical features with prokaryotic orthologues and are preferentially located in mitochondria. Keywords: Ohr/OsmC, Thiol-dependent peroxidases, Phylogeny

  9. Functional and evolutionary characterization of Ohr proteins in eukaryotes reveals many active homologs among pathogenic fungi. (United States)

    Meireles, D A; Domingos, R M; Gaiarsa, J W; Ragnoni, E G; Bannitz-Fernandes, R; da Silva Neto, J F; de Souza, R F; Netto, L E S


    Ohr and OsmC proteins comprise two subfamilies within a large group of proteins that display Cys-based, thiol dependent peroxidase activity. These proteins were previously thought to be restricted to prokaryotes, but we show here, using iterated sequence searches, that Ohr/OsmC homologs are also present in 217 species of eukaryotes with a massive presence in Fungi (186 species). Many of these eukaryotic Ohr proteins possess an N-terminal extension that is predicted to target them to mitochondria. We obtained recombinant proteins for four eukaryotic members of the Ohr/OsmC family and three of them displayed lipoyl peroxidase activity. Further functional and biochemical characterization of the Ohr homologs from the ascomycete fungus Mycosphaerella fijiensis Mf_1 (MfOhr), the causative agent of Black Sigatoka disease in banana plants, was pursued. Similarly to what has been observed for the bacterial proteins, we found that: (i) the peroxidase activity of MfOhr was supported by DTT or dihydrolipoamide (dithiols), but not by β-mercaptoethanol or GSH (monothiols), even in large excess; (ii) MfOhr displayed preference for organic hydroperoxides (CuOOH and tBOOH) over hydrogen peroxide; (iii) MfOhr presented extraordinary reactivity towards linoleic acid hydroperoxides (k=3.18 (±2.13)×10 8 M -1 s -1 ). Both Cys 87 and Cys 154 were essential to the peroxidase activity, since single mutants for each Cys residue presented no activity and no formation of intramolecular disulfide bond upon treatment with hydroperoxides. The pK a value of the Cys p residue was determined as 5.7±0.1 by a monobromobimane alkylation method. Therefore, eukaryotic Ohr peroxidases share several biochemical features with prokaryotic orthologues and are preferentially located in mitochondria. Copyright © 2017. Published by Elsevier B.V.

  10. Diagnóstico sorológico da brucelose bovina em animais adultos vacinados com dose reduzida da cepa 19 de Brucella abortus Serological diagnosis of bovine brucellosis in adult herd vaccinated with Brucella abortus strain 19 reduced dose

    Directory of Open Access Journals (Sweden)

    Gustavo Coelho Jardim


    Full Text Available Com o presente trabalho avaliou-se o uso de dose reduzida da vacina produzida com a amostra 19 de Brucella abortus, em rebanho adulto negativo para a enfermidade, por meio de técnicas de diagnóstico sorológico preconizadas pelo Programa Nacional de Controle e Erradicação da Brucelose e Tuberculose Animal e por um ensaio indireto de imunoadsorção enzimática (ELISA ID. A prova de fixação de complemento detectou 46,77% de positivos, o antígeno acidificado tamponado 67,74%, o 2-mercaptoetanol com soroaglutinação lenta 87,09% e o ELISA ID 100%. A dose reduzida interferiu no diagnóstico sorológico. Nenhuma das técnicas apresentou especificidade adequada para uso em rebanho nestas condições, até 3 meses após a vacinação.The study evaluated the use of a reduced dose of the Brucella abortus strain 19 vaccine, in an adult herd negative for the disease, by serological diagnostic techniques, advocated by the Brazilian Program for Animal Brucellosis and Tuberculosis Control and Eradication, and by an indirect ELISA. The complement fixation test detecteed 46.77% positives, the rose bengal test 67.74%, the mercaptoethanol with standard agglutination test 87.09% and the ELISA ID 100%. The reduced dose influenced the serological diagnosis. None of the techniques reached a suitable specificity for use in the herd under those conditions, up to 3 months after vaccination.

  11. Zoonoses in humans from small rural properties in Jataizinho, Parana, Brazil

    Directory of Open Access Journals (Sweden)

    Daniela Dib Gonçalves


    Full Text Available The aim of this study was to conduct a serological survey for Lyme diseases, brucellosis, leptospirosis and toxoplasmosis and identify the risk variables related to these zoonoses in humans living in the rural area of Jataizinho, state of Parana, Brazil. A total of 63 rural properties were surveyed. Additionally, 207 serum samples collected from these rural area inhabitants were tested for indirect immunofluorescence (IFI and western blots (WB were performed to detect Borrelia burgdorferi (sensu lato; a tamponated acidified antigen test (AAT and 2-mercaptoethanol (2-ME were used to detect antibodies of Brucella abortus; the microscopic agglutination test (MAT was carried out to detect antibodies anti-Leptospira spp. and IFI was used to find antibodies of Toxoplasma gondii. Two of the samples (0.96% were reactive for Lyme borreliosis, three (1.4% for brucellosis, 25 (12.1% for leptospirosis and 143 (69.1% for toxoplasmosis. Although the town of Jataizinho has a human development index (IDH that was considered to be average (0.733 in the state of Parana, the low social, economic and cultural conditions of the population from small rural properties have resulted in lack of basic information on animal health and direct or indirect contact with the various species of domestic animals, wildlife and ticks have probably contributed to the prevalence levels found. These results show the need for additional regional studies in order to determine the epidemiological characteristics of these diseases as well as their respective vectors and reservoirs so that effective prophylaxis can be administered in the human population.

  12. Porphyrin-magnetite nanoconjugates for biological imaging

    LENUS (Irish Health Repository)

    Nowostawska, Malgorzata


    Abstract Background The use of silica coated magnetic nanoparticles as contrast agents has resulted in the production of highly stable, non-toxic solutions that can be manipulated via an external magnetic field. As a result, the interaction of these nanocomposites with cells is of vital importance in understanding their behaviour and biocompatibility. Here we report the preparation, characterisation and potential application of new "two-in-one" magnetic fluorescent nanocomposites composed of silica-coated magnetite nanoparticles covalently linked to a porphyrin moiety. Method The experiments were performed by administering porphyrin functionalised silica-coated magnetite nanoparticles to THP-1 cells, a human acute monocytic leukaemia cell line. Cells were cultured in RPMI 1640 medium with 25 mM HEPES supplemented with heat-inactivated foetal bovine serum (FBS). Results We have synthesised, characterised and analysed in vitro, a new multimodal (magnetic and fluorescent) porphyrin magnetic nanoparticle composite (PMNC). Initial co-incubation experiments performed with THP-1 macrophage cells were promising; however the PMNC photobleached under confocal microscopy study. β-mercaptoethanol (β-ME) was employed to counteract this problem and resulted not only in enhanced fluorescence emission, but also allowed for elongated imaging and increased exposure times of the PMNC in a cellular environment. Conclusion Our experiments have demonstrated that β-ME visibly enhances the emission intensity. No deleterious effects to the cells were witnessed upon co-incubation with β-ME alone and no increases in background fluorescence were recorded. These results should present an interest for further development of in vitro biological imaging techniques.

  13. Preparative two-dimensional gel electrophoresis with agarose gels in the first dimension for high molecular mass proteins. (United States)

    Oh-Ishi, M; Satoh, M; Maeda, T


    A two-dimensional gel electrophoresis (2-DE) method that uses an agarose isoelectric focusing (IEF) gel in the first dimension (agarose 2-DE) was compared with an immobilized pH gradient 2-DE method (IPG-Dalt). The former method was shown to produce significant improvements in the 2-D electrophoretic separation of high molecular mass proteins larger than 150 kDa, up to 500 kDa, and to have a higher loading capacity, as much as 1.5 mg proteins in total for micropreparative runs. The extraction medium found best in this study for agarose 2-DE of mammal tissues was 6 M urea, 1 M thiourea, 0.5% 2-mercaptoethanol, protease inhibitor cocktail (Complete Mini EDTA-free), 1% Triton X-100 and 3% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Trichloroacetic acid (TCA) treatment of the agarose gel after IEF is to be carefully weighed beforehand, because some high molecular mass proteins were less likely to enter the second-dimensional polyacrylamide gel after TCA fixation, and proteins such as mouse skeletal muscle actin gave pseudospots in the agarose 2-DE patterns without TCA fixation. As a good compromise we suggest fixation of proteins in the agarose gel with TCA for one hour or less. The first-dimensional agarose IEF gel containing Pharmalyte as a carrier ampholyte was 180 mm in length and 2.5-4.8 mm in diameter. The gel diameter was shown to determine the loading capacity of the agarose 2-DE, and 1.5 mg liver proteins in total were successfully separated by the use of a 4.8 mm diameter agarose gel.

  14. Heterodimeric l-amino acid oxidase enzymes from Egyptian Cerastes cerastes venom: Purification, biochemical characterization and partial amino acid sequencing

    Directory of Open Access Journals (Sweden)

    A.E. El Hakim


    Full Text Available Two l-amino acid oxidase enzyme isoforms, Cc-LAAOI and Cc-LAAOII were purified to apparent homogeneity from Cerastes cerastes venom in a sequential two-step chromatographic protocol including; gel filtration and anion exchange chromatography. The native molecular weights of the isoforms were 115 kDa as determined by gel filtration on calibrated Sephacryl S-200 column, while the monomeric molecular weights of the enzymes were, 60, 56 kDa and 60, 53 kDa for LAAOI and LAAOII, respectively. The tryptic peptides of the two isoforms share high sequence homology with other snake venom l-amino acid oxidases. The optimal pH and temperature values of Cc-LAAOI and Cc-LAAOII were 7.8, 50 °C and 7, 60 °C, respectively. The two isoenzymes were thermally stable up to 70 °C. The Km and Vmax values were 0.67 mM, 0.135 μmol/min for LAAOI and 0.82 mM, 0.087 μmol/min for LAAOII. Both isoenzymes displayed high catalytic preference to long-chain, hydrophobic and aromatic amino acids. The Mn2+ ion markedly increased the LAAO activity for both purified isoforms, while Na+, K+, Ca2+, Mg2+ and Ba2+ ions showed a non-significant increase in the enzymatic activity of both isoforms. Furthermore, Zn2+, Ni2+, Co2+, Cu2+ and AL3+ ions markedly inhibited the LAAOI and LAAOII activities. l-Cysteine and reduced glutathione completely inhibited the LAAO activity of both isoenzymes, whereas, β-mercaptoethanol, O-phenanthroline and PMSF completely inhibited the enzymatic activity of LAAOII. Furthermore, iodoacitic acid inhibited the enzymatic activity of LAAOII by 46% and had no effect on the LAAOI activity.

  15. Purification and characterization of a novel heparin degrading enzyme from Aspergillus flavus (MTCC-8654). (United States)

    Banga, Jaspreet; Tripathi, C K M


    A heparinase-producing fungus was isolated, and the strain was taxonomically characterized as Aspergillus flavus by morphophysiological and 26S rRNA gene homology studies. The culture produced intracellular heparinase enzyme, which was purified 40.5-fold by DEAE-Sephadex A-50, CM-Sephadex C-50, and Sephadex G-100 column chromatography. Specific activity of the purified enzyme was found to be 44.6 IU/microg protein and the molecular weight of native as well as reduced heparinase was 24 kDa, showing a monomeric unit structure. Peptide mass spectrum showed poor homogeneity with the database in the peptide bank. The enzyme activity was maximum at 30 degrees C in the presence of 300 mM NaCl at pH 7.0. In the presence of Co2+, Mn2+ ions, and reducing agents (beta-mercaptoethanol, dithiothreitol), enzyme activity was enhanced and inhibited by iodoacetic acid. These observations suggested that free sulfohydryl groups of cysteine residues were necessary for catalytic activity of the enzyme. The enzyme was also inhibited by histidine modifier, DEPC, which suggests that along with cysteine, histidine may be present at its active site. The enzyme showed a high affinity for heparin as a substrate with K (m) and V (max) as 2.2 x 10(-5 )M and 30.8 mM min(-1), respectively. The affinity of the enzyme for different glycosaminoglycans studied varied, with high substrate specificity toward heparin and heparin-derived polysaccharides. Depolymerization of heparin and fractionation of the oligosaccharides yielded heparin disaccharides as main product.

  16. A kinetic study of the mechanism of radiation induced agglomeration of ovalbumin in aqueous solution

    International Nuclear Information System (INIS)

    Tuce, Zorana; Janata, Eberhard; Radojcic, Marija; Milosavljevic, B.H.


    The effect of concentration on the protein radiolytic damage resulting in a change in molecular mass was measured in the concentration range from 0.2 to 2 mmolxdm -3 ovalbumin in phosphate buffered solutions saturated with N 2 O. The electrophoretic analysis of samples on discontinuous SDS-polyacrylamide gels in the presence or absence of 5% β-mercaptoethanol showed an expected result, i.e. that the protein scission did not take place in the absence of oxygen. Only ovalbumin agglomerates, bonded by covalent bonds other than S-S bridges, were observed. The G-value for the formation of ovalbumin agglomerates increased linearly from 1.1 to 2.4 by increasing the ovalbumin concentration from 0.2 to 2 mmolxdm -3 . The result is interpreted as to be owing to the competition between ovalbumin agglomeration and some intramolecular reactions which did not lead to the change in the molecular mass. It was also found that the G-value is independent of irradiation dose rate. The result was rationalized as a kinetic evidence that the agglomeration is not a cross-linking process, i.e. it does not occur via recombination of the protein radicals produced in the interaction of ovalbumin and · OH radical. The result suggested that the agglomeration takes place via the process of grafting, i.e. it occurs in the reaction of ovalbumin radical and an intact ovalbumin molecule. The time-resolved light scattering experiments provided an additional proof, supporting the reaction scheme of radiation-induced protein agglomeration. The biological consequences of the proposed mechanism of protein agglomeration are also discussed

  17. Diagnostic Tests in Human Brucellosis

    Directory of Open Access Journals (Sweden)

    Hamid Reza Nouri


    Full Text Available Context: Brucellosis represents a zoonotic bacterial disease, caused by a gram negative bacterium called Brucella. Between the diverses pecies of this bacteria, B. melitensis, B. abortus, B. suis and B. canis consist the main causes of the disease in humans.More than half a million new cases of Brucellosis are reported annually. Consequently, brucellosis is a remarkable threat for the health of society. Because of the multiple nonspecific clinical signs of this infection, such as fever (60% of cases, night sweating, insomnia and anorexia, which are similar to other diseases, the detection of brucellosis is time-consuming and needs more scrutiny. Evidence Acquisition: Blood culture is considered the gold standard for the detection of brucellosis and the sensitivity of this test in the acute form is high. However, for the chronic type of disease, it is remarkably low, in addition, in some cases, it needs long reaction times. Nevertheless, today, some kinds of tests like automatic culturing system and serological methods, such as Rose Bengal (RB test, serum agglutination test (SAT, 2-mercaptoethanol (2ME and coombs, which are operated based on agglutination, are useful for the problems mentioned earlier. Conclusion: Although serological methods are common for the diagnosis of brucellosis, false results are observable for several methods, such as the SAT method. Tests like the enzyme-linked immunosorbent assay (ELISA, for the screening of specific traits, although confirmed, have their advantages and defects. The lateral flow assay (LFA shows promising evidence to be effective in the diagnosis of brucellosis. The polymerase chain reaction (PCR is more prevalent than other common tests, according to sensitivity and fast answering potency in case of molecular diagnosis. Also, PCR is proper for patients' follow-up during the period of treatment and crimination of relapse by this method is easier compared to others.

  18. Pure zinc sulfide quantum dot as highly selective luminescent probe for determination of hazardous cyanide ion

    Energy Technology Data Exchange (ETDEWEB)

    Shamsipur, Mojtaba, E-mail: [Department of Chemistry, Razi University, Kermanshah (Iran, Islamic Republic of); Rajabi, Hamid Reza, E-mail: [Chemistry Department, Yasouj University, Yasouj 75918-74831 (Iran, Islamic Republic of)


    A rapid and simple fluorescence method is presented for selective and sensitive determination of hazardous cyanide ion in aqueous solution based on functionalized zinc sulfide (ZnS) quantum dot (QD) as luminescent prob. The ultra-small ZnS QDs were synthesized using a chemical co-precipitation method in the presence of 2-mercaptoethanol (ME) as an efficient capping agent. The prepared pure ZnS QDs was applied as an optical sensor for determination of cyanide ions in aqueous solutions. ZnS nanoparticles have exhibited a strong fluorescent emission at about 424 nm. The fluorescence intensity of QDs is linearly proportional to the cyanide ion concentration in the range 2.44 × 10{sup −6} to 2.59 × 10{sup −5} M with a detection limit of 1.70 × 10{sup −7} M at pH 11. The designed fluorescent sensor possesses remarkable selectivity for cyanide ion over other anions such as Cl{sup −}, Br{sup −}, F{sup −}, I{sup −}, IO{sub 3}{sup −}, ClO{sub 4}{sup −}, BrO{sub 3}{sup −}, CO{sub 3}{sup 2−}, NO{sub 2}{sup −}, NO{sub 3}{sup −}, SO{sub 4}{sup 2−}, S{sub 2}O{sub 4}{sup 2−}, C{sub 2}O{sub 4}{sup 2−}, SCN{sup −}, N{sub 3}{sup −}, citrate and tartarate with negligible influences on the cyanide detection by fluorescence spectroscopy. - Highlights: • Preparation of functionalized ZnS quantum dots in aqueous media • Highly selective quantum dot based luminescent probe for determination of cyanide • Fast and sensitive determination of hazardous CN{sup −} by fluorescence quenching.

  19. Western blot analysis of antigens specifically recognized by natural immune responses of patients with Japanese encephalitis infections. (United States)

    Patarapotikul, J; Pothipunya, S; Wanotayan, R; Hongyantarachai, A; Tharavanij, S


    Specific recognition of antigenic proteins of Japanese encephalitis virus (JEV) by JE patients was investigated by using non-reducing and reducing Western immunoblot analysis. Under non-reducing conditions, the profile of JEV proteins recognized comprised E (52 kDa), NS1 (45 and 41 kDa), NS3 (66.2 kDa) and NS5 (103 and 97.4 kDa). When recognition patterns of sera from JE and dengue patients were compared, only slight differences between JE and dengue sera were found (under non-reducing conditions), involving only the 66.2 kDa protein: to this protein, JE sera exhibited greater reactivity, but not in greater frequency, than did dengue sera. In contrast, cerebrospinal fluid (CSF) from JE patients showed more differences from JE sera: CSF antibody lacked recognition of the 41 kDa protein and had lower frequencies, as well as less reactivities to several other proteins. These results suggested that restricted populations of lymphocytes were localized in the central nervous system of JE patients. The effect of reducing agent (2 beta-mercaptoethanol) on the recognition patterns of those groups of sera was also analysed: the reducing agent affected all the proteins mentioned above, however, the effects were not uniform. It is proposed that JE and dengue sera may recognize different epitopes on some or all of these proteins. Such differences cannot be detected by Western immunoblot analysis, but it would be feasible to test this hypothesis using epitope mapping with synthetic peptides in a multi-pin ELISA. Analysis in this fine detail is essential for designing improved JE vaccines.

  20. Porphyrin-magnetite nanoconjugates for biological imaging

    Directory of Open Access Journals (Sweden)

    Conroy Jennifer


    Full Text Available Abstract Background The use of silica coated magnetic nanoparticles as contrast agents has resulted in the production of highly stable, non-toxic solutions that can be manipulated via an external magnetic field. As a result, the interaction of these nanocomposites with cells is of vital importance in understanding their behaviour and biocompatibility. Here we report the preparation, characterisation and potential application of new "two-in-one" magnetic fluorescent nanocomposites composed of silica-coated magnetite nanoparticles covalently linked to a porphyrin moiety. Method The experiments were performed by administering porphyrin functionalised silica-coated magnetite nanoparticles to THP-1 cells, a human acute monocytic leukaemia cell line. Cells were cultured in RPMI 1640 medium with 25 mM HEPES supplemented with heat-inactivated foetal bovine serum (FBS. Results We have synthesised, characterised and analysed in vitro, a new multimodal (magnetic and fluorescent porphyrin magnetic nanoparticle composite (PMNC. Initial co-incubation experiments performed with THP-1 macrophage cells were promising; however the PMNC photobleached under confocal microscopy study. β-mercaptoethanol (β-ME was employed to counteract this problem and resulted not only in enhanced fluorescence emission, but also allowed for elongated imaging and increased exposure times of the PMNC in a cellular environment. Conclusion Our experiments have demonstrated that β-ME visibly enhances the emission intensity. No deleterious effects to the cells were witnessed upon co-incubation with β-ME alone and no increases in background fluorescence were recorded. These results should present an interest for further development of in vitro biological imaging techniques.

  1. Prevalence and risk factors for bovine brucellosis in the State of Santa Catarina, Brazil

    Directory of Open Access Journals (Sweden)

    Karina Diniz Baumgarten


    Full Text Available A study was conducted to verify that the State of Santa Catarina has maintained a low prevalence of bovine brucellosis, which would allow the state to move forward with implementing strategies for disease eradication. The state was divided into five regions. In each region, a predetermined number of randomly selected properties was sampled. In each property, blood samples were collected from randomly selected cows with ages equal to or greater than 24 months. Sera from the animals were submitted to a serial testing protocol, with screening by the buffered acidified antigen test and confirmation by the 2-mercaptoethanol test. In each property, a questionnaire was used to identify the risk factors associated with the disease. In the state, the prevalence rate of infected herds was 0.912% [0.297 - 2.11] and infected animals was 1.21% [0.09 - 4.97]. Relative to the earlier study in 2002, there was no difference. The risk factors associated with the condition of a herd infected with brucellosis were as follows: herd size ? 12 cows (OR = 7.47 [2.14 - 34.34] and the presence of flooded areas (OR = 5.68 [1.62 - 26.13]. In view of the low prevalence, it is recommended that the state proceed with the implementation of eradication strategies that are based on a surveillance system structured to detect and eliminate infected herds, and supported by an effective compensation fund for the replacement of seropositive animals. Additionally, the State should make a significant effort to educate and supervise producers to ensure the testing of breeding animals for brucellosis before introducing them into their properties.

  2. Characterization of a major 31-kilodalton peptidoglycan-bound protein of Legionella pneumophila

    International Nuclear Information System (INIS)

    Butler, C.A.; Hoffman, P.S.


    A 31-kilodalton (kDa) protein was solubilized from the peptidoglycan (PG) fraction of Legionella pneumophila after treatment with either N-acetylmuramidase from the fungus Chalaropsis sp. or with mutanolysin from Streptomyces globisporus. The protein exhibited a ladderlike banding pattern by autoradiography when radiolabeled [(35S]cysteine or [35S]methionine) PG material was extensively treated with hen lysozyme. The banding patterns ranging between 31 and 45 kDa and between 55 and 60 kDa resolved as a single 31-kDa protein when the material was subsequently treated with N-acetylmuramidase. Analysis of the purified 31-kDa protein for diaminopimelic acid by gas chromatography revealed 1 mol of diaminopimelic acid per mol of protein. When outer membrane PG material containing the major outer membrane porin protein was treated with N-acetylmuramidase or mutanolysin, both the 28.5-kDa major outer membrane protein and the 31-kDa protein were solubilized from the PG material under reducing conditions. In the absence of 2-mercaptoethanol, a high-molecular-mass complex (100 kDa) was resolved. The results of this study indicate that a 31-kDa PG-bound protein is a major component of the cell wall of L. pneumophila whose function may be to anchor the major outer membrane protein to PG. Finally, a survey of other Legionella species and other serogroups of L. pneumophila suggested that PG-bound proteins may be a common feature of this genus

  3. Purification and partial characterization of Phaseolus vulgaris seed aminopeptidase

    Directory of Open Access Journals (Sweden)

    Abdala A.P.


    Full Text Available The aminopeptidase activity of Phaseolus vulgaris seeds was measured using L-Leu-p-nitroanilide and the L-aminoacyl-ß-naphthylamides of Leu, Ala, Arg and Met. A single peak of aminopeptidase activity on Leu-ß-naphthylamide was eluted at 750 µS after gradient elution chromatography on DEAE-cellulose of the supernatant of a crude seed extract. The effluent containing enzyme activity was applied to a Superdex 200 column and only one peak of aminopeptidase activity was obtained. SDS-polyacrylamide gel electrophoresis (10% presented only one protein band with molecular mass of 31 kDa under reducing and nonreducing conditions. The aminopeptidase has an optimum pH of 7.0 for activity on all substrates tested and the highest Vmax/KM ratio for L-Leu-ß-naphthylamide. The enzyme activity was increased 40% by 0.15 M NaCl, inhibited 94% by 2.0 mM Zn2+, inhibited 91% by sodium p-hydroxymercuribenzoate and inhibited 45% by 0.7 mM o-phenanthroline and 30 µM EDTA. Mercaptoethanol (3.3 mM, dithioerythritol (1.7 mM, Ala, Arg, Leu and Met (70 µM, p-nitroaniline (0.25 mM and ß-naphthylamine (0.53 mM had no effect on enzyme activity when assayed with 0.56 mM of substrate. Bestatin (20 µM inhibited 18% the enzyme activity. The aminopeptidase activity in the seeds decayed 50% after two months when stored at 4oC and room temperature. The enzyme is leucyl aminopeptidase metal- and thiol group-dependent.

  4. Production and Characterization of Organic Solvent-Tolerant Cellulase from Bacillus amyloliquefaciens AK9 Isolated from Hot Spring. (United States)

    Irfan, Muhammad; Tayyab, Ammara; Hasan, Fariha; Khan, Samiullah; Badshah, Malik; Shah, Aamer Ali


    A cellulase-producing bacterium, designated as strain AK9, was isolated from a hot spring of Tatta Pani, Azad Kashmir, Pakistan. The bacterium was identified as Bacillus amyloliquefaciens through 16S rRNA sequencing. Cellulase from strain AK9 was able to liberate glucose from soluble cellulose and carboxymethyl cellulose (CMC). Enzyme was purified through size exclusion chromatography and a single band of ∼47 kDa was observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was purified with recovery of 35.5%, 3.6-fold purity with specific activity of 31 U mg -1 . The purified cellulase retained its activity over a wide range of temperature (50-70 °C) and pH (3-7) with maximum stability at 60 °C and pH 5.0. The activity inhibited by ethylenediaminetetraacetic acid (EDTA), suggested that it was metalloenzyme. Diethyl pyrocarbonate (DEPC) and β-mercaptoethanol significantly inhibited cellulase activity that revealed the essentiality of histidine residues and disulfide bonds for its catalytic function. It was stable in non-ionic surfactants, in the presence of various metal ions, and in water-insoluble organic solvents. Approximately 9.1% of reducing sugar was released after enzymatic saccharification of DAP-pretreated agro-residue, compared to a very low percentage by autohydrolysis treatment. Hence, it is concluded that cellulase from B. amyloliquefaciens AK9 can potentially be used in bioconversion of lignocellulosic biomass to fermentable sugars.

  5. [Solid state isotope hydrogen exchange for deuterium and tritium in human gene-engineered insulin]. (United States)

    Zolotarev, Yu A; Dadayan, A K; Kozik, V S; Gasanov, E V; Nazimov, I V; Ziganshin, R Kh; Vaskovsky, B V; Murashov, A N; Ksenofontov, A L; Haribin, O N; Nikolaev, E N; Myasoedov, N F


    The reaction of high temperature solid state catalytic isotope exchange in peptides and proteins under the action of catalyst-activated spillover hydrogen was studied. The reaction of human gene-engineered insulin with deuterium and tritium was conducted at 120-140° C to produce insulin samples containing 2-6 hydrogen isotope atoms. To determine the distribution of the isotope label over tritium-labeled insulin's amino acid residues, oxidation of the S-S bonds of insulin by performic acid was performed and polypeptide chains isolated; then their acid hydrolysis, amino acid analysis and liquid scintillation counts of tritium in the amino acids were conducted. The isotope label was shown to be incorporated in all amino acids of the protein, with the peptide fragment FVNQHLCGSHLVE of the insulin β-chain showing the largest incorporation. About 45% of the total protein isotope label was incorporated in His5 and His10 of this fragment. For the analysis of isotope label distribution in labeled insulin's peptide fragments, the recovery of the S-S bonds by mercaptoethanol, the enzymatic hydrolysis by glutamyl endopeptidase from Bacillus intermedius and HPLC division of the resulting peptides were carried out. Attribution of the peptide fragments formed due to hydrolysis at the Glu-X bond in the β-chain was accomplished by mass spectrometry. Mass spectrometry analysis data of the deuterium-labeled insulin samples' isotopomeric composition showed that the studied solid state isotope exchange reaction equally involved all the protein molecules. Biological studying of tritium-labeled insulin showed its physiological activity to be completely retained.

  6. Radical-Scavenging Activity of Dietary Phytophenols in Combination with co-Antioxidants Using the Induction Period Method

    Directory of Open Access Journals (Sweden)

    Seiichiro Fujisawa


    Full Text Available The radical-scavenging activity of dietary phytophenols has been investigated by many researches due to their antioxidant, anti-inflammatory and anticancer property but the radical-scavenging effect of 2-phytophenol and the phytophenol:co-antioxidants, vitamin C and thiol combination under nearly anaerobic conditions still remains unknown. The radical-scavenging activity for seventeen phytophenols and for six synthetic phenols (positive controls was investigated using the induction period method in the polymerization of methyl methacrylates (MMA initiated by thermal decomposition of benzoyl peroxide (BPO by monitoring differential scanning calorimetery (DSC. The kinh for the phytophenols was likely with the range 0.5 × 103 M−1s−1−2.2 × 103 M−1s−1, whereas that for synthetic phenols, hydroquinone and galvinoxyl, was with the range 7 × 103 M−1s−1−8 × 103 M−1s−1. Also, the additive scavenging effect of the (−-epigallocatechin (EGC:(−-epicatechin (EC and the (+-catechin:epicatechin (EC combination was observed at 1:1 molar ratio, whereas that of the EC:quercetin combination showed the cancel (prooxidative effect. Furthermore, the EGC:ASDB (L-ascorbyl 2,6-dibutylate or 2-ME (2-mercaptoethanol combination showed the prooxidative effect. Such enhancement of prooxidation in the combination may increase their toxic effects due to their cooxidation. Also, the synergic, additive or cancel effects of the flavonoid:vitamins E combination on the induction period in the BPO (a PhCOO* radical and 2,2′-azobisisobutyronitrile (AIBN, an R* radical systems are discussed.

  7. A simple and efficient protocol for isolation of high quality functional RNA from different tissues of turmeric (Curcuma longa L.). (United States)

    Deepa, K; Sheeja, T E; Santhi, R; Sasikumar, B; Cyriac, Anu; Deepesh, P V; Prasath, D


    Many experiments in plant molecular biology require processing of a large number of RNA samples and in some cases large quantities are required for a single application. In turmeric, a major spice and medicinal plant, a protocol for RNA isolation is not available. The major difficulty encountered while using other popular protocols is the low yield and quality of RNA which hampers the downstream applications like qRT-PCR, cDNA synthesis and micro RNA isolation. Commercial kits though available are costly and were found to be unsuccessful in case of rhizomes and root tissues that are rich in polyphenols, polysaccharides and alkaloids. It was thus felt that a quick, handy and cheap protocol of total RNA isolation from different tissues of turmeric was required for day to day working in our lab. The new protocol utilizes SDS based extraction buffer including β-mercaptoethanol and PVP with sequential acid phenol:chloroform extraction to remove polyphenols and proteins, followed by the purification with sodium acetate to eliminate polysaccharides. The protocol is simple and can be completed in less than 3 h. The RNA yield from rhizome was higher by more than fivefold with both A260/280 and A260/230 ratio in the range of 1.8-2.0. The protocol worked well with leaf, rhizome, pseudostem and root tissues with RIN >7.0 and the isolated RNA could be successfully used for cDNA synthesis, RT-PCR, qRT-PCR and small RNA isolation including microRNA.

  8. Distribution of small proteoglycans and glycosaminoglycans in humerus-related articular cartilage of chickens

    Directory of Open Access Journals (Sweden)

    E.D. Rodrigues


    Full Text Available The expression of components present in the cartilaginous extracellular matrix is related to development, gender, and genotype, as well as to the biomechanical properties of each type of cartilage. In the present study, we analyzed small proteoglycans and glycosaminoglycans present in different cartilages of the chicken wing after extraction with guanidine hydrochloride or papain. Quantitative analysis of glycosaminoglycans showed a larger amount in humeral cartilage (around 200 mg/g tissue than in articular cartilage of the radius and ulna, with 138 and 80 mg/g tissue, respectively. Non-collagenous proteins isolated were predominantly from cartilage in the proximal regions of the humerus and radius. D4 fractions obtained by ultracentrifugation were separated by DEAE-Sephacel and Octyl-Sepharose chromatography and analyzed by SDS-PAGE. Two bands of 57 and 70-90 kDa were observed for all samples treated with ß-mercaptoethanol. Immunoblotting of these proteins was positive for the small proteoglycans fibromodulin and decorin, respectively. Apparently, the 57-kDa protein is present in macromolecular complexes of 160 and 200 kDa. Chondroitin sulfate was detected in all regions. HPLC analysis of the products formed by chondroitinase AC and ABC digestion mainly revealed ß-D-glucuronic acid and N-acetyl ß-D-galactosamine residues. The 4-sulfation/6-sulfation ratio was close to 3, except for the proximal cartilage of the radius (2.5. These results suggest functional differences between the scapula-humerus, humerus-ulna, and humerus-radius joints of the chicken wing. This study contributes to the understanding of the physiology of cartilage and joints of birds under different types of mechanical stress.

  9. Amino Acid Transport Associated to Cluster of Differentiation 98 Heavy Chain (CD98hc) Is at the Cross-road of Oxidative Stress and Amino Acid Availability. (United States)

    de la Ballina, Laura R; Cano-Crespo, Sara; González-Muñoz, Elena; Bial, Susanna; Estrach, Soline; Cailleteau, Laurence; Tissot, Floriane; Daniel, Hannelore; Zorzano, Antonio; Ginsberg, Mark H; Palacín, Manuel; Féral, Chloé C


    CD98hc functions as an amino acid (AA) transporter (together with another subunit) and integrin signaling enhancer. It is overexpressed in highly proliferative cells in both physiological and pathological conditions. CD98hc deletion induces strong impairment of cell proliferation in vivo and in vitro Here, we investigate CD98hc-associated AA transport in cell survival and proliferation. By using chimeric versions of CD98hc, the two functions of the protein can be uncoupled. Although recovering the CD98hc AA transport capacity restores the in vivo and in vitro proliferation of CD98hc-null cells, reconstitution of the integrin signaling function of CD98hc is unable to restore in vitro proliferation of those cells. CD98hc-associated transporters (i.e. xCT, LAT1, and y(+)LAT2 in wild-type cells) are crucial to control reactive oxygen species and intracellular AA levels, thus sustaining cell survival and proliferation. Moreover, in CD98hc-null cells the deficiency of CD98hc/xCT cannot be compensated, leading to cell death by ferroptosis. Supplementation of culture media with β-mercaptoethanol rescues CD98hc-deficient cell survival. Under such conditions null cells show oxidative stress and intracellular AA imbalance and, consequently, limited proliferation. CD98hc-null cells also present reduced intracellular levels of branched-chain and aromatic amino acids (BCAAs and ARO AAs, respectively) and induced expression of peptide transporter 1 (PEPT1). Interestingly, external supply of dipeptides containing BCAAs and ARO AAs rescues cell proliferation and compensates for impaired uptake of CD98hc/LAT1 and CD98hc/y(+)LAT2. Our data establish CD98hc as a master protective gene at the cross-road of redox control and AA availability, making it a relevant therapeutic target in cancer. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Characterization of Salt-Regulated Mannitol-1-Phosphate Dehydrogenase in the Red Alga Caloglossa continua1 (United States)

    Iwamoto, Koji; Kawanobe, Hideaki; Ikawa, Tomoyoshi; Shiraiwa, Yoshihiro


    Mannitol-1-phosphate (M1P) dehydrogenase (M1PDH; EC, an enzyme catalyzing the reduction of Fru-6-phosphate (F6P) to M1P in algal mannitol biosynthesis, was purified to homogeneity from a cell homogenate of the eulittoral red alga Caloglossa continua (Okamura) King et Puttock. The enzyme was a monomer with an apparent molecular mass of 53 kD, as determined by gel filtration and SDS-PAGE, and exhibited an pI of approximately 5.5. The substrate specificity was very high toward F6P and M1P for respective reductive and oxidative reactions. The enzyme was found to be a sulfhydryl-type, because its activity was inhibited by N-ethylmaleimide and p-hydroxymercuribenzoate, and the inhibition by p-hydroxymercuribenzoate was rescued by 2-mercaptoethanol. Some unknown factors in the extract may also have inhibited the activity, because the total activity was greatly increased through the purification procedure. The optimum pH for F6P reduction was changed from 6.0 or lower to 7.2 by the addition of 200 mm NaCl. The reduction of F6P showed strong substrate inhibition above 0.5 mm. However, Km(F6P) of M1PDH was increased eight times by the addition of 200 mm NaCl, whereas Vmax was in a similar range with the avoidance of substrate inhibition by F6P. These results indicate that the enzyme was finely and directly regulated by the salt concentration without the requirement for gene expression. M1PDH can therefore be a key enzyme for regulating mannitol biosynthesis when the alga is stressed by a salinity change. PMID:12972650

  11. Metabolic behavior of cell surface biotinylated proteins

    International Nuclear Information System (INIS)

    Hare, J.F.; Lee, E.


    The turnover of proteins on the surface of cultured mammalian cells was measured by a new approach. Reactive free amino or sulfhydryl groups on surface-accessible proteins were derivatized with biotinyl reagents and the proteins solubilized from culture dishes with detergent. Solubilized, biotinylated proteins were then adsorbed onto streptavidin-agarose, released with sodium dodecyl sulfate and mercaptoethanol, and separated on polyacrylamide gels. Biotin-epsilon-aminocaproic acid N-hydroxysuccinimide ester (BNHS) or N-biotinoyl-N'-(maleimidohexanoyl)hydrazine (BM) were the derivatizing agents. Only 10-12 bands were adsorbed onto streptavidin-agarose from undervatized cells or from derivatized cells treated with free avidin at 4 degrees C. Two-dimensional isoelectric focusing-sodium dodecyl sulfate gel electrophoresis resolved greater than 100 BNHS-derivatized proteins and greater than 40 BM-derivatized proteins. There appeared to be little overlap between the two groups of derivatized proteins. Short-term pulse-chase studies showed an accumulation of label into both groups of biotinylated proteins up until 1-2 h of chase and a rapid decrease over the next 1-5 h. Delayed appearance of labeled protein at the cell surface was attributed to transit time from site of synthesis. The unexpected and unexplained rapid disappearance of pulse-labeled proteins from the cell surface was invariant for all two-dimensionally resolved proteins and was sensitive to temperature reduction to 18 degrees C. Long-term pulse-chase experiments beginning 4-8 h after the initiation of chase showed the disappearance of derivatized proteins to be a simple first-order process having a half-life of 115 h in the case of BNHS-derivatized proteins and 30 h in the case of BM-derivatized proteins

  12. Identification and further characterization of the specific cell binding fragment from sponge aggregation factor. (United States)

    Gramzow, M; Bachmann, M; Uhlenbruck, G; Dorn, A; Müller, W E


    Monoclonal antibodies (McAbs) were raised against the aggregation factor (AF) from the marine sponge Geodia cydonium. Two clones were identified that secrete McAbs against the cell binding protein of the AF complex. Fab fragments of McAbs: 5D2-D11 completely abolished the activity of the AF to form secondary aggregates from single cells. The McAbs were determined to react with the AF in vitro; this interaction was prevented by addition of the aggregation receptor, isolated and purified from the same species. After dissociation of the AF by sodium dodecyl sulfate and 2-mercaptoethanol, followed by electrophoretical fractionation, a 47-kD protein was identified by immunoblotting which interacted with the McAbs: 5D2-D11. During this dissociation procedure, the sunburst structure of the AF was destroyed. In a second approach, the 47-kD protein was isolated by immunoprecipitation; 12 molecules of this protein species were calculated to be associated with the intact AF particle. The 47-kD AF fragment bound to dissociated Geodia cells with a high affinity (Ka of 7 X 10(8) M-1) even in the absence of Ca++ ions; the number of binding sites was approximately 4 X 10(6)/cell. This interaction was prevented by addition of the aggregation receptor to the 47-kD protein in the homologous cell system. Moreover, it was established that this binding occurs species-specifically. The 47-kD fragment of the AF was localized only extracellularly by indirect immunofluorescence staining in cryostat slices. These data suggest that the 47-kD protein is the cell binding molecule of the AF from Geodia.

  13. A study of the cytoplasmic expression of a form of human prolactin and of its solubilization and renaturation from bacterial inclusion bodies

    International Nuclear Information System (INIS)

    Affonso, Regina


    Different vector elements, that can determine a high expression level of a form of human prolactin (taghPrl) in bacterial cytoplasm, were studied. Expression conditions were first optimized for a reference vector, which was used to transform different strains of E. coli: HB2151, RRI and RB791. The highest expression level (113 ±16 μg/mL.A 600 ) was obtained in HB2151, after activation with only 0.1 mM IPTG. At this point the influence of the transcription terminator (g32 from bacteriophage T4), of the translation enhancer (g10 from bacteriophage T7), of the promoter (λP L or tac) and of the antibiotic resistance gene (amp r or kan r ) were studied. The first three elements did not show any significant influence, at least in our systems. On the contrary, the analysis of the influence of amp r and kan r genes showed, unexpectedly, that the presence of the last one provides an approximately 5-fold higher expression for taghPrl in E. coli cytoplasm. Finally, an appropriate extraction, solubilization, renaturation and purification process, able to provide a monomeric form of taghPrl, was studied. A method utilizing urea and mercaptoethanol as solubilizing agents and a dialysis as a renaturation procedure, provided with some modifications, one of the highest yields ever reported in the literature: 35.4 ± 4.5% of total recovery. Moreover, the biological activity of the taghPrl obtained, when tested in the Nb2 cell proliferation assay, was of the same order of that shown by the International Standard of human prolactin of pituitary origin. These data show that the cytoplasmic expression system here described, which can provide an expression efficiency 50-100 - fold higher than the periplasmic expression, can represent a valid alternative for the production of this and of other hormones of pharmaceutical interest and grade. (author)


    Pickering, Andrew. M.; Davies, Kelvin. J. A.


    Proteins are sensitive to oxidation, and oxidized proteins are excellent substrates for degradation by proteolytic enzymes such as the Proteasome and the mitochondrial Lon protease. Protein labeling is required for studies of protein turnover. Unfortunately, most labeling techniques involve 3H or 14C methylation which is expensive, exposes researchers to radioactivity, generates large amounts of radioactive waste, and allows only single-point assays because samples require acid-precipitation. Alternative labeling methods, have largely proven unsuitable, either because the probe itself is modified by the oxidant(s) being studied, or because the alternative labeling techniques are too complex or too costly for routine use. What is needed is a simple, quick, and cheap labeling technique that uses a non-radioactive marker, that binds strongly to proteins, is resistant to oxidative modification, and emits a strong signal. We have devised a new reductive method for labeling free carboxyl groups of proteins with the small fluorophore 7-amino-4-methycoumarin (AMC). When bound to target proteins, AMC fluoresces very weakly but when AMC is released by proteinases, proteases, or peptidases, it fluoresces strongly. Thus, without acid-precipitation, the proteolysis of any target protein can be studied continuously, in multiwell plates. In direct comparisons, 3H-labeled proteins and AMC-labeled proteins exhibited essentially identical degradation patterns during incubation with trypsin, cell extracts, and purified proteasome. AMC-labeled proteins are well-suited to study increased proteolytic susceptibility following protein modification, since the AMC-protein bond is resistant to oxidizing agents such as hydrogen peroxide and peroxynitrite, and is stable over time and to extremes of pH, temperature (even boiling), freeze-thawing, mercaptoethanol, and methanol. PMID:21988844

  15. OFFGEL isoelectric focusing and polyacrylamide gel electrophoresis separation of platinum-binding proteins. (United States)

    Mena, Ma Luz; Moreno-Gordaliza, Estefanía; Moraleja, Irene; Cañas, Benito; Gómez-Gómez, Ma Milagros


    In this work a 2D electrophoretic separation procedure able to maintain the integrity of platinum-protein bonds has been developed. The method is based on the use of sequential OFFGEL isoelectric focussing (IEF) and PAGE. A systematic study of the reagents used for PAGE, for OFFGEL-IEF separation, and post-separation treatment of gels (such as enzymatic digestion and sample preparation for MS analysis) was tackled regarding their suitability for the identification of platinum binding proteins using standard proteins incubated with cisplatin. The distribution of platinum in high and low molecular weight fractions (separated by cut-off filters) was determined by ICP-MS, which allows evaluating platinum-protein bond stability under the conditions studied. SDS-PAGE in the absence of β-mercaptoethanol or dithiotreitol preserved the platinum-protein bonds. In addition, neither the influence of the electric field during the electrophoretic separation, nor the processes of fixing, staining and destaining of proteins in the gel did result in the loss of platinum from platinum binding proteins. SDS-PAGE under non-reducing conditions provides separation of platinum-binding proteins in very narrow bands with quantitative recoveries. Different amounts of platinum-bound proteins covering the range 0.3-2.0 μg were separated and mineralised for platinum determination, showing good platinum linearity. Limits of detection for a mixture of five standard proteins incubated with cisplatin were between the range of 2.4 and 13.9 pg of platinum, which were satisfactory for their application to biological samples. Regarding OFFGEL-IEF, a denaturing solution without thiourea and without dithiotreitol is recommended. The suitability of the OFFGEL-IEF for the separation of platinum binding proteins of a kidney cytosol was demonstrated. Copyright © 2011 Elsevier B.V. All rights reserved.

  16. ELISA Cut-off Point for the Diagnosis of Human Brucellosis; a Comparison with Serum Agglutination Test

    Directory of Open Access Journals (Sweden)

    Anahita Sanaei Dashti


    Full Text Available Background: Brucellosis is a world-wide disease, which has a diverse clinical manifestation, and its diagnosis has to be proven by laboratory data. Serum agglutination test (SAT is the most-widely used test for diagnosing brucellosis. The enzyme linked immunosorbent assay (ELISA can also determine specific antibody classes against brucella. It is a sensitive, simple and rapid test, which could be an acceptable alternative to SAT with fewer limitations, however, like any other new test it should be further evaluated and standardized for various populations. This study was planned to determine an optimal cut-off point, for ELISA which would offer maximum sensitivity and specificity for the test when compared to SAT.Methods: Four hundred and seven patients with fever and other compatible symptoms of brucellosis were enrolled in the study. Serum agglutination test, 2-Mercaptoethanol test, and ELISA were performed on their sera. Results: The cut-off point of 53 IU/ml of ELISA-IgG yielded the maximal sensitivity and specificity comparing to the other levels of ELISA-IgG, and was considered the best cut off-point of ELISA-IgG to diagnose acute brucellosis. At this cut-off, the sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, and negative likelihood ratio were 84.09%, 85.38%, 62.20, 94.90, 5.75, 0.18, respectively.Conclusion: The best cut-off point of ELISA-IgG is 53 IU/ml, which yields the maximal sensitivity and specificity to diagnose acute brucellosis.

  17. Evaluation of CD4+ CD25+ FoxP3+ regulatory T cells during treatment of patients with brucellosis. (United States)

    Hasanjani Roushan, M R; Bayani, M; Soleimani Amiri, S; Mohammadnia-Afrouzi, M; Nouri, H R; Ebrahimpour, S


    Cell-mediated immunity (CMI) plays a critical role in the control of brucellosis. Regulatory T cells (Tregs) have a functional character in modulating the balance between host immune response and tolerance, which can eventually lead to chronic infection or relapse. The aim of this study was to assess the alteration of Tregs in cases of brucellosis before and after treatment. Thirty cases of acute brucellosis with the mean age of 41.03±15.15 years (case group) and 30 healthy persons with the mean age of 40.63±13.95 years (control group) were selected and assessed. Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of all individuals. We analyzed the alteration of Treg cell count using flow cytometry for CD4, CD25, and FoxP3 markers. The level of CD4+ CD25+ FoxP3+ Treg cells was increased in active patients compared with controls (2.5±0.99% vs 1.6±0.84%, p= 0.0004), but it had declined in the treated cases (1.83±0.73%, p=0.02). The level of Tregs was elevated in three relapsed cases. The frequency of Tregs and Treg/Teff (effector T cell) ratio was correlated with inverse serum agglutination test (SAT) and, 2-mercaptoethanol (2-ME) titers as markers of treatment in brucellosis. Based on our findings, we suggest that regulatory cells, such as CD4+ CD25+ FoxP3+ Treg cells, may contribute to the development of infection processes involving immune responses in brucellosis, and evaluation of regulatory T-cell levels may be a potential diagnostic strategy for the treatment outcome in chronic and relapsed cases of brucellosis.

  18. Study of the viability of technetium-99m labeling of whole antimyosin antibody and its fragment: development of radiopharmaceutical for cardiac survey

    International Nuclear Information System (INIS)

    Carvalho, Guilherme Luiz de Castro


    In the acute myocardium infarction, the myocytes cell membrane loses its integrity, allowing the influx of extracellular macromolecules such as circulating antibody into the damaged cell. The use of the specific antibodies against cardiac myosin labeled with 99m Tc allows to determine the localization and extension of myocardial infarction. The purpose of this work was to study the viability of labeling of the antimyosin monoclonal antibody and its fragment F(ab')2 with 99m Tc. Because of the high cost of antimyosin antibody, others antibodies were used to optimize the methodology and the best condition was used for antimyosin antibody. The intact antibody was cleaved by pepsin to produce F(ab') 2 fragment. The F(ab') 2 and the intact antibody were reduced by treatment with Dithiothreitol (DTT) and 2-Mercaptoethanol (2-ME) and labeled with 99m Tc by direct method. Different concentrations of reductant, mixing conditions and incubation times were studied. In the standard condition, incubation at molar ratio 1:1000 (antibody:reducing agent) at room temperature for 30 minutes with continuous rotation (850 rpm), 13.28 - SH groups were formed per molecule. It was studied the influence of p H, of the concentration of stannous chloride (Sn 2+ ) and incubation time in the labeling condition. The better radiochemical yield (90.06 +- 1.53%) was obtained using 2.5 μg of Sn 2+ in p H 4.5 for 60 minutes. The labeling of the fragment F(ab') 2 did not present satisfactory results because of the low yield of the digestion. After purification by PD-10, the biodistribution study was performed and showed that the intact antimyosin antibody labeled with 99m Tc presented fast kinetic compatible with the biodistribution of an intact antibody labeled with 99m Tc. Scintigraphy image of the animal with myocardial infarction was obtained and compared with the image of a normal animal. The studies allow to conclude that the use of fragment F(ab') 2 are not viable, but the use of the labeled

  19. Evaluation of ovostatin and ovostatin assay (United States)

    Moriarity, Debra M.


    Ovostatin is a 780,000 MW protein, originally isolated from chicken egg white, which is active as a protease inhibitor. Structural studies indicate that the protein is a tetramer of identical subunits of 165,000 MW which can be separated upon reduction with beta- mercaptoethanol. Chicken ovostatin is an inhibitor of metalloproteases such as collagenase and thermolysin, and of acid proteases such as pepsin and rennin. Ovostatin isolated from duck eggs and from crocodile eggs appears to be similar to chicken egg ovostatin, but with significant differences in structure and function. Duck ovostatin contains a reactive thiol ester which is not found in the chicken protein, and duck and crocodile ovostatin inhibit serine protease such as trypsin and chymotrypsin, while chicken ovostatin does not. Electron microscopy of ovostatin indicates that two subunits associated near the middle of each polypeptide to form a dimer with four arms. Two of these dimers then associate to produce a tetramer with eight arms, with the protease binding site near the center of the molecule. Upon binding of the protease, a conformational change causes all eight arms to curl toward the center of the molecule, effectively trapping the protease and sterically hindering access of the substrates to its active site. The structural organization and mechanism of action proposed for ovostatin are nearly identical to that proposed for alpha(sub 2)- macroglobulin, a serum protease inhibitor which may play an important role in regulation of proteases in animal tissues. Although the general arrangement of subunits appears to be the same for all ovostatins studied, some differences have been observed, with chicken ovostatin more closely resembling reptilian ovostatin than the duck protein. This is a surprising result, given the evolutionary relatedness of chickens and ducks. It is possible that the differences in structures may be due to deformed subunit arrangements which occur during the processing and

  20. Molecular characterization of tlyA gene product, Rv1694 of Mycobacterium tuberculosis: A non-conventional hemolysin and a ribosomal RNA methyl transferase

    Directory of Open Access Journals (Sweden)

    Ahmed Neesar


    Full Text Available Abstract Background Mycobacterium tuberculosis is a virulent bacillus causing tuberculosis, a disease responsible for million deaths each year worldwide. In order to understand its mechanism of pathogenesis in humans and to help control tuberculosis, functions of numerous Mycobacterium tuberculosis genes are being characterized. In this study we report the dual functionality of tlyA gene product of Mycobacterium tuberculosis annotated as Rv1694, a 268 amino acid long basic protein. Results The recombinant purified Rv1694 protein was found to exhibit hemolytic activity in vitro. It showed concentration and time-dependent hemolysis of rabbit and human erythrocytes. Multiple oligomeric forms (dimers to heptamers of this protein were seen on the membranes of the lysed erythrocytes. Like the oligomers of conventional, well-known, pore-forming toxins, the oligomers of Rv1694 were found to be resistant to heat and SDS, but were susceptible to reducing agents like β-mercaptoethanol as it had abolished the hemolytic activity of Rv1694 indicating the role of disulfide bond(s. The Rv1694 generated de novo by in vitro transcription and translation also exhibited unambiguous hemolysis confirming the self assembly and oligomerization properties of this protein. Limited proteolytic digestion of this protein has revealed that the amino terminus is susceptible while in solution but is protected in presence of membrane. Striking feature of Rv1694 is its presence on the cell wall of E. coli as visualized by confocal microscopy. The surface expression is consistent with the contact dependent haemolytic ability of E. coli expressing this protein. Also, immune serum specific to this protein inhibits the contact dependent hemolysis. Moreover, Rv1694 protein binds to and forms stable oligomers on the macrophage phagosomal membranes. In addition to all these properties, E. coli expressing Rv1694 was found to be susceptible to the antibiotic capreomycin as its growth

  1. Occurrence of oxidative stress in dairy cows seropositives for Brucella abortus. (United States)

    Perin, Géssica; Fávero, Juscivete F; Severo, Diego R T; Silva, Anielen D; Machado, Gustavo; Araújo, Hugo L; Lilenbaum, Walter; Morsch, Vera M; Schetinger, Maria Rosa C; Jordão, Ricardo S; Stefani, Lenita M; Bottari, Nathieli B; Da Silva, Aleksandro S


    Bovine brucellosis is an important zoonotic disease caused by the bacterium Brucella abortus that leads to economic losses due to animal discard and commercial restrictions. Since positive animals for brucellosis are culled, little is known about the pathogenesis of this disease. Therefore, the aims of this study were to evaluate possible changes in the activity of deaminase adenosine (ADA) and the oxidative stress in cows seropositives for brucellosis (Experiment I), and to evaluate the seroprevalence of B. abortus in dairy cows from the Western state of Santa Catarina, Southern Brazil (Experiment II). The Experiment I evaluated 20 pregnant cows: ten seropositives for B. abortus and ten seronegatives that were used as controls. The ADA activity and markers of oxidative stress (TBARS, catalase (CAT) and superoxide dismutase (SOD)) were evaluated in these animals. A reduction in the activity of ADA and catalase enzymes in seropositive animals was observed (p < 0.001). Conversely, there was an increase in TBARS levels and superoxide dismutase activity in cows infected by B. abortus (p < 0.001). The presence of oxidative stress and a reduction of ADA might be related to the modulation of the inflammatory response. The experiment II was performed due to a high number of herds with restrictions imposed by cases of brucellosis in the state of Santa Catarina in the last two years, and thus, the seroprevalence for B. abortus was evaluated in 1242 serum samples of cows of 69 herds. The serodiagnosis was performed using two tests: buffered acidified antigen and 2-mercaptoethanol. However, none of the serum samples were positive for B. abortus. Although we did not find seropositive animals for brucellosis in our study, the disease still requires continued surveillance, due to its economic impact, and to the oxidative stress caused by it, which may have contributed to cases of abortion in three seropositive cows (Experiment I) in the final third of the gestation

  2. Insights into the biological features of the antigenic determinants recognized by four monoclonal antibodies in redia and adult stages of the liver fluke Fasciola hepatica. (United States)

    Alba, Annia; Sánchez, Jorge; Hernández, Hilda; Mosqueda, Maryani; Rodríguez, Suanel Y; Capó, Virginia; Otero, Oscar; Alfonso, Carlos; Marcet, Ricardo; Sarracent, Jorge


    Fasciola hepatica is a digenean trematode which infects a wide variety of domestic animals and also humans. Previous studies have demonstrated that four monoclonal antibodies (Mabs) against the total extract of F. hepatica redia (named as 1E4, 6G11, 4E5 and 4G11) also recognized the excretion - secretion antigens (ES Ag) of adult parasites, which is a biologically-relevant mixture of molecules with functional roles during infection and immune evasion on definitive hosts. In the present report we describe the partial characterization of the epitopes recognized by these Mabs by heat treatment, mercaptoethanol reduction, pronase proteolysis and sodium peryodate oxidation, which suggested their predominant protein and conformational nature. Also, a comparative study using immunodetection assays on crude extracts and on histological sections of both rediae and adults of F. hepatica were performed to explore the expression pattern of the antigenic determinants in these developmental stages. From these experiments it was found that the Mabs reacted most likely with the same proteins of approximately 64 and 105 kDa present on both rediae and adult's extracts. However, the 1E4, 6G11 and 4E5 Mabs also recognized other molecules of the total extract of F. hepatica adults, a fact that constitutes an evidence of the antigenic variation between both stages and points at a certain biological relevance of the recognized antigenic determinants. Immunolocalization studies on histological sections revealed that all Mabs reacted with the tegument of F. hepatica in both rediae and adults stages, while the epitopes recognized by 1E4, 6G11 and 4E5 antibodies were also preferentially localized in the intestinal caeca and in different organs of the reproductive system of adult specimens. The immunogenicity of these antigenic determinants, their conserved status among different stages of the life cycle of F. hepatica and their presence in both tegument and ES Ag of adult parasites

  3. Cloning, Characterization and Expression Pattern Analysis of a Cytosolic Copper/Zinc Superoxide Dismutase (SaCSD1 in a Highly Salt Tolerant Mangrove (Sonneratia alba

    Directory of Open Access Journals (Sweden)

    Enze Yang


    Full Text Available Mangroves are critical marine resources for their remarkable ability to tolerate seawater. Antioxidant enzymes play an especially significant role in eliminating reactive oxygen species and conferring abiotic stress tolerance. In this study, a cytosolic copper/zinc superoxide dismutase (SaCSD1 cDNA of Sonneratia alba, a mangrove species with high salt tolerance, was successfully cloned and then expressed in Escherichia coli Rosetta-gami (designated as SaCSD1. SaCSD1 comprised a complete open reading frame (ORF of 459 bp which encoded a protein of 152 amino acids. Its mature protein is predicted to be 15.32 kDa and the deduced isoelectric point is 5.78. SaCSD1 has high sequence similarity (85%–90% with the superoxide dismutase (CSD of some other plant species. SaCSD1 was expressed with 30.6% yield regarding total protein content after being introduced into the pET-15b (Sma I vector for expression in Rosetta-gami and being induced with IPTG. After affinity chromatography on Ni-NTA, recombinant SaCSD1 was obtained with 3.2-fold purification and a specific activity of 2200 U/mg. SaCSD1 showed good activity as well as stability in the ranges of pH between 3 and 7 and temperature between 25 and 55 °C. The activity of recombinant SaCSD1 was stable in 0.25 M NaCl, Dimethyl Sulphoxide (DMSO, glycerol, and chloroform, and was reduced to a great extent in β-mercaptoethanol, sodium dodecyl sulfate (SDS, H2O2, and phenol. Moreover, the SaCSD1 protein was very susceptive to pepsin digestion. Real-time Quantitative Polymerase Chain Reaction (PCR assay demonstrated that SaCSD1 was expressed in leaf, stem, flower, and fruit organs, with the highest expression in fruits. Under 0.25 M and 0.5 M salt stress, the expression of SaCSD1 was down-regulated in roots, but up-regulated in leaves.

  4. Bioanalytical Applications of Real-Time ATP Imaging Via Bioluminescence

    Energy Technology Data Exchange (ETDEWEB)

    Gruenhagen, Jason Alan [Iowa State Univ., Ames, IA (United States)


    The research discussed within involves the development of novel applications of real-time imaging of adenosine 5'-triphosphate (ATP). ATP was detected via bioluminescence and the firefly luciferase-catalyzed reaction of ATP and luciferin. The use of a microscope and an imaging detector allowed for spatially resolved quantitation of ATP release. Employing this method, applications in both biological and chemical systems were developed. First, the mechanism by which the compound 48/80 induces release of ATP from human umbilical vein endothelial cells (HUVECs) was investigated. Numerous enzyme activators and inhibitors were utilized to probe the second messenger systems involved in release. Compound 48/80 activated a G{sub q}-type protein to initiate ATP release from HUVECs. Ca2+ imaging along with ATP imaging revealed that activation of phospholipase C and induction of intracellular Ca2+ signaling were necessary for release of ATP. Furthermore, activation of protein kinase C inhibited the activity of phospholipase C and thus decreased the magnitude of ATP release. This novel release mechanism was compared to the existing theories of extracellular release of ATP. Bioluminescence imaging was also employed to examine the role of ATP in the field of neuroscience. The central nervous system (CNS) was dissected from the freshwater snail Lymnaea stagnalis. Electrophysiological experiments demonstrated that the neurons of the Lymnaea were not damaged by any of the components of the imaging solution. ATP was continuously released by the ganglia of the CNS for over eight hours and varied from ganglion to ganglion and within individual ganglia. Addition of the neurotransmitters K+ and serotonin increased release of ATP in certain regions of the Lymnaea CNS. Finally, the ATP imaging technique was investigated for the study of drug release systems. MCM-41-type mesoporous nanospheres were loaded with ATP and end-capped with mercaptoethanol

  5. The use of comet assay to assess DNA integrity of boar spermatozoa following liquid preservation at 5 degrees C and 16 degrees C.

    Directory of Open Access Journals (Sweden)

    J Strzezek


    Full Text Available The comet assay, under neutral conditions, allows the assessment of DNA integrity influenced by sperm ageing, which is manifested in DNA double-strand breaks. Here, we attempted to use a modified neutral comet assay test (single-cell gel electrophoresis, to our knowledge for the first time, to assess DNA integrity of boar spermatozoa during liquid storage for 96 h at 5 degrees C and 16 degrees C. In this comet assay protocol we used 2% beta-mercaptoethanol prior to the lysis procedure, to aid in removing nuclear proteins. Ejaculates from 3 boars (designated A, C and G were diluted with a standard semen extender, Kortowo-3 (K-3, which was supplemented with lipoprotein fractions extracted from hen egg yolk (LPFh or ostrich egg yolk (LPFo. Irrespective of the extender type, the percentage of comet-detected spermatozoa with damaged DNA increased gradually during prolonged storage at 5 degrees C and 16 degrees C. Spermatozoa stored in K-3 extender exhibited elevated levels of DNA damage at both storage temperatures. Significant differences in DNA damage among the boars were more pronounced during storage in LPF-based extenders at 5 degrees C: spermatozoa of boars A and G were less susceptible to DNA damage. The percent of tail DNA in comets was lower in LPF-based extenders, and there were individual variations among the boars. We observed that changes in DNA integrity were dependent on the extender type and storage temperature. A higher level of DNA instability was observed in K-3 extended semen compared with K-3/LPFh or K-3/LPFo extended semen during storage at 5 degrees C. No significant difference in the level of DNA damage between K-3/LPFh and K-3/LPFo was observed. It seems that a long-term storage can affect genomic integrity of boar spermatozoa. The modified neutral comet assay can be used to detect low levels of DNA damage in boar spermatozoa during liquid preservation. Therefore, screening for sperm DNA damage may be used as an additional

  6. Aerobiological and immunochemical studies on Carica papaya L. pollen: an aeroallergen from India. (United States)

    Chakraborty, P; Ghosh, D; Chowdhury, I; Roy, I; Chatterjee, S; Chanda, S; Gupta-Bhattacharya, S


    Carica papaya L. is a fruit yielding tree, wildly grown or cultivated in the tropics and subtropics. Its pollen grain has been reported to be airborne and cause immunoglobulin E (IgE)-mediated hypersensitivity. To conduct long-term aerobiological study on Carica pollen, along with aeroallergenic particles originating from it and to identify vis-a-vis characterize an important IgE-reactive component present in this pollen. The seasonal and diurnal periodicities of airborne C. papaya pollen were recorded in a 5-year survey using a Burkard volumetric sampler. The allergenic potential was studied by skin prick tests, IgE-enzyme-linked immunosorbent assay (ELISA) and also by aeroallergen immunoblotting. The total pollen extract was fractionated by Sephacryl S-200 column, and out of the eluted five fractions, the maximum IgE-reactive fraction (as found in ELISA inhibition) was resolved into five major subfractions in reverse-phase high-performance liquid chromatography (RP-HPLC). The subfraction with optimum IgE reactivity was studied by activity gel, native and nonreducing sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The homogeneity of the isolated protein fraction was checked by crossed immunoelectrophoresis with rabbit antisera and IgE reactivity was confirmed by ELISA inhibition and immunoblotting using individual patient sera. The Carica pollen occurred in the air round the year with peaks during January and September-October. Among a patient population of 1000, skin-test results showed 27.8% +1 level and 5.6% +2/+3 level reactions. In aeroallergen immunoblotting of exposed Burkard tape segments, the detected allergen spots showed a significant correlation with airborne pollen count recorded. The pollen extract elicited loss of IgE reactivity when treated with reducing agent-like beta-mercaptoethanol and heat, but showed six IgE-reactive components in nonreducing IgE-immunoblot. The fraction 1 eluted from Sephacryl S-200 column showed

  7. Determination of methylmercury in marine sediment samples: Method validation and occurrence data

    International Nuclear Information System (INIS)

    Carrasco, Luis; Vassileva, Emilia


    Highlights: • A method for MeHg determination at trace level in marine sediments is completely validated. • Validation is performed according to ISO-17025 and Eurachem guidelines. • The extraction efficiency of four sample preparation procedures is evaluated. • The uncertainty budget is used as a tool for evaluation of main uncertainty contributors. • Comparison with independent methods yields good agreement within stated uncertainty. - Abstract: The determination of methylmercury (MeHg) in sediment samples is a difficult task due to the extremely low MeHg/THg (total mercury) ratio and species interconversion. Here, we present the method validation of a cost-effective fit-for-purpose analytical procedure for the measurement of MeHg in sediments, which is based on aqueous phase ethylation, followed by purge and trap and hyphenated gas chromatography–pyrolysis–atomic fluorescence spectrometry (GC–Py–AFS) separation and detection. Four different extraction techniques, namely acid and alkaline leaching followed by solvent extraction and evaporation, microwave-assisted extraction with 2-mercaptoethanol, and acid leaching, solvent extraction and back extraction into sodium thiosulfate, were examined regarding their potential to selectively extract MeHg from estuarine sediment IAEA-405 certified reference material (CRM). The procedure based on acid leaching with HNO 3 /CuSO 4 , solvent extraction and back extraction into Na 2 S 2 O 3 yielded the highest extraction recovery, i.e., 94 ± 3% and offered the possibility to perform the extraction of a large number of samples in a short time, by eliminating the evaporation step. The artifact formation of MeHg was evaluated by high performance liquid chromatography coupled to inductively coupled plasma mass spectrometry (HPLC–ICP–MS), using isotopically enriched Me 201 Hg and 202 Hg and it was found to be nonexistent. A full validation approach in line with ISO 17025 and Eurachem guidelines was followed

  8. Reversible inactivation of CO dehydrogenase with thiol compounds

    Energy Technology Data Exchange (ETDEWEB)

    Kreß, Oliver [Department of Microbiology, University of Bayreuth, 95440 Bayreuth (Germany); Gnida, Manuel [Department of Chemistry, University of Paderborn, 33098 Paderborn (Germany); Pelzmann, Astrid M. [Department of Microbiology, University of Bayreuth, 95440 Bayreuth (Germany); Marx, Christian [Institute of Biochemistry and Biophysics, Friedrich-Schiller-University of Jena, 07745 Jena (Germany); Meyer-Klaucke, Wolfram [Department of Chemistry, University of Paderborn, 33098 Paderborn (Germany); Meyer, Ortwin, E-mail: [Department of Microbiology, University of Bayreuth, 95440 Bayreuth (Germany)


    Highlights: • Rather large thiols (e.g. coenzyme A) can reach the active site of CO dehydrogenase. • CO- and H{sub 2}-oxidizing activity of CO dehydrogenase is inhibited by thiols. • Inhibition by thiols was reversed by CO or upon lowering the thiol concentration. • Thiols coordinate the Cu ion in the [CuSMo(=O)OH] active site as a third ligand. - Abstract: Carbon monoxide dehydrogenase (CO dehydrogenase) from Oligotropha carboxidovorans is a structurally characterized member of the molybdenum hydroxylase enzyme family. It catalyzes the oxidation of CO (CO + H{sub 2}O → CO{sub 2} + 2e{sup −} + 2H{sup +}) which proceeds at a unique [CuSMo(=O)OH] metal cluster. Because of changing activities of CO dehydrogenase, particularly in subcellular fractions, we speculated whether the enzyme would be subject to regulation by thiols (RSH). Here we establish inhibition of CO dehydrogenase by thiols and report the corresponding K{sub i}-values (mM): L-cysteine (5.2), D-cysteine (9.7), N-acetyl-L-cysteine (8.2), D,L-homocysteine (25.8), L-cysteine–glycine (2.0), dithiothreitol (4.1), coenzyme A (8.3), and 2-mercaptoethanol (9.3). Inhibition of the enzyme was reversed by CO or upon lowering the thiol concentration. Electron paramagnetic resonance spectroscopy (EPR) and X-ray absorption spectroscopy (XAS) of thiol-inhibited CO dehydrogenase revealed a bimetallic site in which the RSH coordinates to the Cu-ion as a third ligand ([Mo{sup VI}(=O)OH{sub (2)}SCu{sup I}(SR)S-Cys]) leaving the redox state of the Cu(I) and the Mo(VI) unchanged. Collectively, our findings establish a regulation of CO dehydrogenase activity by thiols in vitro. They also corroborate the hypothesis that CO interacts with the Cu-ion first. The result that thiol compounds much larger than CO can freely travel through the substrate channel leading to the bimetallic cluster challenges previous concepts involving chaperone function and is of importance for an understanding how the sulfuration step in

  9. Epidemiological situation of bovine brucellosis in Settlement Nossa Senhora Aparecida at the Municipality of Mariluz in the State of Paraná, Brazil

    Directory of Open Access Journals (Sweden)

    Marcos Y. Kanashiro


    - TAT and 2-mercaptoethanol - 2-Me. In a simple analysis it was found that the prevalence of outbreaks and infected animals were, respectively, 16.31% and 2.6%. Among the producers questioned, 90% said they buy animals for reproduction, and among them, 57% do not conduct examinations of brucellosis in animals purchased. 18% rent pasture.

  10. Seroprevalence and risk factors for bovine brucellosis in Minas Gerais State, Brazil

    Directory of Open Access Journals (Sweden)

    Luciana Faria de Oliveira


    Full Text Available This cross-sectional study was conducted in the state of Minas Gerais, Brazil in 2011 to estimate the seroprevalence of bovine brucellosis (Brucella abortus in properties and cattle, and to identify potential infection risk factors for herds with cows of reproductive age. The state was divided into seven regions: 1. Noroeste, Norte and Nordeste; 2. Leste; 3. Central; 4. Zona da Mata; 5. Sul and Sudoeste; 6. Alto Paranaíba; and 7. Triângulo Mineiro. Random, two-stage sampling was performed of both properties and cattle from each property. Blood samples were collected from 18,990 cows of reproductive age from 2,185 properties. The serologic tests used for the detection of anti-B. abortus antibodies included the buffered, acidified plate antigen test, as a screening test, with the 2-mercaptoethanol reduction, test as a confirmatory test, as recommended by the Programa Nacional de Controle e Erradicação da Brucelose e Tuberculose Animal - PNCEBT (National Program for the Control and Eradication of Animal Brucellosis and Tuberculosis. The seroprevalence of bovine brucellosis on the surveyed properties and in cattle were 3.59% (95% confidence interval [CI]: 2.76 4.42% and 0.81% (95% CI: 0.05-1.10%, respectively. Multiple logistic regression analysis identified the total number of cows in the property as a risk factor for the presence of anti-B. abortus antibodies, with an odds ratio (OR of 1.93 (95% CI: 1.12 - 3.34 for herds with 30 to 210 cows and 7.81 (95% CI: 3.72-16.38 for those with more than 210 cows, relative to the risk in herds with less than 30 cows, the base category. While these results demonstrate a significant reduction in the prevalence of properties with bovine brucellosis, the disease is still present in Minas Gerais, with properties with higher numbers of cows at higher risk for infection.

  11. Hematologic changes in dogs naturally infected Leptospira spp., Brucella abortus and Brucella canis

    Directory of Open Access Journals (Sweden)

    Jacqueline Ribeiro de Castro


    Full Text Available ABSTRACT. Castro J.R., Silva C.B., Souza M.A., Salaberry S.R.S., Guimarães E.C., Mundim A.V. & Lima-Ribeiro A.M.C. [Hematologic changes in dogs naturally infected Leptospira spp., Brucella abortus and Brucella canis.] Altera- ções hematológicas em cães naturalmente infectados por Leptospira spp., Brucella abortus e Brucella canis. Revista Brasileira de Medicina Veterinária, 36(1:49-54, 2014. Laboratório de Doenças Infectocontagiosas, Faculdade de Medicina Veterinária, Universidade Federal de Uberlândia, Av. Ceará s/n, Bloco 2D, Sala 33, Campus Umuarama, Uberlândia, MG 38400-902, Brasil. E-mail: The investigations of leptospirosis and brucellosis canine act as sanitary control in public health and zoonoses because they were established by close contact between dog and human. The aim was to determine the main hematological reagents in asymptomatic dogs against Leptospira spp. Brucella abortus and Brucella canis naturally infected, living in urban areas in the city of Uberlandia, Minas Gerais. We examined 140 blood samples from clinically healthy dogs, males and females and different ages. Leptospirosis was diagnosed by microscopic agglutination test (MAT, with a collection of twelve serovars, whereas, brucellosis was identified through the tests of Agar Gel Immunodiffusion (AGID for B. canis and buffered acidified antigen (TAA confirmed 2-Mercaptoethanol (2-ME for B. abortus. The results were analyzed using descriptive statistics with the calculation of simple percentages, mean and standard deviation. He applied and short sample t test for two independent samples to assess whether there were significant differences (p<0.05 between hematological parameters obtained. Dogs evaluated, 15% (21/140 and 2.85% (4/140 were reactive to Leptospira spp. and B. abortus, respectively. There was no sample reagent against B. canis. It was concluded that although no specific thrombocytopenia may be a significant finding in dogs

  12. Activity-Based Protein Profiling of Ammonia Monooxygenase in Nitrosomonas europaea

    Energy Technology Data Exchange (ETDEWEB)

    Bennett, Kristen; Sadler, Natalie C.; Wright, Aaron T.; Yeager, Chris; Hyman, Michael R.; Löffler, F. E.


    Nitrosomonas europaeais an aerobic nitrifying bacterium that oxidizes ammonia (NH3) to nitrite (NO2) through the sequential activities of ammonia monooxygenase (AMO) and hydroxylamine dehydrogenase (HAO). Many alkynes are mechanism-based inactivators of AMO, and here we describe an activity-based protein profiling method for this enzyme using 1,7-octadiyne (17OD) as a probe. Inactivation of NH4+-dependent O2uptake byN. europaeaby 17OD was time- and concentration-dependent. The effects of 17OD were specific for ammonia-oxidizing activity, andde novoprotein synthesis was required to reestablish this activity after cells were exposed to 17OD. Cells were reacted with Alexa Fluor 647 azide using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) (click) reaction, solubilized, and analyzed by SDS-PAGE and infrared (IR) scanning. A fluorescent 28-kDa polypeptide was observed for cells previously exposed to 17OD but not for cells treated with either allylthiourea or acetylene prior to exposure to 17OD or for cells not previously exposed to 17OD. The fluorescent polypeptide was membrane associated and aggregated when heated with β-mercaptoethanol and SDS. The fluorescent polypeptide was also detected in cells pretreated with other diynes, but not in cells pretreated with structural homologs containing a single ethynyl functional group. The membrane fraction from 17OD-treated cells was conjugated with biotin-azide and solubilized in SDS. Streptavidin affinity-purified polypeptides were on-bead trypsin-digested, and amino acid sequences of the peptide fragments were determined by liquid chromatography-mass spectrometry (LC-MS) analysis. Peptide fragments from AmoA were the predominant peptides detected in 17OD-treated samples. In-gel digestion and

  13. The effect of FF-MAS on porcine cumulus-oocyte complex maturation, fertilization and pronucleus formation in vitro. (United States)

    Faerge, Inger; Strejcek, Frantisek; Laurincik, Jozef; Rath, Detlef; Niemann, Heiner; Schellander, Karl; Rosenkranz, Christine; Hyttel, Poul Maddox; Grøndahl, Christian


    Follicular fluid meiosis-activating sterol (FF-MAS) has been isolated from the follicular fluid (FF) of several species including man. FF-MAS increases the quality of in vitro oocyte maturation, and thus the developmental potential of oocytes exposed to FF-MAS during in vitro maturation is improved. The aim of the present study was to investigate the effects of FF-MAS on porcine oocyte maturation and pronucleus formation in vitro. Porcine cumulus-oocyte complexes (COCs) were isolated from abattoir ovaries and in vitro matured for 48 h in NCSU 37 medium supplemented with 1 mg/l cysteine, 10 ng/ml epidermal growth factor and 50 microM 2-mercaptoethanol with or without 10% porcine follicular fluid (pFF). For the first 22 h, 1 mM db-cAMP and 10 I.E PMSG/hCG was added. The medium was supplemented with 1 microM, 3 microM, 10 microM, 30 microM or 100 microM FF-MAS dissolved in ethanol. After maturation the COCs were denuded mechanically using a fine glass pipette under constant pH and in vitro fertilized with fresh semen (5 x 10(5) spermatozoa/ml). The presumptive zygotes were evaluated 18 h after fertilization. The addition of pFF increased the monospermic as well as the polyspermic penetration of oocytes. In the absence of pFF, the addition of FF-MAS decreased the polyspermic penetration rate, whereas FF-MAS in combination with pFF decreased monospermic and increased polyspermic penetration. The degeneration rate of ova decreased in the presence of FF-MAS irrespective of the presence or absence of pFF. In the absence of pFF, FF-MAS at 3-10 microM increased the number of zygotes with advanced maternal pronuclear stages. In supraphysiological doses, i.e. 30-100 microM, FF-MAS dose-dependently and reversibly inhibited nuclear maturation in the absence of pFF.

  14. Perspectives of digestive pest control with proteinase inhibitors that mainly affect the trypsin-like activity of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae

    Directory of Open Access Journals (Sweden)

    M.E. Pereira


    Full Text Available The present study describes the main characteristics of the proteolytic activities of the velvetbean caterpillar, Anticarsia gemmatalis Hübner, and their sensitivity to proteinase inhibitors and activators. Midguts of last instar larvae reared on an artificial diet were homogenized in 0.15 M NaCl and centrifuged at 14,000 g for 10 min at 4ºC and the supernatants were used in enzymatic assays at 30ºC, pH 10.0. Basal total proteolytic activity (azocasein hydrolysis was 1.14 ± 0.15 absorbance variation min-1 mg protein-1, at 420 nm; basal trypsin-like activity (N-benzoyl-L-arginine-p-nitroanilide, BApNA, hydrolysis was 0.217 ± 0.02 mmol p-nitroaniline min-1 mg protein-1. The maximum proteolytic activities were observed at pH 10.5 using azocasein and at pH 10.0 using BApNA, this pH being identical to the midgut pH of 10.0. The maximum trypsin-like activity occurred at 50ºC, a temperature that reduces enzyme stability to 80 and 60% of the original, when pre-incubated for 5 and 30 min, respectively. Phenylmethylsulfonyl fluoride inhibited the proteolytic activities with an IC50 of 0.39 mM for azocasein hydrolysis and of 1.35 mM for BApNA hydrolysis. Benzamidine inhibited the hydrolysis with an IC50 of 0.69 and 0.076 mM for azocasein and BApNA, respectively. The absence of cysteine-proteinases is indicated by the fact that 2-mercaptoethanol and L-cysteine did not increase the rate of azocasein hydrolysis. These results demonstrate the presence of serine-proteinases and the predominance of trypsin-like activity in the midgut of Lepidoptera insects, now also detected in A. gemmatalis, and suggest this enzyme as a major target for pest control based on disruption of protein metabolism using proteinase inhibitors.

  15. Isolation and properties of polyphenol oxidase from basidiocarps of Lactarius pergamenus Fr. (Fr. fungi

    Directory of Open Access Journals (Sweden)

    M. V. Tsivinska


    Full Text Available Fresh juice of basidiocarps of Lactarius pergamenus Fr. (Fr. fungi was subjected to ion exchange chromatography with used DEAE-toyopearl and CM-cellulose columns, as well as preparative electrophoresis in 7.5% polyacrylamide gels (pH 8.6. Three isoforms of polyphenol oxidase (PPO were discovered and two isoforms (1-1 and 1-2 were purified with a release of protein 0.42 mg/kg and 0.15 mg/kg of basidiocarps, respectively. These isoforms differ in the mobility at disc-electrophoresis in 7.5% PAGE in alkaline buffer system (pH 8.6. Specific activity of isoform 1-2 is 4.8 times higher than that of the isoforms 1-1. The molecular weight determination by gel chromatography on the Toyopearl HW-55 demonstrated that both isoforms 1-1 and 1-2 have the same 64 ± 2 kDa molecular mass. Electrophoresis in 15% PAGE in the presence of sodium dodecylsulphate and β-mercaptoethanol revealed one band with molecular mass of 64 ± 1 kDa which suggests the presence of one polypeptide chain in the molecule of the enzyme. The enzyme has demonstrated the highest activity at pH 6.0 and temperature +10 ºC, and at +70 ºC the enzyme was inactivated. The PPO activity was the highest in young mushrooms and it decreased with their age and positively correlated with the content of the milky juice. Ortho-aminophenol was most effective among all the tested substrates to determine the activity of PPO (o-, m– and p-aminophenol, catechol, tyrosine, resorcinol, phloroglucinol and its relative activity was 129% of the activity of catechol. Ascorbic acid was the most effective inhibitor of the polyphenol oxidase activity which was completely blocked at 1 mM concentration, whereas the same concentration of thiourea and sodium sulphite decreased the enzymatic activity by 40-45%. The PPO in L. pergamenus fungi basidiocarps was mainly localized in the mushroom milky juice where its high activity may be associated with protection of basidiocarps against various pathogens.

  16. Candidates of trichocyst matrix proteins of the dinoflagellate Oxyrrhis marina. (United States)

    Rhiel, Erhard; Wöhlbrand, Lars; Rabus, Ralf; Voget, Sonja


    Trichocysts are a common cell organelle of ciliates and dinoflagellates. They are composed of trichocyst matrix proteins and have been intensely investigated and characterized in ciliates. Here, for the first time, data have been obtained for trichocyst matrix proteins of a dinoflagellate. A DELTA-BLAST search using 14 available and complete amino acid sequences of mature trichocyst matrix proteins of the ciliate Paramecium tetraurelia resulted in 16 hits for the dinoflagellate Oxyrrhis marina when the E values and bit values to be scored were 40. They code for proteins with acidic pI values and exceeded the precursors of the trichocyst matrix proteins of the ciliate approximately twofold in length. The values calculated for coverage, identity, and positives ranged from 76 to 100, 21.5 to 28.3, and 44.9 to 53.9%, respectively. Protein conformation predictions indicate coiled-coil domains which are a common feature of mature ciliate trichocyst matrix proteins. As often several EST sequences of O. marina matched with a queried mature trichocyst matrix protein of P. tetraurelia, a multigene family can be assumed for trichocyst proteins in this dinophyte, too. Trichocyst-enriched fractions of O. marina were isolated and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. When samples were incubated with loading buffer without a reducing agent, the banding pattern was mainly composed of three regions in the range of >90, 75-60, and 50-35 kDa, with each region consisting of four to five bands. Tryptic in gel digestion of proteins excised from these three gel regions followed by mass spectrometry confirmed that up to 14 of the 16 predicted proteins were present within the trichocyst-enriched fractions. When the samples were reduced with either ß-mercaptoethanol or dithiothreitol, the proteins of the three regions disappeared almost completely and proteins in the range of 27 to 15 kDa became the dominating bands. Up to 12 of the predicted proteins

  17. Purification and characterization of neutral alpha-mannosidase from hen oviduct: studies on the activation mechanism of Co2+. (United States)

    Yamashiro, K; Itoh, H; Yamagishi, M; Natsuka, S; Mega, T; Hase, S


    Neutral alpha-mannosidase was purified to homogeneity from hen oviduct. The molecular mass of the enzyme was 480 kDa on gel filtration, and the 110-kDa band on SDS-PAGE in the presence of 2-mercaptoethanol indicated that it is composed of four subunits. The activated enzyme hydrolyzed both p-nitrophenyl alpha-D-mannoside and high mannose-type sugar chains. This substrate specificity is almost the same as that reported for the neutral a-mannosidase from Japanese quail oviduct [Oku and Hase (1991) J. Biochem. 110, 982-989]. Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) Manbeta1-4GlcNAc (Km =0.44 mM) was hydrolyzed four times faster than Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) Manbeta1-4GIcNAcbeta1-4GlcNAc, and Manalpha1-6(Manalpha1-2Manalpha1-2Manalpha1-3)++ +Manbeta1-4GlcNAc was obtained as the end product from Man9GlcNAc on digestion with the activated alpha-mannosidase. The enzyme was activated 24-fold on preincubation with Co2+. The activation with other metal ions, like Mn2+, Ca2+, Fe2+, Fe3+, and Sr2+, was less than 5-fold, and Zn2+, Cu2+, and Hg2+ inhibited the enzyme activity. The optimum pHs for both the enzyme activity and activation with Co2+ were around 7. The cobalt ion contents of the purified, EDTA-treated, and Co2+-activated enzymes were 1.5, 0.0, and 3.9, respectively, per molecule. Since the Co2+-activated enzyme gradually lost its activity on incubation with EDTA and the activity was restored promptly on the addition of Co2+, the binding of Co2+ to the enzyme seems to be essential for its activation. The results obtained with protease inhibitors together with those of the SDS-PAGE before and after activation, showed that the proteolytic cleavage reported for the activation of monkey brain alpha-mannosidase seems not to be involved.

  18. Simple method for Shiga toxin 2e purification by affinity chromatography via binding to the divinyl sulfone group.

    Directory of Open Access Journals (Sweden)

    Hideyuki Arimitsu

    Full Text Available Here we describe a simple affinity purification method for Shiga toxin 2e (Stx2e, a major causative factor of edema disease in swine. Escherichia coli strain MV1184 transformed with the expression plasmid pBSK-Stx2e produced Stx2e when cultivated in CAYE broth containing lincomycin. Stx2e bound to commercial D-galactose gel, containing α-D-galactose immobilized on agarose resin via a divinyl sulfone linker, and was eluted with phosphate-buffered saline containing 4.5 M MgCl2. A small amount of Stx2e bound to another commercial α-galactose-immobilized agarose resin, but not to β-galactose-immobilized resin. In addition, Stx2e bound to thiophilic adsorbent resin containing β-mercaptoethanol immobilized on agarose resin via a divinyl sulfone, and was purified in the same manner as from D-galactose gel, but the Stx2e sample contained some contamination. These results indicate that Stx2e bound to D-galactose gel mainly through the divinyl sulfone group on the resin and to a lesser extent through α-D-galactose. With these methods, the yields of Stx2e and attenuated mutant Stx2e (mStx2e from 1 L of culture were approximately 36 mg and 27.7 mg, respectively, and the binding capacity of the D-galactose gel and thiophilic adsorbent resin for Stx2e was at least 20 mg per 1 ml of resin. In addition, using chimeric toxins with prototype Stx2 which did not bind to thiophilic adsorbent resin and some types of mutant Stx2e and Stx2 which contained inserted mutations in the B subunits, we found that, at the least, asparagine (amino acid 17 of the B subunits was associated with Stx2e binding to the divinyl sulfone group. The mStx2e that was isolated exhibited vaccine effects in ICR mice, indicating that these methods are beneficial for large-scale preparation of Stx2e toxoid, which protects swine from edema disease.

  19. Isolation, production, purification and characterization of an organic-solvent-thermostable alkalophilic cellulase from Bacillus vallismortis RG-07. (United States)

    Gaur, Rajeeva; Tiwari, Soni


    The rising concerns about the scarcity of fossil fuels, the emission of green house gasses and air pollution by incomplete combustion of fossil fuel have also resulted in an increasing focus on the use of cellulases to perform enzymatic hydrolysis of the lignocellulosic materials for the generation of bioethanol. The aim of this study was to isolate a potential thermo-solvent tolerant cellulase producing bacterium from natural resources, and then applied for purification and characterization. The purified enzyme was to be accessible for the bioethanol production as well as industrial exploitation (discuss in our next study). It is the first instance when thermo-solvent tolerant cellulase producing bacterium was isolated from soil sample. The culture was identified as Bacillus vallismortis RG-07 by 16S rDNA sequence analysis. Bacillus vallismortis RG-07 reported maximum cellulase production from sugarcane baggase (4105 U ml(-1)) used as agro-waste carbon source. The cellulase enzyme produced by the Bacillus sp. was purified by (NH4)2SO4 precipitation, ion exchange and gel filtration chromatography, with overall recovery of 28.8%. The molecular weight of purified cellulase was 80 kDa as revealed by SDS-PAGE and activity gel analysis. The optimum temperature and pH for enzyme activity was determined as 65°C and 7.0 and it retained 95 and 75% of activity even at 95°C, and 9.0 respectively. The enzyme activity was enhanced in the presence of organic solvents (30%) n-dodecane, iso-octane, n-decane, xylene, toluene, n-haxane, n-butanol, and cyclohexane, after prolonged incubation (7 days). The enzyme activity was also stimulated by Ca(2+), mercaptoethanol, Tween-60, and Sodium hypochloride whereas strongly inhibited by Hg. Kinetic analysis of purified enzyme showed the Km and Vmax to be 1.923 mg ml(-1) and 769.230 μg ml(-1) min(-1), respectively. The unique property of solvent-thermostable-alkalophilic, nature proves the potential candidature of this isolate for

  20. Synthesis and characterizations of ultra-small ZnS and Zn(1-x)Fe(x)S quantum dots in aqueous media and spectroscopic study of their interactions with bovine serum albumin. (United States)

    Khani, Omid; Rajabi, Hamid Reza; Yousefi, Mohammad Hasan; Khosravi, Ali Azam; Jannesari, Mohammad; Shamsipur, Mojtaba


    This work reports a new experimental methodology for the synthesis of ultra small zinc sulfide and iron doped zinc sulfide quantum dots in aqueous media. The nanoparticles were obtained using a simple procedure based on the precipitation of ZnS in aqueous solution in the presence of 2-mercaptoethanol as a capping agent, at room temperature. The effect of Fe(3+) ion concentration as dopant on the optical properties of ZnS was studied. The size of quantum dots was determined to be about 1nm, using scanning tunneling microscopy. The synthesized nanoparticles were characterized by X-ray diffraction, UV-Vis absorption and photoluminescence emission spectroscopies. The presence and amount of iron impurity in the structure of Zn((1-x))Fe(x)S nanocrystals were confirmed by atomic absorption spectrometry. A blue shift in band-gap of ZnS was observed upon increasing incorporation of Fe(3+) ion in the iron doped zinc sulfide quantum dots. The photoluminescence investigations showed that, in the case of iron doped ZnS nanoparticles, the emission band of pure ZnS nanoparticles at 427nm shifts to 442nm with appearance of a new sharp emission band around 532nm. The X-ray diffraction analysis indicated that the iron doped nanoparticles are crystalline, with cubic zinc blend structure, having particle diameters of 1.7±022nm. Finally, the interaction of the synthesized nanoparticles with bovine serum albumin was investigated at pH 7.2. The UV-Vis absorption and fluorescence spectroscopic methods were applied to compare the optical properties of pure and iron doped ZnS quantum dots upon interaction with BSA. It was proved that, in both cases, the fluorescence quenching of BSA by the quantum dots is mainly a result of the formation of QDs-BSA complex in solution. In the steady-state fluorescence studies, the interaction parameters including binding constants (K(a)), number of binding sites (n), quenching constants ( [Formula: see text] ), and bimolecular quenching rate constants (k

  1. Production of Nα-acetylated thymosin α1 in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Fang Hongqing


    Full Text Available Abstract Background Thymosin α1 (Tα1, a 28-amino acid Nα-acetylated peptide, has a powerful general immunostimulating activity. Although biosynthesis is an attractive means of large-scale manufacture, to date, Tα1 can only be chemosynthesized because of two obstacles to its biosynthesis: the difficulties in expressing small peptides and obtaining Nα-acetylation. In this study, we describe a novel production process for Nα-acetylated Tα1 in Escherichia coli. Results To obtain recombinant Nα-acetylated Tα1 efficiently, a fusion protein, Tα1-Intein, was constructed, in which Tα1 was fused to the N-terminus of the smallest mini-intein, Spl DnaX (136 amino acids long, from Spirulina platensis, and a His tag was added at the C-terminus. Because Tα1 was placed at the N-terminus of the Tα1-Intein fusion protein, Tα1 could be fully acetylated when the Tα1-Intein fusion protein was co-expressed with RimJ (a known prokaryotic Nα-acetyltransferase in Escherichia coli. After purification by Ni-Sepharose affinity chromatography, the Tα1-Intein fusion protein was induced by the thiols β-mercaptoethanol or d,l-dithiothreitol, or by increasing the temperature, to release Tα1 through intein-mediated N-terminal cleavage. Under the optimal conditions, more than 90% of the Tα1-Intein fusion protein was thiolyzed, and 24.5 mg Tα1 was obtained from 1 L of culture media. The purity was 98% after a series of chromatographic purification steps. The molecular weight of recombinant Tα1 was determined to be 3107.44 Da by mass spectrometry, which was nearly identical to that of the synthetic version (3107.42 Da. The whole sequence of recombinant Tα1 was identified by tandem mass spectrometry and its N-terminal serine residue was shown to be acetylated. Conclusions The present data demonstrate that Nα-acetylated Tα1 can be efficiently produced in recombinant E. coli. This bioprocess could be used as an alternative to chemosynthesis for the production

  2. Cloning and expression of the xynA1 gene encoding a xylanase of the GH10 group in Caulobacter crescentus. (United States)

    Graciano, Luciana; Corrêa, Juliana Moço; Vieira, Fabíola Giovanna Nesello; Bosetto, Adilson; Loth, Eduardo Alexandre; Kadowaki, Marina Kimiko; Gandra, Rinaldo Ferreira; Simão, Rita de Cássia Garcia


    Caulobacter crescentus (NA1000 strain) are aquatic bacteria that can live in environments of low nutritional quality and present numerous genes that encode enzymes involved in plant cell wall deconstruction, including five genes for β-xylosidases (xynB1-xynB5) and three genes for xylanases (xynA1-xynA3). The overall activity of xylanases in the presence of different agro-industrial residues was evaluated, and it was found that the residues from the processing of corn were the most efficient in inducing bacterial xylanases. The xynA1 gene (CCNA_02894) encoding a predicted xylanase of group 10 of glyco-hydrolases (GH10) that was efficiently overexpressed in Escherichia coli LMG194 using 0.02 % arabinose, after cloning into the vector pJet1.2blunt and subcloning into the expression vector pBAD/gIII, provided a fusion protein that contained carboxy-terminal His-tags, named XynA1. The characterization of pure XynA1 showed an enzymatic activity of 18.26 U mL(-1) and a specific activity of 2.22 U mg-(1) in the presence of xylan from beechwood as a substrate. XynA1 activity was inhibited by EDTA and metal ions such as Cu(2+) and Mg(2+). By contrast, β-mercaptoethanol, dithiothreitol (DTT), and Ca(2+) induced recombinant enzyme activity. Kinetic data for XynA1 revealed K M and V max values of 3.77 mg mL-(1) and 10.20 μM min-(1), respectively. Finally, the enzyme presented an optimum pH of 6 and an optimum temperature of 50 °C. In addition, 80 % of the activity of XynA1 was maintained at 50 °C for 4 h of incubation, suggesting a thermal stability for the biotechnological processes. This work is the first study concerning the cloning, overexpression, and enzymatic characterization of C. crescentus xylanase.

  3. Caracterização e hidrólise in vitro da globulina principal de grão-de-bico (Cicer arietinum L., var. IAC-Marrocos Characterization and in vitro tryptic hydrolysis of the major globulin from chickpea (Cicer arietinum L.

    Directory of Open Access Journals (Sweden)

    Valdir A. Neves


    Full Text Available No presente estudo procedeu-se ao isolamento e caracterização da fração globulina majoritária (11 S de grão-de-bico, var. IAC-Marrocos. A globulina majoritária extraída foi isolada por cromatografia de filtração em gel e de troca-iônica mostrando apenas uma banda de proteína na eletroforese em gel de poliacrilamida. A globulina majoritária, após passagem em coluna de Sephadex, revelou duas bandas protéicas de 55 e 52,5kDa e três bandas menores em gel de poliacrilamida dodecilsulfato de sódio. Na presença de 2-mercaptoetanol 6 polipeptídios na faixa de 18 a 42kDa foram revelados na eletroforese. A globulina isolada foi submetida à ação da tripsina e quimotripsina onde a forma nativa mostrou-se resistente à ação enzimática enquanto o aquecimento (96 e 121°C/15min não foi suficiente para aumentar a susceptibilidade à hidrólise, significativamente. Adição de NaCl 0,3M levou a um aumento da estabilidade estrutural com menor susceptibilidade à digestão proteolítica, fato em parte perdido com o aquecimento. As hidrólises foram acompanhadas por eletroforese em gel de poliacrilamida dodecilsulfato de sódio.The isolation and characterization of the major globulin fraction (11 S from Chickpea, vc IAC-Marrocos, were evaluated. The major globulin was extracted, isolated by gel filtration and ion-exchange chromatography showing only one protein band on PAGE. The globulin, after Sephadex elution, revealed two protein bands of 55 and 52.5kDa and three minor bands on SDS-PAGE. In the presence of 2-mercaptoethanol six polypeptides were revealed on SDS-PAGE in the range of 18 to 42kDa. The isolated native globulin shown to be resistant to trypsin and chymotrypsin however heating at 96 and 121ºC/15min was not sufficient to increase the hydrolysis significantly. The proteolytic susceptibility of the enzymes was reduced by 0.3M NaCl addition at the assay. The salt concentration was sufficient to stabilize the native protein

  4. Redox-dependent dimerization of p38α mitogen-activated protein kinase with mitogen-activated protein kinase kinase 3. (United States)

    Bassi, Rekha; Burgoyne, Joseph R; DeNicola, Gian F; Rudyk, Olena; DeSantis, Vittorio; Charles, Rebecca L; Eaton, Philip; Marber, Michael S


    The kinase p38α MAPK (p38α) plays a pivotal role in many biological processes. p38α is activated by canonical upstream kinases that phosphorylate the activation region. The purpose of our study was to determine whether such activation may depend on redox-sensing cysteines within p38α. p38α was activated and formed a disulfide-bound heterodimer with MAP2K3 (MKK3) in rat cardiomyocytes and isolated hearts exposed to H 2 O 2 This disulfide heterodimer was sensitive to reduction by mercaptoethanol and was enhanced by the thioredoxin-reductase inhibitor auranofin. We predicted that Cys-119 or Cys-162 of p38α, close to the known MKK3 docking domain, were relevant for these redox characteristics. The C119S mutation decreased whereas the C162S mutation increased the dimer formation, suggesting that these two Cys residues act as vicinal thiols, consistent with C119S/C162S being incapable of sensing H 2 O 2 Similarly, disulfide heterodimer formation was abolished in H9C2 cells expressing both MKK3 and p38α C119S/C162S and subjected to simulated ischemia and reperfusion. However, the p38α C119S/C162S mutants did not exhibit appreciable alteration in activating dual phosphorylation. In contrast, the anti-inflammatory agent 10-nitro-oleic acid (NO 2 -OA), a component of the Mediterranean diet, reduced p38α activation and covalently modified Cys-119/Cys-162, probably obstructing MKK3 access. Moreover, NO 2 -OA reduced the dephosphorylation of p38α by hematopoietic tyrosine phosphatase (HePTP). Furthermore, steric obstruction of Cys-119/Cys-162 by NO 2 -OA pretreatment in Langendorff-perfused murine hearts prevented the p38-MKK3 disulfide dimer formation and attenuated H 2 O 2 -induced contractile dysfunction. Our findings suggest that cysteine residues within p38α act as redox sensors that can dynamically regulate the association between p38 and MKK3. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Determinação da Cinética Ruminal da Proteína de Vários Alimentos Utilizando o Método de Inibidores In Vitro Determination of Ruminal Protein Kinetics of Feedstuffs Using an Inhibitor in vitro Method

    Directory of Open Access Journals (Sweden)

    Fernando Iván Londoño Hernández


    Full Text Available O objetivo do presente trabalho foi estimar os parâmetros cinéticos da degradação dos compostos nitrogenados de 24 alimentos concentrados e 14 volumosos, por intermédio do método de inibidores in vitro, utilizando-se o sistema Kjeldahl. Foi utilizado líquido ruminal oriundo de bovino recebendo dieta com 60% de volumoso e 40% de concentrado. Na preparação de 1000 mL do meio fermentador, utilizaram-se 800 mL do inóculo, 2 g de NaHCO3 em 50 mL de H2O destilada, 3,2 g de pectina em 100 mL de solução McDougall, 0,234 mL de mercaptoethanol, 0,195 g de sulfato de hidrazina em 25 mL de solução McDougall e 0,045 g de cloranfenicol em 25 mL de solução McDougall. Ao meio fermentador adicionaram-se 3,2 g de amido, 3,2 g de xilose e 0,16 mL de antiespumante (Antifoam 204, Sigma Chemical Co. A-6426. Foi determinado o desaparecimento dos compostos nitrogenados dos alimentos nos tempos 0 e 2 horas, incubados na quantidade de, aproximadamente, 1,875 mg de N para cada tubo de ensaio. As estimativas referentes às taxas de degradação mostraram que os alimentos farelo de glúten de milho, caseína, grão de amendoim moído, cama de frango contendo casca de café como material absorvente e raspa de mandioca possuem proteínas de rápida degradação, observando-se as mais lentas taxas de degradação para o fubá de milho, a farinha de carne e ossos, a cama de frango contendo capim-elefante como material absorvente, a levedura de cana-de-açúcar e a farinha de penas. De maneira geral, os parâmetros da degradação apresentaram resultados semelhantes aos reportados in situ. O método dos inibidores in vitro permitiu uma avaliação rápida e eficiente da cinética de degradação da proteína bruta dos alimentos concentrados. As taxas de degradação de alguns alimentos volumosos foram subestimadas.The objective of this work was to evaluate the kinetics parameters of nitrogen compounds degradation for 24 concentrate feedstuffs and 14 grasses, by

  6. Investigation of glutathione peroxidase activity in chicken meat under different experimental conditions Investigação da atividade de glutationa peroxidase em carne de frango submetida a diferentes condições experimentais

    Directory of Open Access Journals (Sweden)

    Alexandre José Cichoski


    Full Text Available Due to the fact that previous studies on the enzymatic activity of Glutathione peroxidase (GSH-Px diverge widely in their methodology and results, this study aimed to investigate the influence of different analytical conditions on GSH-Px activity in chicken thighs from broilers that were fed different diets with different sources and concentrations of selenium. GSH-Px activity was evaluated six hours after slaughter and 120 days after frozen storage at -18 ºC. The different analytical conditions included time of pre-incubation (0, 10 and 30 minutes, reaction medium, types of substrate (H2O2 (0.72 mM, 7.2 mM, and 72 mM and Terc-butil hydroperoxide 15 mM, and different buffer concentrations (buffer 1, potassium phosphate 50 mM pH 7.0 + EDTA 1 mM + mercaptoethanol 1 mM, and buffer 2, tris-HCl 50 mM pH 7.6 + EDTA 1 mM + mercapthanol 5 mM. The results show that the highest GSH-Px activity was observed when enzyme and substrate were in contact at 22 ºC without any pre-incubation, and that, when used at concentrations above 0.72 mM, hydrogen peroxide saturated the GSH-Px enzyme and inhibited its activity. The enzyme presented higher affinity to hydrogen peroxide when compared to terc-butil peroxide, and the addition of a buffer containing mercaptoethanol did not increase GSH-Px enzymatic activity. The activity of GSH-Px was not influenced by the source and concentration of selenium in the diet either. The obtained results allowed the determination of the best temperature of contact between the enzyme and substrate (22 ºC, the optimum concentration, and the type of substrate and buffer to be used. This information is extremely useful for future studies on GSH-Px activity in meat due to the divergence and little information found in the literature.Uma vez que estudos anteriores sobre a atividade enzimática da glutationa peroxidase (GSH-Px divergem acerca da metodologia e dos resultados, este estudo teve por objetivo investigar a influência de

  7. Avaliação do teste do anel em leite na vigilância epidemiológica da brucelose bovina em rebanhos e em laticínios Evaluation of the ring test in an epidemiological surveillance of bovine brucellosis in herds and dairies

    Directory of Open Access Journals (Sweden)

    F.F. Silva Júnior


    Full Text Available Analisaram-se 464 amostras individuais de leite e 54 amostras de leite de latões, oriundos de leite dos mesmos animais, por meio do teste do anel do leite (TAL visando à sua aplicação no diagnóstico individual e de rebanhos da brucelose bovina. Foram também avaliadas 464 amostras de soro sangüíneo por meio de provas do antígeno tamponado acidificado (ATA, soroaglutinação lenta em tubos (SAL e 2-mercaptoetanol (2-ME, todas para brucelose. Cento e vinte e três amostras (26,5% testadas pelo TAL apresentaram resultados positivos. Dessas, 30 resultaram positivas ao ATA, 28 ao ATA, à SAL e ao 2-ME e 18 à SAL. Das amostras positivas ao TAL, 95 pertenciam a animais sorologicamente negativos ao 2-ME, caracterizando 77,2% (95/123 das reações falso-positivas; dos resultados negativos ao TAL, 4 pertenciam a animais sorologicamente positivos, caracterizando 1,2% (4/341 de reações falso-negativas no TAL individual. Das 54 amostras de leite de latões analisadas pelo TAL, 17 foram consideradas positivas, das quais uma foi caracterizada como falso-positivo, pois todos os animais que a compunham foram negativos ao 2-ME. De 37 latões considerados negativos ao TAL, três continham leite de animais positivos ao 2-ME, caracterizando 8,1% de falso-negativos. O TAL individual demonstrou elevado percentual de resultados falso-positivos, enquanto o TAL em amostras de leite obtidas em latões detectou 84,2% de latões contaminados e 75% de rebanhos infectados.The ring test (RT was analyzed regarding its application for the individual and herd bovine brucellosis diagnoses. Individual samples of milk from 464 cows and 54 composite samples of milk bucket from these animals were evaluated. The results were analyzed considering the serological results obtained by the rose bengal (RBT, tube agglutination (TAT and 2-mercaptoethanol (2-ME tests. From the 464 individual milk samples analyzed by the RT, 123 (26.5% presented positive results. From those, 30 were

  8. Comparative studies of antibody anti-CD20 labeled with 188Re

    International Nuclear Information System (INIS)

    Dias, Carla Roberta de Barros Rodrigues


    Nuclear Medicine is an unique and important modality in oncology and the development of new tumor-targeted radiopharmaceuticals for both diagnosis and therapy is an area of interest for researchers. Rituximab (RTX) is a quimeric monoclonal antibody (mAb) (IgG 1) that specifically binds to CD20 antigen with high affinity and has been successfully used for the treatment of Non-Hodgkin Lymphoma (NHL) of cell B. The CD20 antigen is expressed over more than 90% of cell B NHL. Technetium-99m ( 99m Tc) and rhenium-188 ( 188 Re) are an attractive radionuclide pair for clinical use due to their favorable decay properties for diagnosis ( 99m Tc: T 1/2 = 6 h, γ radiation = 140 keV) and therapy ( 188 Re: T 1/2 = 17 h, maximum β energy = 2.12 MeV) and to their availability in the form of 99 Mo/ 99 mTc and 188 W/ 188 Re generators. The radionuclides can be conjugated to mAb using similar chemical procedures. The aim of this work was to study the labeling of anti-CD20 mAb (RTX) with 188 Re using two techniques: the direct labeling method [ 188 Re(V)] and the labeling method via the carbonyl nucleus [ 188 Re(I)]. Besides the quality control, the radiolabeled mAb was submitted to in vivo, in vitro and ex vivo biological studies. For the direct labeling, RTX was reducing by incubation with 2-mercaptoethanol for generating sulphydryl groups (-SH) and further labeled with 188 Re(V), in a study of several parameters in order to reach an optimized formulation. The labeling via the carbonyl nucleus both 99 mTc and 188 Re were employed through 2 different procedures: (1) labeling of intact RTX with 99 mTc(I) and (2) reduced RTX (RTX red ) labeled with 99 mTc(I)/ 188 Re(I). Also a parameter study was performed to obtain an optimized formulation. The quality control method for evaluating the radiochemical purity showed a good labeling yield (93%) for the direct method. The labeling method via carbonyl group, the results showed that the - SH groups of RTX red are a possible way of labeling

  9. Prolactin variants in ram adenohypophyses vary with season (United States)

    Stroud, C. M.; Deaver, D. R.; Peters, J. L.; Loeper, D. C.; Toth, B. E.; Derr, J. A.; Hymer, W. C.


    Secretion of PRL in sheep is affected by photoperiod being highest during the spring and summer, lowest in fall and winter. The objectives of this study were to determine if 1) the production of variant forms of PRL, and 2) immuno- and bioactivities of PRL (iPRL and bPRL) differ during times of the year selected to represent periods of low, transitional and high PRL secretion. Twelve mature rams were maintained on pasture and killed in October, December, and April (n = 4/month). Individual pituitary glands were dispersed, cells obtained, and fixed for immunocytochemical flow cytometry, extracted with 0.01 N NaHCO3 or cultured in serum-free, defined media. The Mr of PRL extracted from cells immediately following dispersion ranged from 14-140K, with significantly more bands greater than 40K being detected from rams sacrificed in December than from those killed in October and April (P less than 0.01). No bands of PRL greater than 25K were observed when samples were reduced with beta-mercaptoethanol prior to electrophoresis, indicating that the high Mr forms were disulfide-linked aggregates. Culture media from October and April contained variants of PRL that ranged from 22-40K but those greater than 25K were generally not observed from cells harvested during December. Extracts of cells after 24 h in culture contained fewer high Mr species during December than had been present in initial extracts from that month. In contrast, during April more high Mr intracellular forms were present after culture than had been detected prior to culture during that month. The percentage of lactotrophs averaged 50.0 +/- 2.5, 47.4 +/- 5.7, and 59.4 +/- 5.5 for October, December, and April, respectively. Initial lactotroph content (pg/lactotroph) of iPRL was higher (P = 0.06) in April (46.0 +/- 17.0) when compared to October and December (8.0 +/- 2.0 and 20.0 +/- 10.0, respectively). In contrast, the bPRL content of initial extracts was higher (P = 0.05) in December (267.0 +/- 68.0) than

  10. Inibição da tripsina de bicho-mineiro do cafeeiro por um fator não-protéico presente em extratos de folhas de mamona Coffee leaf miner trypsin inhibition with castor bean leaf extracts mediated by a non-protein agent

    Directory of Open Access Journals (Sweden)

    Guilherme Duarte Rossi


    insects is a control form whose efficacy was verified by different authors. In order to observe the efficiency of castor bean leaf extracts in inhibiting trypsin-like enzymes of the coffee leaf miner, an experiment was carried out with the purpose of observing an "in vitro" inhibition phenomenon. The results of the trypsin inhibition tests with normal and boiled with and without β-mercaptoethanol 0.2% (v/v castor bean leaf extracts and the results of the acetone precipitation process indicated that the inhibitor is a heat-resistant molecule and it is not a protein. This way, the purification process was made by adsorption chromatography with later analysis in mass spectrometer. The reached results indicated that the presence of a trypsin inhibitor of the coffee leaf miner in the castor bean leaf extracts is capable of inhibiting 2.48 + 0.15 UTI, which stands for about 40% of inhibition. Tests performed with bovine trypsin indicated that the castor bean leaf extract have no inhibiting power on this enzyme.

  11. Selective Permeation and Organic Extraction of Recombinant Green Fluorescent Protein (gfpuv from Escherichia coli

    Directory of Open Access Journals (Sweden)

    Ishii Marina


    Full Text Available Abstract Background Transformed cells of Escherichia coli DH5-α with pGFPuv, induced by IPTG (isopropyl-β-d-thiogalactopyranoside, express the green fluorescent protein (gfpuv during growth phases. E. coli subjected to the combination of selective permeation by freezing/thawing/sonication cycles followed by the three-phase partitioning extraction (TPP method were compared to the direct application of TPP to the same culture of E. coli on releasing gfpuv from the over-expressing cells. Material and Methods Cultures (37°C/100 rpm/ 24 h; μ = 0.99 h-1 - 1.10 h-1 of transformed (pGFP Escherichia coli DH5-α, expressing the green fluorescent protein (gfpuv, absorbance at 394 nm and emission at 509 nm were sonicated in successive intervals of sonication (25 vibrations/pulse to determine the maximum amount of gfpuv released from the cells. For selective permeation, the transformed previously frozen (-75°C cells were subjected to three freeze/thaw (-20°C/ 0.83°C/min cycles interlaid by sonication (3 pulses/ 6 seconds/ 25 vibrations. The intracellular permeate with gfpuv in extraction buffer (TE solution (25 mM Tris-HCl, pH 8.0, 1 mM β-mercaptoethanol β-ME, 0.1 mM PMSF was subjected to the three-phase partitioning (TPP method with t-butanol and 1.6 M ammonium sulfate. Sonication efficiency was verified on the application to the cells previously treated by the TPP method. The intra-cell releases were mixed and eluted through methyl HIC column with a buffer solution (10 mM Tris-HCl, 10 mM EDTA, pH 8.0. Results The sonication maximum released amount obtained from the cells was 327.67 μg gfpuv/mL (20.73 μg gfpuv/mg total proteins – BSA, after 9 min of treatment. Through the selective permeation by three repeated freezing/thawing/sonication cycles applied to the cells, a close content of 241.19 μg gfpuv/mL (29.74 μg gfpuv/mg BSA was obtained. The specific mass range of gfpuv released from the same cultures, by the three-phase partitioning (TPP

  12. Caracterização de alfagalactosidase e sua relação com a germinação das sementes de Caesalpinia peltophoroides (Leguminosae Caesalpinioideae Alphagalactosidase characterization and its relationship with seed germination of Caesalpinia peltophoroides (Legumonosae Caesalpinioideae

    Directory of Open Access Journals (Sweden)

    Eduardo Euclydes de Lima e Borges


    the micropyle cellular wall. The content of arabinose was higher in the micropyle region, with a tendency to increase during imbibition. Activity of alphagalactosidase was detected in dry seeds in the embryonic axis, as well as in the cotyledons, having increased 24 hours after imbibition. The increase of the activity in the cotyledons was observed only after 120 hours of imbibition. The protein content decreased continuously in the embryonic axis after 24 hours of imbibition, however it kept stable in cotyledons. The activity of the alphagalactosidase was raised by the temperature, being maximum at 55ºC for embryonic axis and at 50ºC for cotyledons. Enzyme activity was favored by pH ranging from 5.5 to 6.0 for the embryonic axis and 4.5 to 5.0 for cotyledons. The effectors SDS, CuSO4, galactose and melibiose inhibited enzyme activity of embryonic axis and cotyledons. Mercaptoethanol showed little stimulating effect on alphagalactosidase in cotyledons. The K M of the embryonic axis and the cotyledons were 1.74 and 2.64, respectively.

  13. An optimized method for extraction and detection of Coconut cadang-cadang viroid(CCCVd) from oil palm. (United States)

    Mohammadi, M R; Vadamalai, G; Joseph, H


    Coconut cadong-cadong viroid (CCCVd) causes the Lethal cadang-cadang disease of coconut palms in the Philippines and it is recently reported to be associated with the orange spotting disease on oil palm in Malaysia. The low concentration of the viroid RNA in oil palm as well as the high content of polyphenols and polysaccharides in this plant which interfere with the purification steps makes it difficult to extract and detect this viroid from oil palm. A previously described method was modified and optimized for extraction and detection of CCCVd from infected oil palms. Briefly, 7 g of leaf material was homogenized in a mortar or a blender using liquid nitrogen. 10 ml of extraction buffer (100 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM EDTA) along with 100 mM 2-mercaptoethanol and 10 ml water saturated phenol was added to the frozen powder. After centrifuging at 4 degrees C, 4000 g for 30 min, the aqueous phase was extracted once more with phenol then once with chloroform-isoamyl alcohol (24:1). After adding sodium acetate, pH 5.6 to 200 mM, the mixture was precipitated with 2.5 vol ethanol overnight in -20 freezer and then the pellet was washed with 70% ethanol and air-dried. One milliliter of 8 M LiCl was added to the dried pellet and after shaking overnight at 4 degrees C and another centrifugation step the supernatant was collected and precipitated again with ethanol and then the resulting pellet was washed and air-dried. To carry out northern blotting, samples equivalent to 40 g of plant tissue were mixed with formamide buffer and loaded onto a 12% polyacrylamide gel containing 7 M urea and after separation by electrophoresis, were electroblotted onto membrane and fixed by UV cross-linking. Pre-hybridization and hybridization using hybridization buffer (50% formamide, 25%SSPE, 0.1% Ficol and PVP, 0.1 % SDS, 0.02 % DNA (5mg/ml)) was carried out at 45 degrees C for 90 min and 16 h, respectively followed by two low stringency washes (0.5 X SSC, 0.1% SDS, at room

  14. Stemcell Information: SKIP000251 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available line derived from Pompe disease patient. Same patient as HPS0175 and HPS0176. ポンペ病患者由来iPS細胞... SKIP000251 ... Diseased HPS0177 HPS0177 ... ポンペ病 E740 Pompe disease 232300 ... -- -- ... No No iPS cell...CQC/TSdeltaF ... Yes MEF (X-rays:5000R or MMC) 3-5x10^(5) cells/60mm dish DMEM/HamF12(2mM L-Glutamine)+20%KSR+0....1mM NEAA+0.1mM 2-Mercaptoethanol+5ng/mL human bFGF ... Riken Bio Resource Center 理研BRC cellこのメールアドレスはスパムボットから保護されています。閲覧するにはJavaScriptを有効にする必要があります。 document.getElementById('cloak86a027676488730bfd30a062b3b10f3d').innerHTML = ''; var prefix = 'ma' + 'il' + 'to'; var path = 'hr' + 'ef' + '='; var addy86a027676488730bfd30a062b3b10f3d = '...ips' + '@'; addy86a027676488730bfd30a062b3b10f3d = addy86a027676488730bfd30a062b3b10f3d + 'brc' + '.' + 'riken' + '.' + 'jp'; var addy_text86a027676488730bfd30a062b3b10f3d = '...ips' + '@' + 'brc' + '.' + 'riken' + '.' + 'jp';document.getElementById('cloak86a027676488730bfd30a062b3b10f3d').innerHTML += ''+addy_text86a027676488730bfd30a062b3b10f3d+''; cellこのメールアドレスはスパムボットから保護されています。閲覧するにはJavaScriptを有効にする必要があります。 document.getElementById('cloake3a09ced5284db348e72a9acb32934e0').innerHTML = ''; var prefix = 'ma' + 'il' + 'to'; var path = 'hr' + 'ef' + '='; var addye3a09ced5284db348e72a9acb32934e0 = 'ips' + '@'; addye3a09ced5284db348e72a9acb32934e0 = addye3a09ced5284db348e72a9acb32934e0 + 'brc' + '.' + 'riken' + '.' + 'jp'; var addy_texte3a09ced5284db348e72a9acb32934e0 = 'ips' + '@' + 'brc' + '.' + 'riken' + '.' + 'jp';document.getElementById('cloake3a09ced5284db348e72a9acb32934e0').innerHTML += ''+addy_texte3a09ced5284db348e72a9acb32934e0+''; Available Riken Bio Resource Center

  15. Production and properties of the cellulase-free xylanase from Thermomyces lanuginosus IOC-4145 Produção e propriedades de xilanase livre de celulase de Thermomyces lanuginosus IOC-4145

    Directory of Open Access Journals (Sweden)

    Mônica Caramez Triches Damaso


    Full Text Available In recent years, xylanases have expanded their use in many processing industries, such as pulp and paper, food and textile. Thermomyces lanuginosus IOC-4145 was able to produce a very high level of cellulase-free xylanase in shaken cultures using corncob as substrate (500 U/mL. An optimization of the medium composition in submerged fermentation was carried out aiming at a low cost medium composition for enzyme production. Statistical experiment design was employed for this purpose, pointing out corncob as the most important parameter, which affects enzyme production. Additionally, the influence of several chemicals on xylanase activity was investigated in the crude extract. A slight stimulation of the enzyme (5-15% was achieved with NaCl and urea, both at 3 and 5 mM of concentration. On the other hand, dithiothreitol and beta-mercaptoethanol at a molarity of 5mM have caused a strong stimulation of the enzyme (40-53%. The crude xylanase displayed appreciable thermostability, retaining almost 50% of activity during 24 hours of incubation at 50ºC; about 50% of activity was present at 60ºC even after 4 hours of incubation. The enzyme also exhibited good storage stability at -20ºC without any stabilizing agent.Nos últimos anos tem crescido o uso de xilanases em muitas indústrias, tais como polpa e papel, alimentos e têxtil. Thermomyces lanuginosus IOC-4145 foi capaz de produzir um alto nível de xilanase livre de celulase em culturas agitadas usando sabugo de milho como substrato (500 U/mL. Procedeu-se, inicialmente, à otimização da composição do meio de produção em fermentação submersa, com o intuito de alcançar uma composição de meio de produção de baixo custo para produção da enzima. Para este propósito, utilizou-se planejamento estatístico de experimentos. O sabugo de milho revelou-se como a mais importante variável que afeta a produção enzimática. Adicionalmente, a influência de vários reagentes na atividade xilan


    Directory of Open Access Journals (Sweden)

    I. S. Malakhov


    infection value of stated vaccines. According to information from literature, N-terminus of the L2 protein can induce non strain-specific neutralizing antibody that protects organism from papillomavirus challenge. E7 protein is a virus oncogene, its function is unlimited proliferation of infected cells that cause malignization in chronic course of disease. This protein is a very attractive target for therapeutic vaccines because of its necessity both for virus life cycle and sustenance of malignant phenotype in cancer cells. So, in this research the design of immunogen on the base of proteins HPV L2 and E7 is selected, vaccine on the base of which will avoid the disadvantages of Gardasil and Cervarix listed above. The stain-producer of protein on the base of cells E. coli was created. The protein was purified in denaturing reducing conditions by metal-affine chromatography and refold by sequential remove of urea and 2-mercaptoethanol.

  17. Teste de hemaglutinação na sorologia da malária humana empregando hemácias parasitadas pelo Plasmodium berghei

    Directory of Open Access Journals (Sweden)

    Maria Carmen Arroyo Sanchez-Ruiz


    the hemagglutination test could result from its reactivity with IgM antibodies. After 2-mercaptoethanol treatment, all serafrom patients with patent parasitemia were non-reactive. The hemagglutination test, with reference to the IgG-immunofluorescence test, showed a 0.989 co-positivity for patients with a patent parasitemia and a 0.969 co- negativity for non-malarialpatients. For residents in endemic areas a co-positivity of 0.528 and a co- negativity of0.833 were observed.

  18. Synthesis and characterization of CdS nanoparticle/polymer composites (United States)

    Lama, Bimala

    Pure, sulfur rich and wurtzite phase CdS nanoparticles with average size ~4.7 nm were prepared in aqueous solution using thioglycerol as a capping ligand. Approximately 542 molecules of thioglycerol molecules were present on the surface of each CdS-TG nanoparticle. CdS-TG nanoparticles can trap a large amount of water molecules and the chemical shift of the trapped water molecules are dependent on the environment and the amount of water trapped. The presence of sodium ions in CdS-TG increases the order of thioglycerol molecules due to the interaction with ions. Relaxation values indicated the interaction between TSLi molecule and thioglycerol. Formation of the hydrophobic monolayer of TSLi on the outer surface of CdS-TG nanoparticles were confirmed by 2D-HETCOR studies. At the interface, cations are far from the aromatic ring and thioglycerol molecules and remain in water pockets with some motions. Pure, sulfur rich, wurtzite phase CdS-TEG nanoparticles with average size of ~4.5 nm were prepared using 2-mercaptoethanol (also known as thioethyleneglycol, TEG) as a capping ligand. Grafting of aromatic ring containing sulfonyl chloride with CdS-TEG nanoparticles through sulfonate ester was studied using benzene sulfonyl chloride in the basic aqueous medium. NMR studies confirmed the feasibility of the reaction and indicated that the rate of esterification reaction increased with increase in concentration of benzene sulfonyl chloride. Naphthalene sulfonyl chloride with CdS-TEG nanoparticles were used to study the photoluminescence behavior before and after the reaction. Quenching of the light observed in the naphthalene rings bonded to the nanoparticles and confirmed that electron or energy transfer took place easily in covalently bonded aromatic rings and nanoparticles. Grafting of polystyrene chain was done by changing polystyrene sulfonic acid to the polystyrene sulfonyl chloride. Some aromatic rings in polymer were bonded with nanoparticles through ester bond and

  19. Mycelial glucoamylases produced by the thermophilic fungus Scytalidium thermophilum strains 15.1 and 15.8: purification and biochemical characterization Glucoamilases miceliais produzidas pelas linhagens 15.1 e 15.8 do fungo termofílico Scytalidium thermophilum: purificação e caracterização bioquímica

    Directory of Open Access Journals (Sweden)

    M.S. Ferreira-Nozawa


    Full Text Available Two strains (15.1 and 15.8 of the thermophilic fungus Scytalidium thermophilum produced high levels of intracellular glucoamylases, with potential for industrial applications. The isoform I of the glucoamylase produced by 15.1 strain was sequentially submitted to DEAE-Cellulose and CM-Cellulose chromatography, and purified 141-fold, with 5.45% recovery. The glucoamylase of strain 15.8 was purified 71-fold by CM-Cellulose and Concanavalin A-Sepharose chromatography, with 7.38% recovery. Temperature and pH optima were in the range of 50-60ºC and 5.0-6.0, respectively, using starch and maltose as substrates. The glucoamylase of S. thermophilum 15.8 was more stable (t50 > 60 min than that of S. thermophilum 15.1 (t50= 11-15 min, at 60ºC. The glucoamylase activities were enhanced by several ions (e.g. Mn2+ and Ca2+ and inhibited by β-mercaptoethanol. The glucoamylase from 15.1 strain showed a Km of 0.094 mg/ml and 0.029 mg/ml and Vmax of 202 U/mg prot and 109 U/mg prot, for starch and maltose, respectively. The hydrolysis products of starch and maltose, analyzed by TLC, demonstrated glucose as end product and confirming the character of the enzyme as glucoamylase. Differences were observed in relation to the products formed with maltose as substrate between the two strains studied. S. thermophilum 15.8 formed maltotriose in contrast with S. thermophilum 15.1.Duas linhagens (15.1 e 15.8 do fungo termofílico Scytalidium thermophilum se mostraram produtoras de grandes quantidades de glucoamilases, com potencial aplicação industrial. A isoforma I de glucoamilase produzida pela linhagem 15.1 foi submetida seqüencialmente a cromatografia em colunas de DEAE-celulose e CM-celulose, sendo purificada 141 vezes com porcentagem de recuperação de 5,45%. A glucoamilase da linhagem 15.8 foi purificada 71 vezes através do uso de colunas de cromatografia de CM-celulose e Concanavalina A-sepharose com porcentagem de recuperação de 7,38%. Temperatura e pH

  20. Situação epidemiológica da brucelose bovina no Estado de Sergipe Epidemiological situation of bovine brucellosis in the State of Sergipe, Brazil

    Directory of Open Access Journals (Sweden)

    V.G.S.O. Silva


    that could be associated with the risk of infection. The serum samples were screened by the Rose-Bengal Test (RBT and positive sera were re-tested by the 2-mercaptoethanol test (2-ME. The herd was considered positive if at least one animal was positive on both RBT and 2-ME tests. The prevalences of infected herds and animals in the State were, respectively: 12.6% [9.2-16.0%] and 3.4% [2.3-4.4%]. The prevalences of infected herds and animals in the regions were, respectively: region 1, 11.1% [7.9-15.0%] and 2.6% [1.6-3.5%]; and region 2, 12.9% [9.1-17.6%] and 6.2% [3.0-9.5%]. The risk factors associated with the presence of the infection were: veterinary assistance (OR= 2.89 [1.15-7.23], herd size ≥30 adult females (OR= 1.88 [1.07-3.28], and use of artificial insemination (OR=1.92 [0.84-4.38].

  1. Detección de anticuerpos anti-Brucella spp. en cerdos mediante técnicas de aglutinación y ELISA indirecto en las provincias de Buenos Aires y La Pampa: Argentina Detection of anti-Brucella spp. antibodies in swine by agglutination techniques and indirect ELISA in the Buenos Aires and La Pampa provinces: Argentina

    Directory of Open Access Journals (Sweden)

    H.A. Castro


    Full Text Available En nuestro país no existe un programa de control sobre brucelosis porcina y su verdadera situación epidemiológica es desconocida. El objetivo de nuestro trabajo fue detectar la presencia de anticuerpos anti-Brucella spp. en porcinos provenientes de criaderos del sudoeste de la provincia de Buenos Aires y del este de la provincia de La Pampa. La toma de muestras de sangre se realizó en el momento del faenado de los animales. La detección de anticuerpos se efectuó mediante las técnicas de aglutinación con antígeno tamponado en placa (BPA, seroaglutinación en tubo (SAT, aglutinación con 2-ME (2-ME y ELISA indirecto, con dos antígenos diferentes: el antígeno CYT (fracción citoplasmática de B. abortus S19 y el antígeno CP (extracto citoplasmático libre de lipopolisacárido. Del total de las muestras analizadas (n=325, el 17,8% fue positivo para BPA, el 13,8% fue positivo para SAT y sólo el 8,0% fue positivo para 2-ME. Mediante ELISA-CYT, este porcentaje se elevó a 21,0%, mientras que a través del ELISA-CP sólo se halló un 10,0% de muestras reactivas. Estos resultados son compatibles con los informados en los escasos reportes previos para todo el país y sugieren la necesidad de extender los estudios a otras zonas, donde sea habitual la cría de cerdos.Porcine brucellosis is one of the most important zoonoses in this country. Currently, there is no control program for porcine brucellosis in Argentina and the epidemiological situation is still unknown. The purpose of our study was to detect anti-Brucella spp. antibodies in swine in the southwest of the Buenos Aires province and the east of the La Pampa province. Blood samples were obtained when animals were slaughtered. The presence of anti-brucella antibodies was studied by the buffered plate agglutination test (BPA, the tube agglutination test (SAT, the 2-mercaptoethanol (2-ME agglutination test and indirect ELISA tests, using the cytosolic fraction from Brucella abortus S19

  2. Extração, purificação e caracterização físico-química da proteína verde fluorescente recombinante (GFPuv expressa em Escherichia coli Extraction, purification and physical-chemical characterization of recombinant green fluorescent protein (GFPuv expressed in Escherichia coli cells

    Directory of Open Access Journals (Sweden)

    Eb Chiarini


    Full Text Available O aumento do uso da proteína verde fluorescente (GFPuv como ferramenta de pesquisas biotecnológicas requer um estudo mais cuidadoso das propriedades bioquímicas e físicas da molécula de GFPuv. Este trabalho teve como objetivo a aplicação de métodos físicos e químicos para o isolamento, a extração da GFPuv de células de Escherichia coli DH5-±, purificação da proteína, e o estudo da estabilidade desta em diferentes valores de pH. Células de E. coli expressando GFPuv foram submetidas a quatro ciclos sucessivos de congelamento e descongelamento intercalados por sonicação (CDS, para promover a permeação seletiva da GFPuv. Os permeados foram submetidos à extração por partição em três fases (TPP e posterior purificação por eluição da proteína em coluna cromatográfica de interação hidrofóbica (HIC.Obteve-se rendimento semelhante em GFPuv no 1º ciclo de permeação seletiva (CDS e por extração (TPP associada à purificação (HIC para os quais impurezas não foram visualizadas por eletroforese. As estruturas moleculares da GFPuv extraída e purificada mostraram-se inalteradas em valores de pH entre 6,0 e 9,8, e foram confirmadas nos espectros de emissão e de excitação.The recombinant green fluorescent protein (GFPuv has been used as a marker in several research fields. The purpose of the present work was to evaluate the influence of the selective physical permeation procedure applied to the cells of Escherichia coli for the extraction of GFPuv in relation to the chemical procedures of extraction and purification. Transformed cells (0.92-1.44 mg/mL of E. coli DH5-a expressing GFPuv were submitted to four cycles (1º, 2º, 3º, 4º of freezing (-20 ºC/ 0.83 ºC/ min/thawing interlaid by sonication (3 pulses/6 s/25 vibrations. The intracellular permeate with GFPuv in buffer solution (Tris-HCl 25 mM pH 8.0 + b-mercaptoethanol (1 mM + PMSF (0.1 mM was submitted to the three-phase partitioning (TPP method and

  3. Situação epidemiológica da brucelose bovina no Estado do Rio de Janeiro Epidemiological situation of bovine brucellosis in the State of Rio de Janeiro, Brazil

    Directory of Open Access Journals (Sweden)

    M.F.C. Klein-Gunnewiek


    focused on herd traits as well as husbandry and sanitary practices that could be associated with the risk of infection. The serum samples were screened for antibodies against Brucella spp. by the Rose-Bengal Test (RBT, and all positive sera were re-tested by the 2-mercaptoethanol test (2-ME. The herd was considered positive if at least one animal was positive on both RBT and 2-ME tests. The prevalences of infected herds and animals in the State were, respectively: 15.4% [12.9-17.9%] and 4.1% [2.8-5.3%]. The prevalences of infected herds and animals in the regions were, respectively: region 1, 13.8% [10.2-18.2%] and 3.0% [1.9-4.1%]; region 2, 15.7% [11.9-20.2%] and 2.3% [1.4-3.2%]; and region 3, 19.6% [15.4-24.4%] and 9.3% [4.5-14.1%]. The risk factors (odds ratio, OR associated with the presence of the infection were: herd size larger than 30 cows (OR=2.33 [1.51-3.07], purchase of animals for breeding (OR= 1.95 [1.13-2.45, and pasture rental practice (R= 1.74 [1.03-2.74].

  4. Situação epidemiológica da brucelose bovina no Estado de Goiás Epidemiological status of bovine brucellosis in the State of Goiás, Brazil

    Directory of Open Access Journals (Sweden)

    W.V. Rocha


    -established number of animals was sampled in each of these herds. A total of 10,744 serum samples from 900 herds were collected. In each herd, it was applied an epidemiological questionnaire focused on herd traits as well as husbandry and sanitary practices that could be associated with the risk of infection. The serum samples were screened for antibodies against Brucella spp. by the Rose-Bengal Test (RBT, and all positive sera were re-tested by the 2-Mercaptoethanol test (2-ME. The herd was considered positive if at least one animal was positive on both RBT and 2-ME tests. For region 1, the herd prevalence was 7.7% [4.7-10.7%] and the animal prevalence was 1.4% [0.99-1.7%]. For region 2, the herd prevalence was 19.5% [15.0-24.0%] and the animal prevalence was 2.6% [2.0-3.1%]. For region 3, the herd prevalence was 21.4% [16.8-26.1%] and the animal prevalence was 4.3% [3.7-5.0%]. For the whole state, the herd prevalence was 17.5% [14.9-20.2%] and the animal prevalence was 3.0% [2.7-3.3%]. The multivariate analysis identified the following risk factors (odds ratio, OR associated with positive herds: purchase of breeding stock from cattle traders (OR = 2.06 [1.12-3.52], occurrence of abortions over the last 12 months (OR = 5.83 [3.86-8.8], and vaccination against brucellosis (OR = 2.07 [1.38-3.09]. Both the abortions and the vaccination are, in this case, a consequence of the herd being infected with brucellosis.

  5. Situação epidemiológica da brucelose bovina no Estado da Bahia Epidemiological situation of bovine brucellosis in the State of Bahia, Brazil

    Directory of Open Access Journals (Sweden)

    A.J.S. Alves


    Full Text Available O trabalho consistiu em estratificar o Estado da Bahia em quatro regiões com características homogêneas (circuitos produtores para que fossem amostradas aleatoriamente, em cada uma delas, 300 propriedades. Em cada propriedade foram escolhidas, de forma aleatória, 10 a 15 fêmeas bovinas adultas, das quais foi obtida uma amostra de sangue. No total, foram amostrados 10.816 animais, provenientes de 1.413 propriedades. O protocolo de testes utilizado foi o da triagem com o teste do antígeno acidificado tamponado (Rosa Bengala e a confirmação dos positivos com o teste do 2-mercaptoetanol. O rebanho foi considerado positivo se pelo menos um animal reagiu às duas provas sorológicas. As prevalências de focos e a de fêmeas adultas soropositivas do Estado foram de 4,2% [3,1-5,3%] e 0,66% [0,41-0,93%], respectivamente. Para os circuitos produtores foram: circuito 1, 5,8% [3,6-8,7%] e 0,86% [0,41-1,3%]; circuito 2, 3,1% [1,5-5,6%] e 1,2% [0,25-2,1%]; circuito 3, 6,3% [4,0-9,3%] e 1,7% [0,66-2,7%]; e circuito 4, 0,60% [0,07-2,2%] e 0,07 [0,00-0,21%]. Para a análise de fatores de riscos associados à doença foi aplicado um questionário epidemiológico em cada propriedade visitada. Os fatores de risco (odds ratio, OR associados à condição de foco foram: compra de reprodutores (OR= 2,27 e presença de áreas alagadiças (OR= 1,76. A vacinação de fêmeas de três até oito meses de idade foi um fator de proteção (OR= 0,53.A study to characterize the epidemiological situation of bovine brucellosis in the State of Bahia was carried out in 2004. The State was divided into four similar production regions, 300 herds were randomly sampled in each region, and 10 to 15 adult bovine females were sampled in each of these herds. A total of 10,816 serum samples from 1,413 herds were collected. The serum samples were screened for antibodies to Brucella spp. by the Rose-Bengal Test (RBT, and all RBT-positive sera were re-tested by the 2-mercaptoethanol

  6. Validação interlaboratorial do teste de polarização fluorescente para o diagnóstico sorológico da brucelose bovina Interlaboratorial validation of the fluorescence polarization assay for the serodiagnosis of bovine brucellosis

    Directory of Open Access Journals (Sweden)

    Luis Antonio Mathias


    , the relative specificity and the reproducibility of the test in four Brazilian laboratories. Serum samples from 1,389 bovines were selected and submitted to the rose Bengal (RBT and 2-mercaptoethanol tests in one of the laboratories. The same samples were tested by the complement fixation (CFT test and by the PFA in the four laboratories participating of the research. The reference population (golden standard used to evaluate the PFA was the combination of the results of RBT, CFT and 2-ME. TG-ROC analysis was used to obtain the cut-off that provided the best combination of sensitivity and specificity. The agreement between laboratories was obtained by the kappa statistic and Pearson correlation coefficient (r. The PFA cut-off values were from 85.2 to 93.6. The sensitivity of the PFA assay varied from 91.7% to 97.3%, and the specificity values varied from 82.6% to 98.3%. When comparing PFA results from the four laboratories, the kappa values was between 0.69 and 0.95, which indicates, in most situations, excellent reproducibility, and the correlation coefficient varied from 0.76 to 0.99. The results showed that the PFA had a good performance, with high sensitivity and specificity. Compared to the conventional tests, the PFA has the advantages of being easy and quick to perform, and it is not prone to the occurrence of prozone, as the CFT or the 2-ME, nor to the occurrence of anti-complementary effect, as the CFT.

  7. Situação epidemiológica da brucelose bovina no Estado do Espírito Santo Epidemiological situation of bovine brucellosis in the State of Espírito Santo, Brazil

    Directory of Open Access Journals (Sweden)

    S.S. Azevedo


    questionnaire focused on herd traits as well as husbandry and sanitary practices that could be associated with the risk of infection. The serum samples were screened for antibodies against Brucella spp. by the Rose-Bengal Test (RBT, and all positive sera were re-tested by the 2-mercaptoethanol test (2-ME. The herd was considered positive if at least one animal was positive on both RBT and 2-ME tests. The prevalence of infected herds and animals in the State were, respectively, 9.0% [7.0-11.6%] and 3.5% [1.9-6.4%]. The prevalence of infected herds and animals in the regions were, respectively: region 1, 6.8% [4.5-10.2%] and 3.4% [1.3-8.6%]; and region 2, 10.9% [7.9-14.8%] and 3.7% [2.1-6.3%]. The risk factors (odds ratio, OR associated with the presence of the infection were: use of artificial insemination (OR = 7.05 [2.51-19.82] and intensive/semi-intensive management systems (OR = 2.98 [1.22-7.26]. Vaccination of heifers from three to eight months of age was a protective factor (OR = 0.03 [0.01-0.1].

  8. Enfermedad de Chagas congenita en la Ciudad de Salta, Argentina Congenital Chagas' disease in Salta, Argentina

    Directory of Open Access Journals (Sweden)

    Mario Zaidenberg


    presence of T. cruzi in blood, explored in fresh smears by serial micro-hematocrite and/or by xenodiagnosis, was the only criterion to define infection in NB. All NB were followed up by direct agglutination (DA with or without 2 mercaptoethanol (DA-w2ME, DA-wo2ME and IIF in order to establish the specific antibody kinetics. Clinical studies on NB with T. cruzi infection include routine laboratory tests. Benznidazole (3 to 7 mg/kg/day and, in 1 case, nifurtimox (15 mg/kg/day were employed as therapeutic agents. T. cruzi infection was confirmed in 149 PW (15.9%, table I. These chagasic mothers delivered 6 chagasic NB (CCHD-NB, (4%. Diagnosis of congenital Chagas' disease accounted for a total of 12 NB out of the 968 studied. 4 out of them were positive by both micro-hematocrite and blood smears and 7 by micro-hematocrite alone. Xenodiagnosis was performed in 2 NB resulting positive in both cases, table II. The most usual clinical findings included hepatomegaly (present in all cases, splenomegaly 8/12, jaundice 10/12 and prematurity 5/12, table 3. Laboratory findings showed anemia to be of hypochromic microcytic type in all cases. Other laboratory findings included lymphocytosis, normal erythrosedimentation, slight to moderate increase of transaminases in all cases, and elevated indirect bilirrubin in cases with jaundice, table 4. Analysis of cerebro spinal fluid in 6 CCh-NB revealed the presence of T. cruzi in 2 cases, plus abnormal cytochemical content in one of them, table 4. The serological reactions of infected and treated NB became negative between 4th and 8th month in all but 1 case that remained positive until 14th, fig. 1. A close correlation was found between DA and IIF. DA-w2ME liter showed a significant drop during the initial phase of the controls. Benznidazole was successful in 11 out of the 12 CCh-NB. The remaining NB was effectively treated with nifurtimox. Therapeutic tolerance was satisfactory for both agents. These observations showed that congenital Chagas

  9. Situação epidemiológica da brucelose bovina no Estado de Minas Gerais Epidemiological status of bovine brucellosis in the State of Minas Gerais, Brazil

    Directory of Open Access Journals (Sweden)

    V.S.P. Gonçalves


    eficaz ao reduzir a prevalência em todo o Estado e em todos os sistemas de produção animal. As autoridades sanitárias devem priorizar o controle da compra de animais para reprodução, que não apresentem garantias sanitárias e incorporar essa medida às ações de educativas.A study to characterize the epidemiological status of brucellosis was carried out in the State of Minas Gerais. The State was divided in seven regions. Three hundred herds were randomly sampled in each region and a pre-established number of animals was sampled in each of these herds. A total of 20,643 serum samples from 2,204 herds were collected. In each herd, it was applied an epidemiological questionnaire focused on herd traits as well as husbandry and sanitary practices that could be associated with the risk of infection. The serum samples were screened for antibodies against Brucella spp. by the Rose-Bengal Test (RBT, and all positive sera were re-tested by the 2-mercaptoethanol test (2-ME. The herd was considered positive if at least one animal was positive on both RBT and 2-ME tests. The prevalence of infected herds and animals in the State were, respectively, 6.0% [5.0-7.1%] and 1.1% [0.78-1.4%]. In the productive regions, the prevalence of infected herds and animals were, respectively: regions 1, 4.7% [2.7-7.7%] and 0.82% [0.06-1.6%]; region 2, 7.2% [4.6-10.6%] and 1.2% [0.53-1.8%]; region 3, 6.8% [4.3-10.0%] and 1.5% [0.47-2.4%]; region 4, 6.5% [4.1-9.8%] and 1.1% [0.39-1.7%]; region 5, 3.8% [2.0-6.5%] and 0.40% [0.11-0.69%]; region 6, 6.2% [3.8-9.6%] and 0.66% [0.29-1.0%]; and region 7, 11.0% [7.7-15.0%] and 1.7% [0.92-2.6%]. The risk factors (odds ratio, OR associated with positive herds were: purchase of breeding stock (OR = 1.66 [1.13-2.44], occurrence of abortions over the last 12 months (OR = 1.81 [1.26-2.60], and the presence of deer in the farm (OR = 1.56 [1.08-2.27]. Vaccination against brucellosis was a protective factor (OR = 0.38 [0.19-0.79]. It can be concluded that the